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Sample records for alleles conferring resistance

  1. Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae.

    PubMed Central

    Génissel, Anne; Augustin, Sylvie; Courtin, Claudine; Pilate, Gilles; Lorme, Philippe; Bourguet, Denis

    2003-01-01

    Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F(2) screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.00045-0.0080) with a detection probability of 90%. These results demonstrate that (i) the F(2) screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates. PMID:12737656

  2. Specific HLA-DRB and -DQB Alleles and Haplotypes Confer Disease Susceptibility or Resistance in Bahraini Type 1 Diabetes Patients

    PubMed Central

    Al-Harbi, Einas M.; Abbassi, Abdul-Jabbar; Tamim, Hala; al-Jenaidi, Fayza; Kooheji, Mariam; Kamal, Madeeha; al-Mahroos, Salwa; al-Nasir, Faisal; Motala, Ayesha A.; Almawi, Wassim Y.

    2004-01-01

    Insofar as genetic susceptibility to type 1 diabetes is associated with HLA class II genes, with certain allelic combinations conferring disease susceptibility or resistance, this study assessed the distributions of HLA-DR and -DQ among 107 unrelated patients with type 1 diabetes and 88 healthy controls from Bahrain, all of Arab origin. The HLA-DRB and -DQB genotypes were determined by PCR-sequence-specific priming. The following alleles showed the strongest association with type 1 diabetes among patients versus controls according to their frequencies: DRB1*030101 (0.430 versus 0.097; P < 0.001), DRB1*040101 (0.243 versus 0.034; P < 0.001), DQB1*0201 (0.467 versus 0.193; P < 0.001), and DQB1*0302 (0.229 versus 0.091; P < 0.001). When the frequencies of alleles in controls were compared to those in patients, negative associations were seen for DRB1*100101 (0.085 versus 0.014; P < 0.001), DRB1*110101 (0.210 versus 0.060; P < 0.001), DQB1*030101 (0.170 versus 0.075; P = 0.006), and DQB1*050101 (0.335 versus 0.121; P < 0.001). In addition, the DRB1*030101-DQB1*0201 (70.1 versus 22.7%; P < 0.001) and DRB1*030101-DQB1*0302 (21.5 versus 0.0%; P < 0.001) genotypes were more prevalent among patients, thereby conferring disease susceptibility, whereas the DRB1*100101-DQB1*050101 (20.5 versus 2.8%; P < 0.001), DRB1*110101-DQB1*030101 (28.4 versus 8.4%; P < 0.001), and DRB1*110101-DQB1*050101 (30.7 versus 0.9%; P < 0.001) genotypes were more prevalent among controls, thus assigning a protective role. These results confirm the association of specific HLA-DR and -DQ alleles and haplotypes with type 1 diabetes and may underline several characteristics that distinguish Bahraini patients from other Caucasians patients. PMID:15013978

  3. Specific HLA-DRB and -DQB alleles and haplotypes confer disease susceptibility or resistance in Bahraini type 1 diabetes patients.

    PubMed

    Al-Harbi, Einas M; Abbassi, Abdul-Jabbar; Tamim, Hala; al-Jenaidi, Fayza; Kooheji, Mariam; Kamal, Madeeha; al-Mahroos, Salwa; al-Nasir, Faisal; Motala, Ayesha A; Almawi, Wassim Y

    2004-03-01

    Insofar as genetic susceptibility to type 1 diabetes is associated with HLA class II genes, with certain allelic combinations conferring disease susceptibility or resistance, this study assessed the distributions of HLA-DR and -DQ among 107 unrelated patients with type 1 diabetes and 88 healthy controls from Bahrain, all of Arab origin. The HLA-DRB and -DQB genotypes were determined by PCR-sequence-specific priming. The following alleles showed the strongest association with type 1 diabetes among patients versus controls according to their frequencies: DRB1*030101 (0.430 versus 0.097; P < 0.001), DRB1*040101 (0.243 versus 0.034; P < 0.001), DQB1*0201 (0.467 versus 0.193; P < 0.001), and DQB1*0302 (0.229 versus 0.091; P < 0.001). When the frequencies of alleles in controls were compared to those in patients, negative associations were seen for DRB1*100101 (0.085 versus 0.014; P < 0.001), DRB1*110101 (0.210 versus 0.060; P < 0.001), DQB1*030101 (0.170 versus 0.075; P = 0.006), and DQB1*050101 (0.335 versus 0.121; P < 0.001). In addition, the DRB1*030101-DQB1*0201 (70.1 versus 22.7%; P < 0.001) and DRB1*030101-DQB1*0302 (21.5 versus 0.0%; P < 0.001) genotypes were more prevalent among patients, thereby conferring disease susceptibility, whereas the DRB1*100101-DQB1*050101 (20.5 versus 2.8%; P < 0.001), DRB1*110101-DQB1*030101 (28.4 versus 8.4%; P < 0.001), and DRB1*110101-DQB1*050101 (30.7 versus 0.9%; P < 0.001) genotypes were more prevalent among controls, thus assigning a protective role. These results confirm the association of specific HLA-DR and -DQ alleles and haplotypes with type 1 diabetes and may underline several characteristics that distinguish Bahraini patients from other Caucasians patients.

  4. Mutated Cadherin Alleles from a Field Population of Helicoverpa armigera Confer Resistance to Bacillus thuringiensis Toxin Cry1Ac▿

    PubMed Central

    Yang, Yajun; Chen, Haiyan; Wu, Yidong; Yang, Yihua; Wu, Shuwen

    2007-01-01

    The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r1) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r2 and r3) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r2 and r3 alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera. PMID:17827322

  5. A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence

    PubMed Central

    Jiao, Yuntong; Xu, Weirong; Duan, Dong; Wang, Yuejin; Nick, Peter

    2016-01-01

    Stilbenes are central phytoalexins in Vitis, and induction of the key enzyme stilbene synthase (STS) is pivotal for disease resistance. Here, we address the potential for breeding resistance using an STS allele isolated from Chinese wild grapevine Vitis pseudoreticulata (VpSTS) by comparison with its homologue from Vitis vinifera cv. ‘Carigane’ (VvSTS). Although the coding regions of both alleles are very similar (>99% identity on the amino acid level), the promoter regions are significantly different. By expression in Arabidopsis as a heterologous system, we show that the allele from the wild Chinese grapevine can confer accumulation of stilbenes and resistance against the powdery mildew Golovinomyces cichoracearum, whereas the allele from the vinifera cultivar cannot. To dissect the upstream signalling driving the activation of this promoter, we used a dual-luciferase reporter system in a grapevine cell culture. We show elevated responsiveness of the promoter from the wild grape to salicylic acid (SA) and to the pathogen-associated molecular pattern (PAMP) flg22, equal induction of both alleles by jasmonic acid (JA), and a lack of response to the cell death-inducing elicitor Harpin. This elevated SA response of the VpSTS promoter depends on calcium influx, oxidative burst by RboH, mitogen-activated protein kinase (MAPK) signalling, and JA synthesis. We integrate the data in the context of a model where the resistance of V. pseudoreticulata is linked to a more efficient recruitment of SA signalling for phytoalexin synthesis. PMID:27702992

  6. Characterization of a New Pm2 Allele Conferring Powdery Mildew Resistance in the Wheat Germplasm Line FG-1.

    PubMed

    Ma, Pengtao; Xu, Hongxng; Li, Lihui; Zhang, Hongxia; Han, Guohao; Xu, Yunfeng; Fu, Xiaoyi; Zhang, Xiaotian; An, Diaoguo

    2016-01-01

    Powdery mildew has a negative impact on wheat production. Novel host resistance increases the diversity of resistance genes and helps to control the disease. In this study, wheat line FG-1 imported from France showed a high level of powdery mildew resistance at both the seedling and adult stages. An F2 population and F2:3 families from the cross FG-1 × Mingxian 169 both fit Mendelian ratios for a single dominant resistance gene when tested against multiple avirulent Blumeria tritici f. sp. tritici (Bgt) races. This gene was temporarily designated PmFG. PmFG was mapped on the multi-allelic Pm2 locus of chromosome 5DS using seven SSR, 10 single nucleotide polymorphism (SNP)-derived and two SCAR markers with the flanking markers Xbwm21/Xcfd81/Xscar112 (distal) and Xbwm25 (proximal) at 0.3 and 0.5 cM being the closest. Marker SCAR203 co-segregated with PmFG. Allelism tests between PmFG and documented Pm2 alleles confirmed that PmFG was allelic with Pm2. Line FG-1 produced a significantly different reaction pattern compared to other lines with genes at or near Pm2 when tested against 49 Bgt isolates. The PmFG-linked marker alleles detected by the SNP-derived markers revealed significant variation between FG-1 and other lines with genes at or near Pm2. It was concluded that PmFG is a new allele at the Pm2 locus. Data from seven closely linked markers tested on 31 wheat cultivars indicated opportunities for marker-assisted pyramiding of this gene with other genes for powdery mildew resistance and additional traits. PMID:27200022

  7. Characterization of a New Pm2 Allele Conferring Powdery Mildew Resistance in the Wheat Germplasm Line FG-1

    PubMed Central

    Ma, Pengtao; Xu, Hongxng; Li, Lihui; Zhang, Hongxia; Han, Guohao; Xu, Yunfeng; Fu, Xiaoyi; Zhang, Xiaotian; An, Diaoguo

    2016-01-01

    Powdery mildew has a negative impact on wheat production. Novel host resistance increases the diversity of resistance genes and helps to control the disease. In this study, wheat line FG-1 imported from France showed a high level of powdery mildew resistance at both the seedling and adult stages. An F2 population and F2:3 families from the cross FG-1 × Mingxian 169 both fit Mendelian ratios for a single dominant resistance gene when tested against multiple avirulent Blumeria tritici f. sp. tritici (Bgt) races. This gene was temporarily designated PmFG. PmFG was mapped on the multi-allelic Pm2 locus of chromosome 5DS using seven SSR, 10 single nucleotide polymorphism (SNP)-derived and two SCAR markers with the flanking markers Xbwm21/Xcfd81/Xscar112 (distal) and Xbwm25 (proximal) at 0.3 and 0.5 cM being the closest. Marker SCAR203 co-segregated with PmFG. Allelism tests between PmFG and documented Pm2 alleles confirmed that PmFG was allelic with Pm2. Line FG-1 produced a significantly different reaction pattern compared to other lines with genes at or near Pm2 when tested against 49 Bgt isolates. The PmFG-linked marker alleles detected by the SNP-derived markers revealed significant variation between FG-1 and other lines with genes at or near Pm2. It was concluded that PmFG is a new allele at the Pm2 locus. Data from seven closely linked markers tested on 31 wheat cultivars indicated opportunities for marker-assisted pyramiding of this gene with other genes for powdery mildew resistance and additional traits. PMID:27200022

  8. Detection of an allele conferring resistance to Bacillus sphaericus binary toxin in Culex quinquefasciatus populations by molecular screening.

    PubMed

    Chalegre, Karlos Diogo de Melo; Romão, Tatiany Patrícia; Amorim, Liliane Barbosa; Anastacio, Daniela Bandeira; de Barros, Rosineide Arruda; de Oliveira, Cláudia Maria Fontes; Regis, Lêda; de-Melo-Neto, Osvaldo Pompílio; Silva-Filha, Maria Helena Neves Lobo

    2009-02-01

    The activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound alpha-glucosidase. A 19-nucleotide deletion in the cqm1 gene (cqm1(REC)) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus-treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1(REC) allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus, comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1(REC) allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus-treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through alpha-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1(REC) allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus. This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations. PMID:19098223

  9. Genetic mapping, marker assisted selection and allelic relationships for the Pu 6 gene conferring rust resistance in sunflower.

    PubMed

    Bulos, Mariano; Vergani, Pablo Nicolas; Altieri, Emiliano

    2014-09-01

    Rust resistance in the sunflower line P386 is controlled by Pu 6 , a gene which was reported to segregate independently from other rust resistant genes, such as R 4 . The objectives of this work were to map Pu 6 , to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu 6 and R 4 . Genetic mapping of Pu 6 with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu 6 was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu 6 and R 4 indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the R adv /R 4 /R 11 / R 13a /R 13b /Pu 6 cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.

  10. Discovery of a Novel er1 Allele Conferring Powdery Mildew Resistance in Chinese Pea (Pisum sativum L.) Landraces.

    PubMed

    Sun, Suli; Fu, Haining; Wang, Zhongyi; Duan, Canxing; Zong, Xuxiao; Zhu, Zhendong

    2016-01-01

    Pea powdery mildew, caused by Erysiphe pisi D.C., is an important disease worldwide. Deployment of resistant varieties is the main way to control this disease. This study aimed to screen Chinese pea (Pisum sativum L.) landraces resistant to E. pisi, and to characterize the resistance gene(s) at the er1 locus in the resistant landraces, and to develop functional marker(s) specific to the novel er1 allele. The 322 landraces showed different resistance levels. Among them, 12 (3.73%), 4 (1.24%) and 17 (5.28%) landraces showed immunity, high resistance and resistance to E. pisi, respectively. The other landraces appeared susceptible or highly susceptible to E. pisi. Most of the immune and highly resistant landraces were collected from Yunnan province. To characterize the resistance gene at the er1 locus, cDNA sequences of PsMLO1 gene were determined in 12 immune and four highly resistant accessions. The cDNAs of PsMLO1 from the immune landrace G0005576 produced three distinct transcripts, characterized by a 129-bp deletion, and 155-bp and 220-bp insertions, which were consistent with those of er1-2 allele. The PsMLO1 cDNAs in the other 15 resistant landraces produced identical transcripts, which had a new point mutation (T→C) at position 1121 of PsMLO1, indicating a novel er1 allele, designated as er1-6. This mutation caused a leucine to proline change in the amino acid sequence. Subsequently, the resistance allele er1-6 in landrace G0001778 was confirmed by resistance inheritance analysis and genetic mapping on the region of the er1 locus using populations derived from G0001778 × Bawan 6. Finally, a functional marker specific to er1-6, SNP1121, was developed using the high-resolution melting technique, which could be used in pea breeding via marker-assisted selection. The results described here provide valuable genetic information for Chinese pea landraces and a powerful tool for pea breeders. PMID:26809053

  11. Discovery of a Novel er1 Allele Conferring Powdery Mildew Resistance in Chinese Pea (Pisum sativum L.) Landraces.

    PubMed

    Sun, Suli; Fu, Haining; Wang, Zhongyi; Duan, Canxing; Zong, Xuxiao; Zhu, Zhendong

    2016-01-01

    Pea powdery mildew, caused by Erysiphe pisi D.C., is an important disease worldwide. Deployment of resistant varieties is the main way to control this disease. This study aimed to screen Chinese pea (Pisum sativum L.) landraces resistant to E. pisi, and to characterize the resistance gene(s) at the er1 locus in the resistant landraces, and to develop functional marker(s) specific to the novel er1 allele. The 322 landraces showed different resistance levels. Among them, 12 (3.73%), 4 (1.24%) and 17 (5.28%) landraces showed immunity, high resistance and resistance to E. pisi, respectively. The other landraces appeared susceptible or highly susceptible to E. pisi. Most of the immune and highly resistant landraces were collected from Yunnan province. To characterize the resistance gene at the er1 locus, cDNA sequences of PsMLO1 gene were determined in 12 immune and four highly resistant accessions. The cDNAs of PsMLO1 from the immune landrace G0005576 produced three distinct transcripts, characterized by a 129-bp deletion, and 155-bp and 220-bp insertions, which were consistent with those of er1-2 allele. The PsMLO1 cDNAs in the other 15 resistant landraces produced identical transcripts, which had a new point mutation (T→C) at position 1121 of PsMLO1, indicating a novel er1 allele, designated as er1-6. This mutation caused a leucine to proline change in the amino acid sequence. Subsequently, the resistance allele er1-6 in landrace G0001778 was confirmed by resistance inheritance analysis and genetic mapping on the region of the er1 locus using populations derived from G0001778 × Bawan 6. Finally, a functional marker specific to er1-6, SNP1121, was developed using the high-resolution melting technique, which could be used in pea breeding via marker-assisted selection. The results described here provide valuable genetic information for Chinese pea landraces and a powerful tool for pea breeders.

  12. Discovery of a Novel er1 Allele Conferring Powdery Mildew Resistance in Chinese Pea (Pisum sativum L.) Landraces

    PubMed Central

    Sun, Suli; Fu, Haining; Wang, Zhongyi; Duan, Canxing; Zong, Xuxiao; Zhu, Zhendong

    2016-01-01

    Pea powdery mildew, caused by Erysiphe pisi D.C., is an important disease worldwide. Deployment of resistant varieties is the main way to control this disease. This study aimed to screen Chinese pea (Pisum sativum L.) landraces resistant to E. pisi, and to characterize the resistance gene(s) at the er1 locus in the resistant landraces, and to develop functional marker(s) specific to the novel er1 allele. The 322 landraces showed different resistance levels. Among them, 12 (3.73%), 4 (1.24%) and 17 (5.28%) landraces showed immunity, high resistance and resistance to E. pisi, respectively. The other landraces appeared susceptible or highly susceptible to E. pisi. Most of the immune and highly resistant landraces were collected from Yunnan province. To characterize the resistance gene at the er1 locus, cDNA sequences of PsMLO1 gene were determined in 12 immune and four highly resistant accessions. The cDNAs of PsMLO1 from the immune landrace G0005576 produced three distinct transcripts, characterized by a 129-bp deletion, and 155-bp and 220-bp insertions, which were consistent with those of er1-2 allele. The PsMLO1 cDNAs in the other 15 resistant landraces produced identical transcripts, which had a new point mutation (T→C) at position 1121 of PsMLO1, indicating a novel er1 allele, designated as er1-6. This mutation caused a leucine to proline change in the amino acid sequence. Subsequently, the resistance allele er1-6 in landrace G0001778 was confirmed by resistance inheritance analysis and genetic mapping on the region of the er1 locus using populations derived from G0001778 × Bawan 6. Finally, a functional marker specific to er1-6, SNP1121, was developed using the high-resolution melting technique, which could be used in pea breeding via marker-assisted selection. The results described here provide valuable genetic information for Chinese pea landraces and a powerful tool for pea breeders. PMID:26809053

  13. Systematic review of allelic exchange experiments aimed at identifying mutations that confer drug resistance in Mycobacterium tuberculosis

    PubMed Central

    Nebenzahl-Guimaraes, Hanna; Jacobson, Karen R.; Farhat, Maha R.; Murray, Megan B.

    2014-01-01

    Background Improving our understanding of the relationship between the genotype and the drug resistance phenotype of Mycobacterium tuberculosis will aid the development of more accurate molecular diagnostics for drug-resistant tuberculosis. Studies that use direct genetic manipulation to identify the mutations that cause M. tuberculosis drug resistance are superior to associational studies in elucidating an individual mutation's contribution to the drug resistance phenotype. Methods We systematically reviewed the literature for publications reporting allelic exchange experiments in any of the resistance-associated M. tuberculosis genes. We included studies that introduced single point mutations using specialized linkage transduction or site-directed/in vitro mutagenesis and documented a change in the resistance phenotype. Results We summarize evidence supporting the causal relationship of 54 different mutations in eight genes (katG, inhA, kasA, embB, embC, rpoB, gyrA and gyrB) and one intergenic region (furA-katG) with resistance to isoniazid, the rifamycins, ethambutol and fluoroquinolones. We observed a significant role for the strain genomic background in modulating the resistance phenotype of 21 of these mutations and found examples of where the same drug resistance mutations caused varying levels of resistance to different members of the same drug class. Conclusions This systematic review highlights those mutations that have been shown to causally change phenotypic resistance in M. tuberculosis and brings attention to a notable lack of allelic exchange data for several of the genes known to be associated with drug resistance. PMID:24055765

  14. Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess.

    PubMed

    Hernández, Eric G; Granados, Julio; Partida-Rodríguez, Oswaldo; Valenzuela, Olivia; Rascón, Edgar; Magaña, Ulises; Escamilla-Tilch, Mónica; López-Reyes, Alberto; Nieves-Ramírez, Miriam; González, Enrique; Morán, Patricia; Rojas, Liliana; Valadez, Alicia; Luna, Alexandra; Estrada, Francisco J; Maldonado, Carmen; Ximénez, Cecilia

    2015-01-01

    Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA.

  15. Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess

    PubMed Central

    Hernández, Eric G.; Granados, Julio; Partida-Rodríguez, Oswaldo; Valenzuela, Olivia; Rascón, Edgar; Magaña, Ulises; Escamilla-Tilch, Mónica; López-Reyes, Alberto; Nieves-Ramírez, Miriam; González, Enrique; Morán, Patricia; Rojas, Liliana; Valadez, Alicia; Luna, Alexandra; Estrada, Francisco J.; Maldonado, Carmen; Ximénez, Cecilia

    2015-01-01

    Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA

  16. Intragenic allele pyramiding combines different specificities of wheat Pm3 resistance alleles.

    PubMed

    Brunner, Susanne; Hurni, Severine; Streckeisen, Philipp; Mayr, Gabriele; Albrecht, Mario; Yahiaoui, Nabila; Keller, Beat

    2010-11-01

    Some plant resistance genes occur as allelic series, with each member conferring specific resistance against a subset of pathogen races. In wheat, there are 17 alleles of the Pm3 gene. They encode nucleotide-binding (NB-ARC) and leucine-rich-repeat (LRR) domain proteins, which mediate resistance to distinct race spectra of powdery mildew. It is not known if specificities from different alleles can be combined to create resistance genes with broader specificity. Here, we used an approach based on avirulence analysis of pathogen populations to characterize the molecular basis of Pm3 recognition spectra. A large survey of mildew races for avirulence on the Pm3 alleles revealed that Pm3a has a resistance spectrum that completely contains that of Pm3f, but also extends towards additional races. The same is true for the Pm3b and Pm3c gene pair. The molecular analysis of these allelic pairs revealed a role of the NB-ARC protein domain in the efficiency of effector-dependent resistance. Analysis of the wild-type and chimeric Pm3 alleles identified single residues in the C-terminal LRR motifs as the main determinant of allele specificity. Variable residues of the N-terminal LRRs are necessary, but not sufficient, to confer resistance specificity. Based on these data, we constructed a chimeric Pm3 gene by intragenic allele pyramiding of Pm3d and Pm3e that showed the combined resistance specificity and, thus, a broader recognition spectrum compared with the parental alleles. Our findings support a model of stepwise evolution of Pm3 recognition specificities.

  17. Cgnz1 allele confers kidney resistance to damage preventing progression of immune complex-mediated acute lupus glomerulonephritis.

    PubMed

    Ge, Yan; Jiang, Chao; Sung, Sun-Sang J; Bagavant, Harini; Dai, Chao; Wang, Hongyang; Kannapell, Carol C; Cathro, Helen P; Gaskin, Felicia; Fu, Shu Man

    2013-10-21

    Cgnz1 and Agnz1 on the distal region of mouse chromosome 1 are associated with chronic glomerulonephritis (cGN) and acute GN (aGN). NZM2328.Lc1R27 (R27) was generated by introgressing a C57L/J region where Cgnz1 is located to NZM2328. R27 female mice developed aGN mediated by immune complex (IC) deposition and complement activation without progression to cGN with severe proteinuria. End stage renal disease (ESRD) was not seen in R27 mice as old as 15 mo. Thus, aGN and cGN are under separate genetic control, and IC-mediated proliferative GN need not progress to cGN and ESRD. NZM2328 and R27 female mice have comparable immune and inflammatory parameters. In contrast to NZM2328, R27 mice were resistant to sheep anti-mouse GBM serum-induced nephritis, supporting the hypothesis that aGN is mediated by autoimmunity and resistance to the development of cGN is mediated by end organ resistance to damage. Thus, autoimmunity should be considered distinct from end organ damage. The Cgnz1 region has been mapped to a 1.34 MB region with 45 genes. Nine candidate genes were identified. Clinical relevance of these observations is supported by case studies. Clinical implications and the significance to human lupus and other diseases are presented.

  18. Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants.

    PubMed

    Stirnweis, Daniel; Milani, Samira D; Brunner, Susanne; Herren, Gerhard; Buchmann, Gabriele; Peditto, David; Jordan, Tina; Keller, Beat

    2014-09-01

    The development of high-yielding varieties with broad-spectrum durable disease resistance is the ultimate goal of crop breeding. In plants, immune receptors of the nucleotide-binding-leucine-rich repeat (NB-LRR) class mediate race-specific resistance against pathogen attack. When employed in agriculture this type of resistance is often rapidly overcome by newly adapted pathogen races. The stacking of different resistance genes or alleles in F1 hybrids or in pyramided lines is a promising strategy for achieving more durable resistance. Here, we identify a molecular mechanism which can negatively interfere with the allele-pyramiding approach. We show that pairwise combinations of different alleles of the powdery mildew resistance gene Pm3 in F1 hybrids and stacked transgenic wheat lines can result in suppression of Pm3-based resistance. This effect is independent of the genetic background and solely dependent on the Pm3 alleles. Suppression occurs at the post-translational level, as levels of RNA and protein in the suppressed alleles are unaffected. Using a transient expression system in Nicotiana benthamiana, the LRR domain was identified as the domain conferring suppression. The results of this study suggest that the expression of closely related NB-LRR resistance genes or alleles in the same genotype can lead to dominant-negative interactions. These findings provide a molecular explanation for the frequently observed ineffectiveness of resistance genes introduced from the secondary gene pool into polyploid crop species and mark an important step in overcoming this limitation.

  19. Nucleotide variation and identification of novel blast resistance alleles of Pib by allele mining strategy.

    PubMed

    Ramkumar, G; Madhav, M S; Devi, S J S Rama; Prasad, M S; Babu, V Ravindra

    2015-04-01

    Pib is one of significant rice blast resistant genes, which provides resistance to wide range of isolates of rice blast pathogen, Magnaporthe oryzae. Identification and isolation of novel and beneficial alleles help in crop enhancement. Allele mining is one of the best strategies for dissecting the allelic variations at candidate gene and identification of novel alleles. Hence, in the present study, Pib was analyzed by allele mining strategy, and coding and non-coding (upstream and intron) regions were examined to identify novel Pib alleles. Allelic sequences comparison revealed that nucleotide polymorphisms at coding regions affected the amino acid sequences, while the polymorphism at upstream (non-coding) region affected the motifs arrangements. Pib alleles from resistant landraces, Sercher and Krengosa showed better resistance than Pib donor variety, might be due to acquired mutations, especially at LRR region. The evolutionary distance, Ka/Ks and phylogenetic analyzes also supported these results. Transcription factor binding motif analysis revealed that Pib (Sr) had a unique motif (DPBFCOREDCDC3), while five different motifs differentiated the resistance and susceptible Pib alleles. As the Pib is an inducible gene, the identified differential motifs helps to understand the Pib expression mechanism. The identified novel Pib resistant alleles, which showed high resistance to the rice blast, can be used directly in blast resistance breeding program as alternative Pib resistant sources.

  20. Identification of alleles conferring resistance to gray leaf spot in maize derived from its wild progenitor species teosinte (Zea mays ssp. parviglumis)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gray Leaf Spot [(GLS), causal agent Cercospora zeae-maydis and Cercospora zeina] is an important maize disease in the United States. Current control methods for GLS include using resistant cultivars, crop rotation, chemical applications, and conventional tillage to reduce inoculum levels. Teosinte ...

  1. Identification of novel alleles of the rice blast resistance gene Pi54

    NASA Astrophysics Data System (ADS)

    Vasudevan, Kumar; Gruissem, Wilhelm; Bhullar, Navreet K.

    2015-10-01

    Rice blast is one of the most devastating rice diseases and continuous resistance breeding is required to control the disease. The rice blast resistance gene Pi54 initially identified in an Indian cultivar confers broad-spectrum resistance in India. We explored the allelic diversity of the Pi54 gene among 885 Indian rice genotypes that were found resistant in our screening against field mixture of naturally existing M. oryzae strains as well as against five unique strains. These genotypes are also annotated as rice blast resistant in the International Rice Genebank database. Sequence-based allele mining was used to amplify and clone the Pi54 allelic variants. Nine new alleles of Pi54 were identified based on the nucleotide sequence comparison to the Pi54 reference sequence as well as to already known Pi54 alleles. DNA sequence analysis of the newly identified Pi54 alleles revealed several single polymorphic sites, three double deletions and an eight base pair deletion. A SNP-rich region was found between a tyrosine kinase phosphorylation site and the nucleotide binding site (NBS) domain. Together, the newly identified Pi54 alleles expand the allelic series and are candidates for rice blast resistance breeding programs.

  2. Host mating system and the spread of a disease-resistant allele in a population

    USGS Publications Warehouse

    DeAngelis, D.L.; Koslow, Jennifer M.; Jiang, J.; Ruan, S.

    2008-01-01

    The model presented here modifies a susceptible-infected (SI) host-pathogen model to determine the influence of mating system on the outcome of a host-pathogen interaction. Both deterministic and stochastic (individual-based) versions of the model were used. This model considers the potential consequences of varying mating systems on the rate of spread of both the pathogen and resistance alleles within the population. We assumed that a single allele for disease resistance was sufficient to confer complete resistance in an individual, and that both homozygote and heterozygote resistant individuals had the same mean birth and death rates. When disease invaded a population with only an initial small fraction of resistant genes, inbreeding (selfing) tended to increase the probability that the disease would soon be eliminated from a small population rather than become endemic, while outcrossing greatly increased the probability that the population would become extinct due to the disease.

  3. Temperature Sensitivity Conferred by ligA Alleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

    PubMed Central

    Pankowski, Jarosław A.; Puckett, Stephanie M.

    2016-01-01

    We have assembled a collection of 13 psychrophilic ligA alleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (TS) phenotype. When these ligA alleles were substituted into Francisella novicida, they conferred a TS phenotype with restrictive temperatures between 33 and 39°C. When the F. novicida ligA hybrid strains were plated above their restrictive temperatures, eight of them generated temperature-resistant variants. For two alleles, the mutations that led to temperature resistance clustered near the 5′ end of the gene, and the mutations increased the predicted strength of the ribosome binding site at least 3-fold. Four F. novicida ligA hybrid strains generated no temperature-resistant variants at a detectable level. These results suggest that multiple mutations are needed to create temperature-resistant variants of these ligA gene products. One ligA allele was isolated from a Colwellia species that has a maximal growth temperature of 12°C, and this allele supported growth of F. novicida only as a hybrid between the psychrophilic and the F. novicida ligA genes. However, the full psychrophilic gene alone supported the growth of Salmonella enterica, imparting a restrictive temperature of 27°C. We also tested two ligA alleles from two Pseudoalteromonas strains for their ability to support the viability of a Saccharomyces cerevisiae strain that lacked its essential gene, CDC9, encoding an ATP-dependent DNA ligase. In both cases, the psychrophilic bacterial alleles supported yeast viability and their expression generated TS phenotypes. This collection of ligA alleles should be useful in engineering bacteria, and possibly eukaryotic microbes, to predictable TS phenotypes. PMID:26773080

  4. Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib

    PubMed Central

    Vasudevan, Kumar; Vera Cruz, Casiana M.; Gruissem, Wilhelm; Bhullar, Navreet K.

    2016-01-01

    Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level. PMID:27446145

  5. Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib.

    PubMed

    Vasudevan, Kumar; Vera Cruz, Casiana M; Gruissem, Wilhelm; Bhullar, Navreet K

    2016-01-01

    Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level. PMID:27446145

  6. Parallel Mapping of Antibiotic Resistance Alleles in Escherichia coli

    PubMed Central

    Mortazavi, Pooneh; Knight, Rob; Gill, Ryan T.

    2016-01-01

    Chemical genomics expands our understanding of microbial tolerance to inhibitory chemicals, but its scope is often limited by the throughput of genome-scale library construction and genotype-phenotype mapping. Here we report a method for rapid, parallel, and deep characterization of the response to antibiotics in Escherichia coli using a barcoded genome-scale library, next-generation sequencing, and streamlined bioinformatics software. The method provides quantitative growth data (over 200,000 measurements) and identifies contributing antimicrobial resistance and susceptibility alleles. Using multivariate analysis, we also find that subtle differences in the population responses resonate across multiple levels of functional hierarchy. Finally, we use machine learning to identify a unique allelic and proteomic fingerprint for each antibiotic. The method can be broadly applied to tolerance for any chemical from toxic metabolites to next-generation biofuels and antibiotics. PMID:26771672

  7. Frequency of Cry1F Non-Recessive Resistance Alleles in North Carolina Field Populations of Spodoptera frugiperda (Lepidoptera: Noctuidae).

    PubMed

    Li, Guoping; Reisig, Dominic; Miao, Jin; Gould, Fred; Huang, Fangneng; Feng, Hongqiang

    2016-01-01

    Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a target species of transgenic corn (Zea mays L.) that expresses single and pyramided Bacillus thuringiensis (Bt) toxin. In 2014, S. frugiperda were collected from a light trap in North Carolina, and a total of 212 F1/F2 isofemale lines of S. frugiperda were screened for resistance to Bt and non-Bt corn. All of the 212 isolines were susceptible to corn tissue expressing Cry1A.105 + Cry2Ab, Cry1F + Cry1A.105 + Cry2Ab, and Cry1F + Cry1Ab + Vip3Aa20. Growth rate bioassays were performed to isolate non-recessive Bt resistance alleles. Seven individuals out of the 212 isofemale lines carried major non-recessive alleles conferring resistance to Cry1F. A pooled colony was created from the seven individuals. This colony was 151.21 times more resistant to Cry1F than a known-susceptible population and was also resistant to Cry1A.105, but was not resistant to Cry2Ab and Vip3Aa20. The results demonstrate that field populations of S. frugiperda collected from North Carolina are generally susceptible to Cry1F, but that some individuals carry resistant alleles. The data generated in this study can be used as baseline data for resistance monitoring. PMID:27119741

  8. Frequency of Cry1F Non-Recessive Resistance Alleles in North Carolina Field Populations of Spodoptera frugiperda (Lepidoptera: Noctuidae)

    PubMed Central

    Li, Guoping; Reisig, Dominic; Miao, Jin; Gould, Fred; Huang, Fangneng; Feng, Hongqiang

    2016-01-01

    Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a target species of transgenic corn (Zea mays L.) that expresses single and pyramided Bacillus thuringiensis (Bt) toxin. In 2014, S. frugiperda were collected from a light trap in North Carolina, and a total of 212 F1/F2 isofemale lines of S. frugiperda were screened for resistance to Bt and non-Bt corn. All of the 212 isolines were susceptible to corn tissue expressing Cry1A.105 + Cry2Ab, Cry1F + Cry1A.105 + Cry2Ab, and Cry1F + Cry1Ab + Vip3Aa20. Growth rate bioassays were performed to isolate non-recessive Bt resistance alleles. Seven individuals out of the 212 isofemale lines carried major non-recessive alleles conferring resistance to Cry1F. A pooled colony was created from the seven individuals. This colony was 151.21 times more resistant to Cry1F than a known-susceptible population and was also resistant to Cry1A.105, but was not resistant to Cry2Ab and Vip3Aa20. The results demonstrate that field populations of S. frugiperda collected from North Carolina are generally susceptible to Cry1F, but that some individuals carry resistant alleles. The data generated in this study can be used as baseline data for resistance monitoring. PMID:27119741

  9. Identification of Coupling and Repulsion Phase DNA Marker Associated With an Allele of a Gene Conferring Host Plant Resistance to Pigeonpea sterility mosaic virus (PPSMV) in Pigeonpea (Cajanus cajan L. Millsp.).

    PubMed

    Daspute, Abhijit; Fakrudin, B

    2015-03-01

    Pigeonpea Sterility Mosaic Disease (PSMD) is an important foliar disease caused by Pigeonpea sterility mosaic virus (PPSMV) which is transmitted by eriophyid mites (Aceria cajani Channabasavanna). In present study, a F2 mapping population comprising 325 individuals was developed by crossing PSMD susceptible genotype (Gullyal white) and PSMD resistant genotype (BSMR 736). We identified a set of 32 out of 300 short decamer random DNA markers that showed polymorphism between Gullyal white and BSMR 736 parents. Among them, eleven DNA markers showed polymorphism including coupling and repulsion phase type of polymorphism across the parents. Bulked Segregant Analysis (BSA), revealed that the DNA marker, IABTPPN7, produced a single coupling phase marker (IABTPPN7414) and a repulsion phase marker (IABTPPN7983) co-segregating with PSMD reaction. Screening of 325 F2 population using IABTPPN7 revealed that the repulsion phase marker, IABTPPN7983, was co-segregating with the PSMD responsive SV1 at a distance of 23.9 cM for Bidar PPSMV isolate. On the other hand, the coupling phase marker IABTPPN7414 did not show any linkage with PSMD resistance. Additionally, single marker analysis both IABTPPN7983 (P<0.0001) and IABTPPN 7414 (P<0.0001) recorded a significant association with the PSMD resistance and explained a phenotypic variance of 31 and 36% respectively in F2 population. The repulsion phase marker, IABTPPN7983, could be of use in Marker-Assisted Selection (MAS) in the PPSMV resistance breeding programmes of pigeonpea. PMID:25774108

  10. Specific HLA-DQB and HLA-DRB1 alleles confer susceptibility to pemphigus vulgaris.

    PubMed Central

    Scharf, S J; Freidmann, A; Steinman, L; Brautbar, C; Erlich, H A

    1989-01-01

    The autoimmune dermatologic disease pemphigus vulgaris (PV) is associated with the serotypes HLA-DR4 and HLA-DRw6. Based on nucleotide sequence and oligonucleotide probe analysis of enzymatically amplified DNA encoding HLA-DR beta chain (HLA-DRB) and HLA-DQ beta chain (HLA-DQB; henceforth HLA is omitted from designations), we showed previously that the DR4 susceptibility was associated with the Dw10 DRB1 allele [encoding the mixed lymphocyte culture (MLC)-defined Dw10 specificity]. The DRw6 susceptibility similarly was shown to be associated with a rare DQB allele (DQB1.3), which differed from another nonsusceptible allele by only a valine-to-aspartic acid substitution at position 57. Given the linkage disequilibrium that characterizes HLA haplotypes, it is difficult to assign disease susceptibility to a specific locus rather than to a closely linked gene(s) on the same haplotype. To address this problem, we have analyzed all of the polymorphic loci of the class II HLA region (DRB1, DRB3, DQA, DQB, and DPB) on the DRw6 haplotypes in patients and controls. In 22 PV patients, 4 different DRw6 haplotypes were found that encode the same DQ beta chain (DQB1.3) but contained silent nucleotide differences at the DQB locus as well as coding sequence differences in the DQA and DRB loci. These results, obtained by using a method for allele-specific polymerase chain reaction amplification, strongly support the hypothesis that the allele DQB1.3 confers susceptibility. This DQB allele is correlated with the MLC-defined Dw9 specificity and is associated with two different DRB1 alleles (the common "6A" associated with DRw13 and the rare "6B" associated with DRw14). Since 86% (19 of 22) of DRw6+ patients contain the DQB1.3 allele (vs. 3% of controls), whereas 64% (14 of 22) contain the DRB1 allele 6B (vs. 6% of the controls), we conclude that most of the DRw6 susceptibility to PV can be accounted for by the DQ beta chain. Images PMID:2503828

  11. Allele Mining Strategies: Principles and Utilisation for Blast Resistance Genes in Rice (Oryza sativa L.).

    PubMed

    Ashkani, Sadegh; Yusop, Mohd Rafii; Shabanimofrad, Mahmoodreza; Azady, Amin; Ghasemzadeh, Ali; Azizi, Parisa; Latif, Mohammad Abdul

    2015-01-01

    Allele mining is a promising way to dissect naturally occurring allelic variants of candidate genes with essential agronomic qualities. With the identification, isolation and characterisation of blast resistance genes in rice, it is now possible to dissect the actual allelic variants of these genes within an array of rice cultivars via allele mining. Multiple alleles from the complex locus serve as a reservoir of variation to generate functional genes. The routine sequence exchange is one of the main mechanisms of R gene evolution and development. Allele mining for resistance genes can be an important method to identify additional resistance alleles and new haplotypes along with the development of allele-specific markers for use in marker-assisted selection. Allele mining can be visualised as a vital link between effective utilisation of genetic and genomic resources in genomics-driven modern plant breeding. This review studies the actual concepts and potential of mining approaches for the discovery of alleles and their utilisation for blast resistance genes in rice. The details provided here will be important to provide the rice breeder with a worthwhile introduction to allele mining and its methodology for breakthrough discovery of fresh alleles hidden in hereditary diversity, which is vital for crop improvement.

  12. Expression of cytokeratin confers multiple drug resistance.

    PubMed Central

    Bauman, P A; Dalton, W S; Anderson, J M; Cress, A E

    1994-01-01

    The cytokeratin network is an extensive filamentous structure in the cytoplasm whose biological function(s) is unknown. Based upon previous data showing the modification of cytokeratin by mitoxantrone, we investigated the ability of cytokeratin networks to influence the survival response of cells to chemotherapeutic agents. We have compared the survival of mouse L fibroblasts lacking cytokeratins with that of L cells transfected with cytokeratins 8 and 18 in the presence of chemotherapeutic drugs. The expression of cytokeratins 8 and 18 conferred a multiple drug resistance phenotype on cells exposed to mitoxantrone, doxorubicin, methotrexate, melphalan, Colcemid, and vincristine. The degree of drug resistance was 5-454 times that of parental cells, depending upon the agent used. Drug resistance could not be attributed to altered growth characteristics, altered drug accumulation, or an altered drug efflux in the transfected cells. Cytokeratin does not confer resistance to ionizing radiation, which damages DNA independently of intracellular transport mechanisms. These data suggest a role for cytokeratin networks in conferring a drug resistance phenotype. Images PMID:7515497

  13. Multiple Avirulence Loci and Allele-Specific Effector Recognition Control the Pm3 Race-Specific Resistance of Wheat to Powdery Mildew[OPEN

    PubMed Central

    Roffler, Stefan; Stirnweis, Daniel; Treier, Georges; Herren, Gerhard; Korol, Abraham B.; Wicker, Thomas

    2015-01-01

    In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3a2/f2 from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 allele-dependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 a2/f2 revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogen-encoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes. PMID:26452600

  14. Dynamics of insecticide resistance alleles in house fly populations from New York and Florida.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The frequency of insecticide resistance alleles for two genes (Vssc1 and CYP6D1) was studied in field collected populations of house flies from two different climates. While the frequency of these resistance alleles in flies at dairies from four states has recently been reported, there is no infor...

  15. A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes

    PubMed Central

    Woodward, Andrew W.; Fleming, Wendell A.; Burkhart, Sarah E.; Ratzel, Sarah E.; Bjornson, Marta; Bartel, Bonnie

    2014-01-01

    Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import. PMID:25008153

  16. A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes.

    PubMed

    Woodward, Andrew W; Fleming, Wendell A; Burkhart, Sarah E; Ratzel, Sarah E; Bjornson, Marta; Bartel, Bonnie

    2014-09-01

    Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.

  17. Effective marker alleles associated with type 2 resistance to Fusarium head blight infection in fields

    PubMed Central

    Li, Tao; Luo, Meng; Zhang, Dadong; Wu, Di; Li, Lei; Bai, Guihua

    2016-01-01

    Molecular markers associated with known quantitative trait loci (QTLs) for type 2 resistance to Fusarium head blight (FHB) in bi-parental mapping population usually have more than two alleles in breeding populations. Therefore, understanding the association of each allele with FHB response is particularly important to marker-assisted enhancement of FHB resistance. In this paper, we evaluated FHB severities of 192 wheat accessions including landraces and commercial varieties in three field growing seasons, and genotyped this panel with 364 genome-wide informative molecular markers. Among them, 11 markers showed reproducible marker-trait association (p < 0.05) in at least two experiments using a mixed model. More than two alleles were identified per significant marker locus. These alleles were classified into favorable, unfavorable and neutral alleles according to the normalized genotypic values. The distributions of effective alleles at these loci in each wheat accession were characterized. Mean FHB severities increased with decreased number of favorable alleles at the reproducible loci. Chinese wheat landraces and Japanese accessions have more favorable alleles at the majority of the reproducible marker loci. FHB resistance levels of varieties can be greatly improved by introduction of these favorable alleles and removal of unfavorable alleles simultaneously at these QTL-linked marker loci. PMID:27436944

  18. Metabolic and target-site mechanisms combine to confer strong DDT resistance in Anopheles gambiae.

    PubMed

    Mitchell, Sara N; Rigden, Daniel J; Dowd, Andrew J; Lu, Fang; Wilding, Craig S; Weetman, David; Dadzie, Samuel; Jenkins, Adam M; Regna, Kimberly; Boko, Pelagie; Djogbenou, Luc; Muskavitch, Marc A T; Ranson, Hilary; Paine, Mark J I; Mayans, Olga; Donnelly, Martin J

    2014-01-01

    The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae.

  19. Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm

    PubMed Central

    Morin, Shai; Biggs, Robert W.; Sisterson, Mark S.; Shriver, Laura; Ellers-Kirk, Christa; Higginson, Dawn; Holley, Daniel; Gahan, Linda J.; Heckel, David G.; Carrière, Yves; Dennehy, Timothy J.; Brown, Judith K.; Tabashnik, Bruce E.

    2003-01-01

    Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. Because inheritance of resistance to the Bt toxins in transgenic crops is typically recessive, DNA-based screening for resistance alleles in heterozygotes is potentially much more efficient than detection of resistant homozygotes with bioassays. Such screening, however, requires knowledge of the resistance alleles in field populations of pests that are associated with survival on Bt crops. Here we report that field populations of pink bollworm (Pectinophora gossypiella), a major cotton pest, harbored three mutant alleles of a cadherin-encoding gene linked with resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Each of the three resistance alleles has a deletion expected to eliminate at least eight amino acids upstream of the putative toxin-binding region of the cadherin protein. Larvae with two resistance alleles in any combination were resistant, whereas those with one or none were susceptible to Cry1Ac. Together with previous evidence, the results reported here identify the cadherin gene as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests. PMID:12695565

  20. Multiple origins of Plasmodium falciparum dihydropteroate synthetase mutant alleles associated with sulfadoxine resistance in India.

    PubMed

    Lumb, Vanshika; Das, Manoj K; Singh, Neeru; Dev, Vas; Khan, Wajihullah; Sharma, Yagya D

    2011-06-01

    With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity (H(e)) around mutant dhps alleles (H(e) = 0.55; n = 81) associated with sulfadoxine resistance was lower (P ≤ 0.05) than the mean H(e) around the wild-type dhps allele (H(e) = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine

  1. Allelic analysis of sheath blight resistance with association mapping in rice.

    PubMed

    Jia, Limeng; Yan, Wengui; Zhu, Chengsong; Agrama, Hesham A; Jackson, Aaron; Yeater, Kathleen; Li, Xiaobai; Huang, Bihu; Hu, Biaolin; McClung, Anna; Wu, Dianxing

    2012-01-01

    Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Global attention has focused on examining individual mapping populations for quantitative trait loci (QTLs) for ShB resistance, but to date no study has taken advantage of association mapping to examine hundreds of lines for potentially novel QTLs. Our objective was to identify ShB QTLs via association mapping in rice using 217 sub-core entries from the USDA rice core collection, which were phenotyped with a micro-chamber screening method and genotyped with 155 genome-wide markers. Structure analysis divided the mapping panel into five groups, and model comparison revealed that PCA5 with genomic control was the best model for association mapping of ShB. Ten marker loci on seven chromosomes were significantly associated with response to the ShB pathogen. Among multiple alleles in each identified loci, the allele contributing the greatest effect to ShB resistance was named the putative resistant allele. Among 217 entries, entry GSOR 310389 contained the most putative resistant alleles, eight out of ten. The number of putative resistant alleles presented in an entry was highly and significantly correlated with the decrease of ShB rating (r = -0.535) or the increase of ShB resistance. Majority of the resistant entries that contained a large number of the putative resistant alleles belonged to indica, which is consistent with a general observation that most ShB resistant accessions are of indica origin. These findings demonstrate the potential to improve breeding efficiency by using marker-assisted selection to pyramid putative resistant alleles from various loci in a cultivar for enhanced ShB resistance in rice.

  2. One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins.

    PubMed

    Tabashnik, B E; Liu, Y B; Finson, N; Masson, L; Heckel, D G

    1997-03-01

    Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected.

  3. One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins

    PubMed Central

    Tabashnik, Bruce E.; Liu, Yong-Biao; Finson, Naomi; Masson, Luke; Heckel, David G.

    1997-01-01

    Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected. PMID:9050831

  4. The wheat durable, multipathogen resistance gene Lr34 confers partial blast resistance in rice.

    PubMed

    Krattinger, Simon G; Sucher, Justine; Selter, Liselotte L; Chauhan, Harsh; Zhou, Bo; Tang, Mingzhi; Upadhyaya, Narayana M; Mieulet, Delphine; Guiderdoni, Emmanuel; Weidenbach, Denise; Schaffrath, Ulrich; Lagudah, Evans S; Keller, Beat

    2016-05-01

    The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain-of-function mutations in an ATP-binding cassette transporter gene. An Lr34-like fungal disease resistance with a similar broad-spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34-expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence-based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad-spectrum disease resistance against the most devastating fungal disease of rice. PMID:26471973

  5. The wheat durable, multipathogen resistance gene Lr34 confers partial blast resistance in rice.

    PubMed

    Krattinger, Simon G; Sucher, Justine; Selter, Liselotte L; Chauhan, Harsh; Zhou, Bo; Tang, Mingzhi; Upadhyaya, Narayana M; Mieulet, Delphine; Guiderdoni, Emmanuel; Weidenbach, Denise; Schaffrath, Ulrich; Lagudah, Evans S; Keller, Beat

    2016-05-01

    The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain-of-function mutations in an ATP-binding cassette transporter gene. An Lr34-like fungal disease resistance with a similar broad-spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34-expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence-based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad-spectrum disease resistance against the most devastating fungal disease of rice.

  6. Co-selection and replacement of resistance alleles to Lysinibacillus sphaericus in a Culex quinquefasciatus colony.

    PubMed

    Chalegre, Karlos Diogo de Melo; Tavares, Daniella A; Romão, Tatiany P; de Menezes, Heverly Suzany G; Nascimento, Nathaly A; de Oliveira, Cláudia Maria F; de-Melo-Neto, Osvaldo P; Silva-Filha, Maria Helena N L

    2015-09-01

    The Cqm1 α-glucosidase, expressed within the midgut of Culex quinquefasciatus mosquito larvae, is the receptor for the Binary toxin (Bin) from the entomopathogen Lysinibacillus sphaericus. Mutations of the Cqm1 α-glucosidase gene cause high resistance levels to this bacterium in both field and laboratory populations, and a previously described allele, cqm1REC, was found to be associated with a laboratory-resistant colony (R2362). This study described the identification of a novel resistance allele, cqm1REC-2, that was co-selected with cqm1REC within the R2362 colony. The two alleles display distinct mutations but both generate premature stop codons that prevent the expression of midgut-bound Cqm1 proteins. Using a PCR-based assay to monitor the frequency of each allele during long-term maintenance of the resistant colony, cqm1REC was found to predominate early on but later was replaced by cqm1REC-2 as the most abundant resistance allele. Homozygous larvae for each allele were then generated that displayed similar high-resistance phenotypes with equivalent low levels of transcript and lack of protein expression for both cqm1REC and cqm1REC-2. In progeny from a cross of homozygous individuals for each allele at a 1 : 1 ratio, analyzed for ten subsequent generations, cqm1REC showed a higher frequency than cqm1REC-2. The replacement of cqm1REC by cqm1REC -2 observed in the R2362 colony, kept for 210 generations, indicates changes in fitness related to traits that are unknown but linked to these two alleles, and constitutes a unique example of evolution of resistance within a controlled laboratory environment. PMID:26131741

  7. The functional importance of sequence versus expression variability of MHC alleles in parasite resistance.

    PubMed

    Axtner, Jan; Sommer, Simone

    2012-12-01

    Understanding selection processes driving the pronounced allelic polymorphism of the major histocompatibility complex (MHC) genes and its functional associations to parasite load have been the focus of many recent wildlife studies. Two main selection scenarios are currently debated which explain the susceptibility or resistance to parasite infections either by the effects of (1) specific MHC alleles which are selected frequency-dependent in space and time or (2) a heterozygote or divergent allele advantage. So far, most studies have focused only on structural variance in co-evolutionary processes although this might not be the only trait subject to natural selection. In the present study, we analysed structural variance stretching from exon1 through exon3 of MHC class II DRB genes as well as genotypic expression variance in relation to the gastrointestinal helminth prevalence and infection intensity in wild yellow-necked mice (Apodemus flavicollis). We found support for the functional importance of specific alleles both on the sequence and expression level. By resampling a previously investigated study population we identified specific MHC alleles affected by temporal shifts in parasite pressure and recorded associated changes in allele frequencies. The allele Apfl-DRB*23 was associated with resistance to infections by the oxyurid nematode Syphacia stroma and at the same time with susceptibility to cestode infection intensity. In line with our expectation, MHC mRNA transcript levels tended to be higher in cestode-infected animals carrying the allele Apfl-DRB*23. However, no support for a heterozygote or divergent allele advantage on the sequence or expression level was detected. The individual amino acid distance of genotypes did not explain individual differences in parasite loads and the genetic distance had no effect on MHC genotype expression. For ongoing studies on the functional importance of expression variance in parasite resistance, allele

  8. Pistil-function breakdown in a new S-allele of European pear, S21*, confers self-compatibility.

    PubMed

    Sanzol, Javier

    2009-03-01

    European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, 'Abugo' and 'Ceremeño', were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S(21), but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S(21), so it was named S(21)*. S-genotypes assigned to 'Abugo' and 'Ceremeño' were S(10)S(21)* and S(21)*S(25) respectively, of which S(25) is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S(21) and S(21)* indicated that both alleles exhibit the same pollen function; however, cultivars bearing S(21)* had impaired pistil-S function as they failed to reject either S(21) or S (21)* pollen. RT-PCR analysis showed absence of S(21)* -RNase gene expression in styles of 'Abugo' and 'Ceremeño', suggesting a possible origin for S(21)* pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S(21)* and indels at the 3'UTR) might explain the different pattern of expression between S(21) and S(21)*. Evaluation of cultivars with unknown S-genotype identified another cultivar 'Azucar Verde' bearing S(21)*, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.

  9. Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites.

    PubMed

    Arias-Sánchez, Flor I; Hall, Alex R

    2016-05-01

    Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger. PMID:27194288

  10. Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

    PubMed Central

    Hall, Alex R.

    2016-01-01

    Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger. PMID:27194288

  11. Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites.

    PubMed

    Arias-Sánchez, Flor I; Hall, Alex R

    2016-05-01

    Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger.

  12. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

  13. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections. PMID:26407876

  14. Correlation between carboxylesterase alleles and insecticide resistance in Culex pipiens complex from China

    PubMed Central

    2011-01-01

    Background In China, large amounts of chemical insecticides are applied in fields or indoors every year, directly or indirectly bringing selection pressure on vector mosquitoes. Culex pipiens complex has evolved to be resistant to all types of chemical insecticides, especially organophosphates, through carboxylesterases. Six resistant carboxylesterase alleles (Ester) were recorded previously and sometimes co-existed in one field population, representing a complex situation for the evolution of Ester genes. Results In order to explore the evolutionary scenario, we analyzed the data from an historical record in 2003 and a recent investigation on five Culex pipiens pallens populations sampled from north China in 2010. Insecticide bioassays showed that these five populations had high resistance to pyrethroids, medium resistance to organophosphates, and low resistance to carbamates. Six types of Ester alleles, EsterB1, Ester2, Ester8, Ester9, EsterB10, and Ester11 were identified, and the overall pattern of their frequencies in geographic distribution was consistent with the report seven years prior to this study. Statistical correlation analysis indicated that Ester8 and Ester9 positively correlated with resistance to four insecticides, and EsterB10 to one insecticide. The occurrences of these three alleles were positively correlated, while the occurrence of EsterB1 was negatively correlated with Ester8, indicating an allelic competition. Conclusion Our analysis suggests that one insecticide can select multiple Ester alleles and one Ester allele can work on multiple insecticides. The evolutionary scenario of carboxylesterases under insecticide selection is possibly "one to many". PMID:22177233

  15. Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

    PubMed Central

    Ibrahim, Sulaiman S.; Riveron, Jacob M.; Bibby, Jaclyn; Irving, Helen; Yunta, Cristina; Paine, Mark J. I.; Wondji, Charles S.

    2015-01-01

    Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val109Ile, Asp335Glu and Asn384Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies. PMID:26517127

  16. Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector.

    PubMed

    Ibrahim, Sulaiman S; Riveron, Jacob M; Bibby, Jaclyn; Irving, Helen; Yunta, Cristina; Paine, Mark J I; Wondji, Charles S

    2015-10-01

    Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val109Ile, Asp335Glu and Asn384Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies. PMID:26517127

  17. Directionally selected cytochrome P450 alleles are driving the spread of pyrethroid resistance in the major malaria vector Anopheles funestus.

    PubMed

    Riveron, Jacob M; Irving, Helen; Ndula, Miranda; Barnes, Kayla G; Ibrahim, Sulaiman S; Paine, Mark J I; Wondji, Charles S

    2013-01-01

    Pyrethroid insecticides are critical for malaria control in Africa. However, resistance to this insecticide class in the malaria vector Anopheles funestus is spreading rapidly across Africa, threatening the success of ongoing and future malaria control programs. The underlying resistance mechanisms driving the spread of this resistance in wild populations remain largely unknown. Here, we show that increased expression of two tandemly duplicated P450 genes, CYP6P9a and CYP6P9b, is the main mechanism driving pyrethroid resistance in Malawi and Mozambique, two southern African countries where this insecticide class forms the mainstay of malaria control. Genome-wide transcription analysis using microarray and quantitative RT-PCR consistently revealed that CYP6P9a and CYP6P9b are the two genes most highly overexpressed (>50-fold; q < 0.01) in permethrin-resistant mosquitoes. Transgenic expression of CYP6P9a and CYP6P9b in Drosophila melanogaster demonstrated that elevated expression of either of these genes confers resistance to both type I (permethrin) and type II (deltamethrin) pyrethroids. Functional characterization of recombinant CYP6P9b confirmed that this protein metabolized both type I (permethrin and bifenthrin) and type II (deltamethrin and Lambda-cyhalothrin) pyrethroids but not DDT. Variability analysis identified that a single allele of each of these genes is predominantly associated with pyrethroid resistance in field populations from both countries, which is suggestive of a single origin of this resistance that has since spread across the region. Urgent resistance management strategies should be implemented in this region to limit a further spread of this resistance and minimize its impact on the success of ongoing malaria control programs.

  18. Directionally selected cytochrome P450 alleles are driving the spread of pyrethroid resistance in the major malaria vector Anopheles funestus

    PubMed Central

    Riveron, Jacob M.; Irving, Helen; Ndula, Miranda; Barnes, Kayla G.; Ibrahim, Sulaiman S.; Paine, Mark J. I.; Wondji, Charles S.

    2013-01-01

    Pyrethroid insecticides are critical for malaria control in Africa. However, resistance to this insecticide class in the malaria vector Anopheles funestus is spreading rapidly across Africa, threatening the success of ongoing and future malaria control programs. The underlying resistance mechanisms driving the spread of this resistance in wild populations remain largely unknown. Here, we show that increased expression of two tandemly duplicated P450 genes, CYP6P9a and CYP6P9b, is the main mechanism driving pyrethroid resistance in Malawi and Mozambique, two southern African countries where this insecticide class forms the mainstay of malaria control. Genome-wide transcription analysis using microarray and quantitative RT-PCR consistently revealed that CYP6P9a and CYP6P9b are the two genes most highly overexpressed (>50-fold; q < 0.01) in permethrin-resistant mosquitoes. Transgenic expression of CYP6P9a and CYP6P9b in Drosophila melanogaster demonstrated that elevated expression of either of these genes confers resistance to both type I (permethrin) and type II (deltamethrin) pyrethroids. Functional characterization of recombinant CYP6P9b confirmed that this protein metabolized both type I (permethrin and bifenthrin) and type II (deltamethrin and Lambda-cyhalothrin) pyrethroids but not DDT. Variability analysis identified that a single allele of each of these genes is predominantly associated with pyrethroid resistance in field populations from both countries, which is suggestive of a single origin of this resistance that has since spread across the region. Urgent resistance management strategies should be implemented in this region to limit a further spread of this resistance and minimize its impact on the success of ongoing malaria control programs. PMID:23248325

  19. Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

    PubMed Central

    Zhang, Haonan; Wu, Shuwen; Yang, Yihua; Tabashnik, Bruce E.; Wu, Yidong

    2012-01-01

    Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins. PMID:23285292

  20. pfmdr1 amplification and fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum in Venezuela.

    PubMed

    Griffing, Sean; Syphard, Luke; Sridaran, Sankar; McCollum, Andrea M; Mixson-Hayden, Tonya; Vinayak, Sumiti; Villegas, Leopoldo; Barnwell, John W; Escalante, Ananias A; Udhayakumar, Venkatachalam

    2010-04-01

    Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: S(tct)VMNT (91%) and S(agt)VMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites.

  1. Phenotypic instability of Arabidopsis alleles affecting a disease Resistance gene cluster

    PubMed Central

    Yi, Hankuil; Richards, Eric J

    2008-01-01

    Background Three mutations in Arabidopsis thaliana strain Columbia – cpr1, snc1, and bal – map to the RPP5 locus, which contains a cluster of disease Resistance genes. The similar phenotypes, gene expression patterns, and genetic interactions observed in these mutants are related to constitutive activation of pathogen defense signaling. However, these mutant alleles respond differently to various conditions. Exposure to mutagens, such as ethyl methanesulfonate (EMS) and γ-irradiation, induce high frequency phenotypic instability of the bal allele. In addition, a fraction of the bal and cpr1 alleles segregated from bal × cpr1 F1 hybrids also show signs of phenotypic instability. To gain more insight into the mechanism of phenotypic instability of the bal and cpr1 mutations, we systematically compared the behavior of these unusual alleles with that of the missense gain-of-function snc1 allele in response to DNA damage or passage through F1 hybrids. Results We found that the cpr1 allele is similar to the bal allele in its unstable behavior after EMS mutagenesis. For both the bal and cpr1 mutants, destabilization of phenotypes was observed in more than 10% of EMS-treated plants in the M1 generation. In addition, exceptions to simple Mendelian inheritance were identified in the M2 generation. Like cpr1 × bal F1 hybrids, cpr1 × snc1 F1 hybrids and bal × snc1 F1 hybrids exhibited dwarf morphology. While only dwarf F2 plants were produced from bal × snc1 F1 hybrids, about 10% wild-type F2 progeny were produced from cpr1 × snc1 F1 hybrids, as well as from cpr1 × bal hybrids. Segregation analysis suggested that the cpr1 allele in cpr1 × snc1 crosses was destabilized during the late F1 generation to early F2 generation. Conclusion With exposure to EMS or different F1 hybrid contexts, phenotypic instability is induced for the bal and cpr1 alleles, but not for the snc1 allele. Our results suggest that the RPP5 locus can adopt different metastable genetic or

  2. Molecular Analysis of Chloroquine and Sulfadoxine-Pyrimethamine Resistance-Associated Alleles in Plasmodium falciparum Isolates from Nicaragua

    PubMed Central

    Sridaran, Sankar; Rodriguez, Betzabe; Mercedes Soto, Aida; Macedo De Oliveira, Alexandre; Udhayakumar, Venkatachalam

    2014-01-01

    Chloroquine (CQ) is used as a first-line therapy for the treatment of Plasmodium falciparum malaria in Nicaragua. We investigated the prevalence of molecular markers associated with CQ and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum isolates obtained from the North Atlantic Autonomous Region of Nicaragua. Blood spots for this study were made available from a CQ and SP drug efficacy trial conducted in 2005 and also from a surveillance study performed in 2011. Polymorphisms in P. falciparum CQ resistance transporter, dihydrofolate reductase, and dihydropteroate synthase gene loci that are associated with resistance to CQ, pyrimethamine, and sulfadoxine, respectively, were detected by DNA sequencing. In the 2005 dataset, only 2 of 53 isolates had a CQ resistance allele (CVIET), 2 of 52 had a pyrimethamine resistance allele, and 1 of 49 had a sulfadoxine resistance allele. In the 2011 dataset, none of 45 isolates analyzed had CQ or SP resistance alleles. PMID:24615126

  3. Molecular analysis of chloroquine and sulfadoxine-pyrimethamine resistance-associated alleles in Plasmodium falciparum isolates from Nicaragua.

    PubMed

    Sridaran, Sankar; Rodriguez, Betzabe; Soto, Aida Mercedes; Macedo De Oliveira, Alexandre; Udhayakumar, Venkatachalam

    2014-05-01

    Chloroquine (CQ) is used as a first-line therapy for the treatment of Plasmodium falciparum malaria in Nicaragua. We investigated the prevalence of molecular markers associated with CQ and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum isolates obtained from the North Atlantic Autonomous Region of Nicaragua. Blood spots for this study were made available from a CQ and SP drug efficacy trial conducted in 2005 and also from a surveillance study performed in 2011. Polymorphisms in P. falciparum CQ resistance transporter, dihydrofolate reductase, and dihydropteroate synthase gene loci that are associated with resistance to CQ, pyrimethamine, and sulfadoxine, respectively, were detected by DNA sequencing. In the 2005 dataset, only 2 of 53 isolates had a CQ resistance allele (CVIET), 2 of 52 had a pyrimethamine resistance allele, and 1 of 49 had a sulfadoxine resistance allele. In the 2011 dataset, none of 45 isolates analyzed had CQ or SP resistance alleles.

  4. Selection and Spread of Artemisinin-Resistant Alleles in Thailand Prior to the Global Artemisinin Resistance Containment Campaign

    PubMed Central

    Talundzic, Eldin; Okoth, Sheila Akinyi; Congpuong, Kanungnit; Plucinski, Mateusz M.; Morton, Lindsay; Goldman, Ira F.; Kachur, Patrick S.; Wongsrichanalai, Chansuda; Satimai, Wichai; Barnwell, John W.; Udhayakumar, Venkatachalam

    2015-01-01

    The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region. PMID:25836766

  5. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.

    PubMed

    Wang, Yanpeng; Cheng, Xi; Shan, Qiwei; Zhang, Yi; Liu, Jinxing; Gao, Caixia; Qiu, Jin-Long

    2014-09-01

    Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator-like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops. PMID:25038773

  6. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.

    PubMed

    Wang, Yanpeng; Cheng, Xi; Shan, Qiwei; Zhang, Yi; Liu, Jinxing; Gao, Caixia; Qiu, Jin-Long

    2014-09-01

    Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator-like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.

  7. Evaluation of molecular-Beacon, TaqMan, and fluorescence resonance energy transfer probes for detection of antibiotic resistance-conferring single nucleotide polymorphisms in mixed Mycobacterium tuberculosis DNA extracts.

    PubMed

    Yesilkaya, Hasan; Meacci, Francesca; Niemann, Stefan; Hillemann, Doris; Rüsch-Gerdes, Sabine; Barer, Michael R; Andrew, Peter W; Oggioni, Marco R

    2006-10-01

    The ability of fluorescence resonance energy transfer, molecular-beacon, and TaqMan probes to detect single nucleotide polymorphism (SNP) in the presence of a wild-type allele was evaluated using drug resistance-conferring SNPs in mixed Mycobacterium tuberculosis DNA. It was found that both the absolute quantity and the ratio of alleles determine the detection sensitivity of the probe systems.

  8. Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate mutagenesis of two G. hirsutum breeding lines, TAM 94L...

  9. Transcriptome and allele specificity associated with a 3BL locus for Fusarium crown rot resistance in bread wheat.

    PubMed

    Ma, Jian; Stiller, Jiri; Zhao, Qiang; Feng, Qi; Cavanagh, Colin; Wang, Penghao; Gardiner, Donald; Choulet, Frédéric; Feuillet, Catherine; Zheng, You-Liang; Wei, Yuming; Yan, Guijun; Han, Bin; Manners, John M; Liu, Chunji

    2014-01-01

    Fusarium pathogens cause two major diseases in cereals, Fusarium crown rot (FCR) and head blight (FHB). A large-effect locus conferring resistance to FCR disease was previously located to chromosome arm 3BL (designated as Qcrs-3B) and several independent sets of near isogenic lines (NILs) have been developed for this locus. In this study, five sets of the NILs were used to examine transcriptional changes associated with the Qcrs-3B locus and to identify genes linked to the resistance locus as a step towards the isolation of the causative gene(s). Of the differentially expressed genes (DEGs) detected between the NILs, 12.7% was located on the single chromosome 3B. Of the expressed genes containing SNP (SNP-EGs) detected, 23.5% was mapped to this chromosome. Several of the DEGs and SNP-EGs are known to be involved in host-pathogen interactions, and a large number of the DEGs were among those detected for FHB in previous studies. Of the DEGs detected, 22 were mapped in the Qcrs-3B interval and they included eight which were detected in the resistant isolines only. The enrichment of DEG, and not necessarily those containing SNPs between the resistant and susceptible isolines, around the Qcrs-3B locus is suggestive of local regulation of this region by the resistance allele. Functions for 13 of these DEGs are known. Of the SNP-EGs, 28 were mapped in the Qcrs-3B interval and biological functions for 16 of them are known. These results provide insights into responses regulated by the 3BL locus and identify a tractable number of target genes for fine mapping and functional testing to identify the causative gene(s) at this QTL. PMID:25405461

  10. Effective marker alleles associated with type II resistance of wheat to Fusarium head blight infection in fields

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular markers associated with known quantitative trait loci (QTLs) for type 2 resistance to Fusarium head blight (FHB) in bi-parental mapping populations usually have more than two alleles in breeding populations. Therefore, understanding the association of each allele with FHB response is parti...

  11. Geographical distribution of pyrethroid resistance allele frequency in head lice (Phthiraptera: Pediculidae) from Argentina.

    PubMed

    Toloza, Ariel Ceferino; Ascunce, Marina S; Reed, David; Picollo, María Inés

    2014-01-01

    The human head louse, Pediculus humanus capitis De Geer (Phthiraptera: Pediculidae), is an obligate ectoparasite that causes pediculosis capitis and has parasitized humans since the beginning of humankind. Head louse infestations are widespread throughout the world and have been increasing since the early 1990s partially because of ineffective pediculicides. In Argentina, the overuse of products containing pyrethroids has led to the development of resistant louse populations. Pyrethroid insecticides act on the nervous system affecting voltage-sensitive sodium channels. Three point mutations at the corresponding amino acid sequence positions M815I, T917I, and L920F in the voltage-gated sodium channel gene are responsible for contributing to knockdown resistance (kdr). The management of pyrethroid resistance requires either early detection or the characterization of the mechanisms involved in head louse populations. In the current study, we estimated the distribution of kdr alleles in 154 head lice from six geographical regions of Argentina. Pyrethroid resistance kdr alleles were found in high frequencies ranging from 67 to 100%. Of these, 131 (85.1%) were homozygous resistant, 13 (8.4%) were homozygous susceptible, and 10 (6.5%) were heterozygous. Exact tests for the Hardy-Weinberg equilibrium for each location showed that genotype frequencies differed significantly from expectation in four of the six sites studied. These results show that pyrethroid resistance is well established reaching an overall frequency of 88%, thus close to fixation. With 30 yr of pyrethroid-based pediculicides use in Argentina, kdr resistance has evolved rapidly among these head louse populations. PMID:24605463

  12. Geographical distribution of pyrethroid resistance allele frequency in head lice (Phthiraptera: Pediculidae) from Argentina.

    PubMed

    Toloza, Ariel Ceferino; Ascunce, Marina S; Reed, David; Picollo, María Inés

    2014-01-01

    The human head louse, Pediculus humanus capitis De Geer (Phthiraptera: Pediculidae), is an obligate ectoparasite that causes pediculosis capitis and has parasitized humans since the beginning of humankind. Head louse infestations are widespread throughout the world and have been increasing since the early 1990s partially because of ineffective pediculicides. In Argentina, the overuse of products containing pyrethroids has led to the development of resistant louse populations. Pyrethroid insecticides act on the nervous system affecting voltage-sensitive sodium channels. Three point mutations at the corresponding amino acid sequence positions M815I, T917I, and L920F in the voltage-gated sodium channel gene are responsible for contributing to knockdown resistance (kdr). The management of pyrethroid resistance requires either early detection or the characterization of the mechanisms involved in head louse populations. In the current study, we estimated the distribution of kdr alleles in 154 head lice from six geographical regions of Argentina. Pyrethroid resistance kdr alleles were found in high frequencies ranging from 67 to 100%. Of these, 131 (85.1%) were homozygous resistant, 13 (8.4%) were homozygous susceptible, and 10 (6.5%) were heterozygous. Exact tests for the Hardy-Weinberg equilibrium for each location showed that genotype frequencies differed significantly from expectation in four of the six sites studied. These results show that pyrethroid resistance is well established reaching an overall frequency of 88%, thus close to fixation. With 30 yr of pyrethroid-based pediculicides use in Argentina, kdr resistance has evolved rapidly among these head louse populations.

  13. The apolipoprotein epsilon4 allele confers additional risk in children with familial hypercholesterolemia.

    PubMed

    Wiegman, Albert; Sijbrands, Eric J G; Rodenburg, Jessica; Defesche, Joep C; de Jongh, Saskia; Bakker, Henk D; Kastelein, John J P

    2003-06-01

    Children with familial hypercholesterolemia (FH) exhibit substantial variance of LDL cholesterol. In previous studies, family members of children with FH were included, which may have influenced results. To avoid such bias, we studied phenotype in 450 unrelated children with FH and in 154 affected sib-pairs. In known families with classical FH, diagnosis was based on plasma LDL cholesterol above the age- and gender-specific 95th percentile. Girls had 0.47 +/- 0.15 mmol/L higher LDL cholesterol, compared with boys (p = 0.002). Also in girls, HDL cholesterol increased by 0.07 +/- 0.03 mmol/L per 5 y (pfor trend = 0.005); this age effect was not observed in boys. The distribution of apolipoprotein (apo) E genotypes was not significantly different between probands, their paired affected siblings, or a Dutch control population. Carriers with or without one epsilon4 allele had similar LDL and HDL cholesterol levels. Within the affected sib-pairs, the epsilon4 allele explained 72.4% of the variance of HDL cholesterol levels (-0.15 mmol/L, 95% confidence interval -0.24 to -0.05, p = 0.003). The effect of apoE4 on HDL cholesterol differed with an analysis based on probands or on affected sib-pairs. The affected sib-pair model used adjustment for shared environment, type of LDL receptor gene mutation, and a proportion of additional genetic factors and may, therefore, be more accurate in estimating effects of risk factors on complex traits. We conclude that the epsilon4 allele was associated with lower HDL cholesterol levels in an affected sib-pair analysis, which strongly suggests that apoE4 influences HDL cholesterol levels in FH children. Moreover, the strong association suggests that apoE4 carries an additional disadvantage for FH children. PMID:12646733

  14. Identification of amino acids conferring high-level resistance to expanded-spectrum cephalosporins in the penA gene from Neisseria gonorrhoeae strain H041.

    PubMed

    Tomberg, Joshua; Unemo, Magnus; Ohnishi, Makoto; Davies, Christopher; Nicholas, Robert A

    2013-07-01

    The recent identification of a high-level-ceftriaxone-resistant (MIC = 2 to 4 μg/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empirical antimicrobial monotherapy. The penA gene from H041 (penA41) is a mosaic penA allele similar to mosaic alleles conferring intermediate-level cephalosporin resistance (Ceph(i)) worldwide but has 13 additional mutations compared to the mosaic penA gene from the previously studied Ceph(i) strain 35/02 (penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MICs for ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction fragment exchange and site-directed mutagenesis, we identified three mutations, A311V, T316P, and T483S, that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. A311V and T316P are close to the active-site nucleophile Ser310 that forms the acyl-enzyme complex, while Thr483 is predicted to interact with the carboxylate of the β-lactam antibiotic. These three mutations have thus far been described only for penA41, but dissemination of these mutations in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.

  15. Suspension Array for Multiplex Detection of Eight Fungicide-Resistance Related Alleles in Botrytis cinerea

    PubMed Central

    Zhang, Xin; Xie, Fei; Lv, Baobei; Zhao, Pengxiang; Ma, Xuemei

    2016-01-01

    A simple and high-throughput assay to detect fungicide resistance is required for large-scale monitoring of the emergence of resistant strains of Botrytis cinerea. Using suspension array technology performed on a Bio-Plex 200 System, we developed a single-tube allele-specific primer extension assay that can simultaneously detect eight alleles in one reaction. These eight alleles include E198 and 198A of the β-Tubulin gene (BenA), H272 and 272Y of the Succinate dehydrogenase iron–sulfur subunit gene (SdhB), I365 and 365S of the putative osmosensor histidine kinase gene (BcOS1), and F412 and 412S of the 3-ketoreductase gene (erg27). This assay was first established and optimized with eight plasmid templates containing the DNA sequence variants BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S. Results indicated that none of the probes showed cross-reactivity with one another. The minimum limit of detection for these genotypes was one copy per test. Four mutant plasmids were mixed with 10 ng/μL wild-type genomic DNA in different ratios. Detection sensitivity of mutant loci was 0.45% for BenA-E198A, BcOS1-I365S, and erg27-F412S, and was 4.5% for SdhB-H272Y. A minimum quantity of 0.1 ng of genomic DNA was necessary to obtain reliable results. This is the first reported assay that can simultaneously detect mutations in BenA, SdhB, BcOS1, and erg27. PMID:27708631

  16. Multiple alleles for resistance and susceptibility modulate the defense response in the interaction of tetraploid potato (Solanum tuberosum) with Synchytrium endobioticum pathotypes 1, 2, 6 and 18.

    PubMed

    Ballvora, Agim; Flath, Kerstin; Lübeck, Jens; Strahwald, Josef; Tacke, Eckhard; Hofferbert, Hans-Reinhard; Gebhardt, Christiane

    2011-12-01

    The obligate biotrophic, soil-borne fungus Synchytrium endobioticum causes wart disease of potato (Solanum tuberosum), which is a serious problem for crop production in countries with moderate climates. S. endobioticum induces hypertrophic cell divisions in plant host tissues leading to the formation of tumor-like structures. Potato wart is a quarantine disease and chemical control is not possible. From 38 S. endobioticum pathotypes occurring in Europe, pathotypes 1, 2, 6 and 18 are the most relevant. Genetic resistance to wart is available but only few current potato varieties are resistant to all four pathotypes. The phenotypic evaluation of wart resistance is laborious, time-consuming and sometimes ambiguous, which makes breeding for resistance difficult. Molecular markers diagnostic for genes for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 would greatly facilitate the selection of new, resistant cultivars. Two tetraploid half-sib families (266 individuals) segregating for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 were produced by crossing a resistant genotype with two different susceptible ones. The families were scored for five different wart resistance phenotypes. The distribution of mean resistance scores was quantitative in both families. Resistance to pathotypes 2, 6 and 18 was correlated and independent from resistance to pathotype 1. DNA pools were constructed from the most resistant and most susceptible individuals and screened with genome wide simple sequence repeat (SSR), inverted simple sequence region (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Bulked segregant analysis identified three SSR markers that were linked to wart resistance loci (Sen). Sen1-XI on chromosome XI conferred partial resistance to pathotype 1, Sen18-IX on chromosome IX to pathotype 18 and Sen2/6/18-I on chromosome I to pathotypes 2,6 and 18. Additional genotyping with 191 single nucleotide polymorphism (SNP) markers confirmed the

  17. Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens

    PubMed Central

    Gould, F.; Anderson, A.; Jones, A.; Sumerford, D.; Heckel, D. G.; Lopez, J.; Micinski, S.; Leonard, R.; Laster, M.

    1997-01-01

    The risk of rapid pest adaptation to an insecticide is highly dependent on the initial frequency of resistance alleles in field populations. Because we have lacked empirical estimates of these frequencies, population–genetic models of resistance evolution have relied on a wide range of theoretical estimates. The recent commercialization of genetically engineered cotton that constitutively produces an insecticidal protein derived from the biocontrol agent, Bacillus thuringiensis (Bt) has raised concern that we lack data needed to quantify the risk of insect pests such as Heliothis virescens rapidly adapting to this ecologically valuable class of toxins. By individually mating over 2,000 male H. virescens moths collected in four states to females of a Bt toxin-resistant laboratory strain, and screening F1 and F2 offspring for tolerance of the toxic protein, we were able to directly estimate the field frequency of alleles for resistance as 1.5 × 10−3. This high initial frequency underscores the need for caution in deploying transgenic cotton to control insect pests. Our single-pair mating technique greatly increases the efficiency of detecting recessive resistance alleles. Because alleles that decrease target site sensitivity to Bt toxins and other insecticides are often recessive, this technique could be useful in estimating resistance allele frequencies in other insects exposed to transgenic insecticidal crops or conventional insecticides. PMID:11038613

  18. Transgenic strategies to confer resistance against viruses in rice plants

    PubMed Central

    Sasaya, Takahide; Nakazono-Nagaoka, Eiko; Saika, Hiroaki; Aoki, Hideyuki; Hiraguri, Akihiro; Netsu, Osamu; Uehara-Ichiki, Tamaki; Onuki, Masatoshi; Toki, Seichi; Saito, Koji; Yatou, Osamu

    2014-01-01

    Rice (Oryza sativa L.) is cultivated in more than 100 countries and supports nearly half of the world’s population. Developing efficient methods to control rice viruses is thus an urgent necessity because viruses cause serious losses in rice yield. Most rice viruses are transmitted by insect vectors, notably planthoppers and leafhoppers. Viruliferous insect vectors can disperse their viruses over relatively long distances, and eradication of the viruses is very difficult once they become widespread. Exploitation of natural genetic sources of resistance is one of the most effective approaches to protect crops from virus infection; however, only a few naturally occurring rice genes confer resistance against rice viruses. Many investigators are using genetic engineering of rice plants as a potential strategy to control viral diseases. Using viral genes to confer pathogen-derived resistance against crops is a well-established procedure, and the expression of various viral gene products has proved to be effective in preventing or reducing infection by various plant viruses since the 1990s. RNA interference (RNAi), also known as RNA silencing, is one of the most efficient methods to confer resistance against plant viruses on their respective crops. In this article, we review the recent progress, mainly conducted by our research group, in transgenic strategies to confer resistance against tenuiviruses and reoviruses in rice plants. Our findings also illustrate that not all RNAi constructs against viral RNAs are equally effective in preventing virus infection and that it is important to identify the viral “Achilles’ heel” gene to target for RNAi attack when engineering plants. PMID:24454308

  19. Mhc supertypes confer both qualitative and quantitative resistance to avian malaria infections in a wild bird population.

    PubMed

    Sepil, Irem; Lachish, Shelly; Hinks, Amy E; Sheldon, Ben C

    2013-05-22

    Major histocompatibility complex (Mhc) genes are believed to play a key role in the genetic basis of disease control. Although numerous studies have sought links between Mhc and disease prevalence, many have ignored the ecological and epidemiological aspects of the host-parasite interaction. Consequently, interpreting associations between prevalence and Mhc has been difficult, whereas discriminating alleles for qualitative resistance, quantitative resistance and susceptibility remains challenging. Moreover, most studies to date have quantified associations between genotypes and disease status, overlooking the complex relationship between genotype and the properties of the Mhc molecule that interacts with parasites. Here, we address these problems and demonstrate avian malaria (Plasmodium) parasite species-specific associations with functional properties of Mhc molecules (Mhc supertypes) in a wild great tit (Parus major) population. We further show that correctly interpreting these associations depends crucially on understanding the spatial variation in risk of infection and the fitness effects of infection. We report that a single Mhc supertype confers qualitative resistance to Plasmodium relictum, whereas a different Mhc supertype confers quantitative resistance to Plasmodium circumflexum infections. Furthermore, we demonstrate common functional properties of Plasmodium-resistance alleles in passerine birds, suggesting this is a model system for parasite-Mhc associations in the wild. PMID:23516242

  20. Mhc supertypes confer both qualitative and quantitative resistance to avian malaria infections in a wild bird population

    PubMed Central

    Sepil, Irem; Lachish, Shelly; Hinks, Amy E.; Sheldon, Ben C.

    2013-01-01

    Major histocompatibility complex (Mhc) genes are believed to play a key role in the genetic basis of disease control. Although numerous studies have sought links between Mhc and disease prevalence, many have ignored the ecological and epidemiological aspects of the host–parasite interaction. Consequently, interpreting associations between prevalence and Mhc has been difficult, whereas discriminating alleles for qualitative resistance, quantitative resistance and susceptibility remains challenging. Moreover, most studies to date have quantified associations between genotypes and disease status, overlooking the complex relationship between genotype and the properties of the Mhc molecule that interacts with parasites. Here, we address these problems and demonstrate avian malaria (Plasmodium) parasite species-specific associations with functional properties of Mhc molecules (Mhc supertypes) in a wild great tit (Parus major) population. We further show that correctly interpreting these associations depends crucially on understanding the spatial variation in risk of infection and the fitness effects of infection. We report that a single Mhc supertype confers qualitative resistance to Plasmodium relictum, whereas a different Mhc supertype confers quantitative resistance to Plasmodium circumflexum infections. Furthermore, we demonstrate common functional properties of Plasmodium-resistance alleles in passerine birds, suggesting this is a model system for parasite–Mhc associations in the wild. PMID:23516242

  1. Allelic Heterogeneity at the Serotonin Transporter Locus (SLC6A4) Confers Susceptibility to Autism and Rigid-Compulsive Behaviors

    PubMed Central

    Sutcliffe, James S.; Delahanty, Ryan J.; Prasad, Harish C.; McCauley, Jacob L.; Han, Qiao; Jiang, Lan; Li, Chun; Folstein, Susan E.; Blakely, Randy D.

    2005-01-01

    Autism is a spectrum of neurodevelopmental disorders with a primarily genetic etiology exhibiting deficits in (1) development of language and (2) social relationships and (3) patterns of repetitive, restricted behaviors or interests and resistance to change. Elevated platelet serotonin (5-HT) in 20%–25% of cases and efficacy of selective 5-HT reuptake inhibitors (SSRIs) in treating anxiety, depression, and repetitive behaviors points to the 5-HT transporter (5-HTT; SERT) as a strong candidate gene. Association studies involving the functional insertion/deletion polymorphism in the promoter (5-HTTLPR) and a polymorphism in intron 2 are inconclusive, possibly because of phenotypic heterogeneity. Nonetheless, mounting evidence for genetic linkage of autism to the chromosome 17q11.2 region that harbors the SERT locus (SLC6A4) supports a genetic effect at or near this gene. We confirm recent reports of sex-biased genetic effects in 17q by showing highly significant linkage driven by families with only affected males. Association with common alleles fails to explain observed linkage; therefore, we hypothesized that preferential transmission of multiple alleles does explain it. From 120 families, most contributing to linkage at 17q11.2, we found four coding substitutions at highly conserved positions and 15 other variants in 5′ noncoding and other intronic regions transmitted in families exhibiting increased rigid-compulsive behaviors. In the aggregate, these variants show significant linkage to and association with autism. Our data provide strong support for a collection of multiple, often rare, alleles at SLC6A4 as imposing risk of autism. PMID:15995945

  2. Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction.

    PubMed

    Do, Ron; Stitziel, Nathan O; Won, Hong-Hee; Jørgensen, Anders Berg; Duga, Stefano; Angelica Merlini, Pier; Kiezun, Adam; Farrall, Martin; Goel, Anuj; Zuk, Or; Guella, Illaria; Asselta, Rosanna; Lange, Leslie A; Peloso, Gina M; Auer, Paul L; Girelli, Domenico; Martinelli, Nicola; Farlow, Deborah N; DePristo, Mark A; Roberts, Robert; Stewart, Alexander F R; Saleheen, Danish; Danesh, John; Epstein, Stephen E; Sivapalaratnam, Suthesh; Hovingh, G Kees; Kastelein, John J; Samani, Nilesh J; Schunkert, Heribert; Erdmann, Jeanette; Shah, Svati H; Kraus, William E; Davies, Robert; Nikpay, Majid; Johansen, Christopher T; Wang, Jian; Hegele, Robert A; Hechter, Eliana; Marz, Winfried; Kleber, Marcus E; Huang, Jie; Johnson, Andrew D; Li, Mingyao; Burke, Greg L; Gross, Myron; Liu, Yongmei; Assimes, Themistocles L; Heiss, Gerardo; Lange, Ethan M; Folsom, Aaron R; Taylor, Herman A; Olivieri, Oliviero; Hamsten, Anders; Clarke, Robert; Reilly, Dermot F; Yin, Wu; Rivas, Manuel A; Donnelly, Peter; Rossouw, Jacques E; Psaty, Bruce M; Herrington, David M; Wilson, James G; Rich, Stephen S; Bamshad, Michael J; Tracy, Russell P; Cupples, L Adrienne; Rader, Daniel J; Reilly, Muredach P; Spertus, John A; Cresci, Sharon; Hartiala, Jaana; Tang, W H Wilson; Hazen, Stanley L; Allayee, Hooman; Reiner, Alex P; Carlson, Christopher S; Kooperberg, Charles; Jackson, Rebecca D; Boerwinkle, Eric; Lander, Eric S; Schwartz, Stephen M; Siscovick, David S; McPherson, Ruth; Tybjaerg-Hansen, Anne; Abecasis, Goncalo R; Watkins, Hugh; Nickerson, Deborah A; Ardissino, Diego; Sunyaev, Shamil R; O'Donnell, Christopher J; Altshuler, David; Gabriel, Stacey; Kathiresan, Sekar

    2015-02-01

    Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. When MI occurs early in life, genetic inheritance is a major component to risk. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families, whereas common variants at more than 45 loci have been associated with MI risk in the population. Here we evaluate how rare mutations contribute to early-onset MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes in which rare coding-sequence mutations were more frequent in MI cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare non-synonymous mutations were at 4.2-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). Approximately 2% of early MI cases harbour a rare, damaging mutation in LDLR; this estimate is similar to one made more than 40 years ago using an analysis of total cholesterol. Among controls, about 1 in 217 carried an LDLR coding-sequence mutation and had plasma LDL cholesterol > 190 mg dl(-1). At apolipoprotein A-V (APOA5), carriers of rare non-synonymous mutations were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol, whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding-sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase and apolipoprotein C-III (refs 18, 19). Combined, these observations suggest that, as well as LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.

  3. Multiple rare alleles at LDLR and APOA5 confer risk for early-onset myocardial infarction

    PubMed Central

    Do, Ron; Stitziel, Nathan O.; Won, Hong-Hee; Jørgensen, Anders Berg; Duga, Stefano; Merlini, Pier Angelica; Kiezun, Adam; Farrall, Martin; Goel, Anuj; Zuk, Or; Guella, Illaria; Asselta, Rosanna; Lange, Leslie A.; Peloso, Gina M.; Auer, Paul L.; Girelli, Domenico; Martinelli, Nicola; Farlow, Deborah N.; DePristo, Mark A.; Roberts, Robert; Stewart, Alexander F.R.; Saleheen, Danish; Danesh, John; Epstein, Stephen E.; Sivapalaratnam, Suthesh; Hovingh, G. Kees; Kastelein, John J.; Samani, Nilesh J.; Schunkert, Heribert; Erdmann, Jeanette; Shah, Svati H.; Kraus, William E.; Davies, Robert; Nikpay, Majid; Johansen, Christopher T.; Wang, Jian; Hegele, Robert A.; Hechter, Eliana; Marz, Winfried; Kleber, Marcus E.; Huang, Jie; Johnson, Andrew D.; Li, Mingyao; Burke, Greg L.; Gross, Myron; Liu, Yongmei; Assimes, Themistocles L.; Heiss, Gerardo; Lange, Ethan M.; Folsom, Aaron R.; Taylor, Herman A.; Olivieri, Oliviero; Hamsten, Anders; Clarke, Robert; Reilly, Dermot F.; Yin, Wu; Rivas, Manuel A.; Donnelly, Peter; Rossouw, Jacques E.; Psaty, Bruce M.; Herrington, David M.; Wilson, James G.; Rich, Stephen S.; Bamshad, Michael J.; Tracy, Russell P.; Cupples, L. Adrienne; Rader, Daniel J.; Reilly, Muredach P.; Spertus, John A.; Cresci, Sharon; Hartiala, Jaana; Tang, W.H. Wilson; Hazen, Stanley L.; Allayee, Hooman; Reiner, Alex P.; Carlson, Christopher S.; Kooperberg, Charles; Jackson, Rebecca D.; Boerwinkle, Eric; Lander, Eric S.; Schwartz, Stephen M.; Siscovick, David S.; McPherson, Ruth; Tybjaerg-Hansen, Anne; Abecasis, Goncalo R.; Watkins, Hugh; Nickerson, Deborah A.; Ardissino, Diego; Sunyaev, Shamil R.; O’Donnell, Christopher J.; Altshuler, David; Gabriel, Stacey; Kathiresan, Sekar

    2014-01-01

    Summary Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance1,2. When MI occurs early in life, the role of inheritance is substantially greater1. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families3–8 whereas common variants at more than 45 loci have been associated with MI risk in the population9–15. Here, we evaluate the contribution of rare mutations to MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes where rare coding-sequence mutations were more frequent in cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare, damaging mutations (3.1% of cases versus 1.3% of controls) were at 2.4-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). This sequence-based estimate of the proportion of early MI cases due to LDLR mutations is remarkably similar to an estimate made more than 40 years ago using total cholesterol16. At apolipoprotein A-V (APOA5), carriers of rare nonsynonymous mutations (1.4% of cases versus 0.6% of controls) were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase15,17 and apolipoprotein C318,19. When combined, these observations suggest that, beyond LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk. PMID:25487149

  4. Bean common mosaic virus Isolate Exhibits a Novel Pathogenicity Profile in Common Bean, Overcoming the bc-3 Resistance Allele Coding for the Mutated eIF4E Translation Initiation Factor.

    PubMed

    Feng, Xue; Myers, James R; Karasev, Alexander V

    2015-11-01

    Resistance against Bean common mosaic virus (BCMV) in Phaseolus vulgaris is governed by six recessive resistance alleles at four loci. One of these alleles, bc-3, is able to protect P. vulgaris against all BCMV strains and against other potyviruses; bc-3 was identified as the eIF4E allele carrying mutated eukaryotic translation initiation factor gene. Here, we characterized a novel BCMV isolate 1755a that was able to overcome bc-2 and bc-3 alleles in common bean. Thus, it displayed a novel pattern of interactions with resistance genes in P. vulgaris, and was assigned to a new pathogroup, PG-VIII. The IVT7214 cultivar supporting the replication of BCMV-1755a was found to have the intact homozygous bc-3 cleaved amplified polymorphic sequences marker and corresponding mutations in the eIF4E allele that confer resistance to BCMV isolates from all other pathogroups as well as to other potyviruses. The VPg protein of 1755a had seven amino acid substitutions relative to VPgs of other BCMV isolates unable to overcome bc-3. The 1755a genome was found to be a recombinant between NL1, US1 (both PG-I), and a yet unknown BCMV strain. Analysis of the recombination patterns in the genomes of NL1 and US1 (PG-I), NY15P (PG-V), US10 and RU1-OR (PG-VII), and 1755a (PG-VIII), indicated that P1/HC-Pro cistrons of BCMV strains may interact with most resistance genes. This is the first report of a BCMV isolate able to overcome the bc-3 resistance allele, suggesting that the virus has evolved mechanisms to overcome multiple resistance genes available in common bean. PMID:26196181

  5. Ancestral QTL Alleles from Wild Emmer Wheat Improve Drought Resistance and Productivity in Modern Wheat Cultivars

    PubMed Central

    Merchuk-Ovnat, Lianne; Barak, Vered; Fahima, Tzion; Ordon, Frank; Lidzbarsky, Gabriel A.; Krugman, Tamar; Saranga, Yehoshua

    2016-01-01

    Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is considered a promising source for improving stress resistances in domesticated wheat. Here we explored the potential of selected quantitative trait loci (QTLs) from wild emmer wheat, introgressed via marker-assisted selection, to enhance drought resistance in elite durum (T. turgidum ssp. durum) and bread (T. aestivum) wheat cultivars. The resultant near-isogenic lines (BC3F3 and BC3F4) were genotyped using SNP array to confirm the introgressed genomic regions and evaluated in two consecutive years under well-watered (690–710 mm) and water-limited (290–320 mm) conditions. Three of the introgressed QTLs were successfully validated, two in the background of durum wheat cv. Uzan (on chromosomes 1BL and 2BS), and one in the background of bread wheat cvs. Bar Nir and Zahir (chromosome 7AS). In most cases, the QTL x environment interaction was validated in terms of improved grain yield and biomass—specifically under drought (7AS QTL in cv. Bar Nir background), under both treatments (2BS QTL), and a greater stability across treatments (1BL QTL). The results provide a first demonstration that introgression of wild emmer QTL alleles can enhance productivity and yield stability across environments in domesticated wheat, thereby enriching the modern gene pool with essential diversity for the improvement of drought resistance. PMID:27148287

  6. Mutations in α-Tubulin Confer Dinitroaniline Resistance at a Cost to Microtubule Function

    PubMed Central

    Ma, Christopher; Li, Catherine; Ganesan, Lakshmi; Oak, Jean; Tsai, Susan; Sept, David

    2007-01-01

    Protozoan microtubules are sensitive to disruption by dinitroanilines, compounds that kill intracellular Toxoplasma gondii parasites without affecting microtubules in vertebrate host cells. We previously isolated a number of resistant Toxoplasma lines that harbor mutations to the α1-tubulin gene. Some of the mutations are localized in or near the M and N loops, domains that coordinate lateral interactions between protofilaments. Other resistance mutations map to a computationally identified binding site beneath the N loop. Allelic replacement of wild-type α1-tubulin with the individual mutations is sufficient to confer dinitroaniline resistance. Some mutations seem to increase microtubule length, suggesting that they increase subunit affinity. All mutations are associated with replication defects that decrease parasite viability. When parasites bearing the N loop mutation Phe52Tyr are grown without dinitroaniline selection, they spontaneously acquired secondary mutations in the M loop (Ala273Val) or in an α-tubulin–specific insert that stabilizes the M loop (Asp367Val). Parasites with the double mutations have both reduced resistance and diminished incidence of replication defects, suggesting that the secondary mutations decrease protofilament affinity to increase parasite fitness. PMID:17881728

  7. ATP7B expression confers multidrug resistance through drug sequestration.

    PubMed

    Moinuddin, F M; Shinsato, Yoshinari; Komatsu, Masaharu; Mitsuo, Ryoichi; Minami, Kentaro; Yamamoto, Masatatsu; Kawahara, Kohich; Hirano, Hirofumi; Arita, Kazunori; Furukawa, Tatsuhiko

    2016-04-19

    We previously reported that ATP7B is involved in cisplatin resistance and ATP7A confers multidrug resistance (MDR) in cancer cells.In this study, we show that ATP7B expressing cells also are resistant to doxorubicin, SN-38, etoposide, and paclitaxel as well as cisplatin.In ATP7B expressing cells, doxorubicin relocated from the nuclei to the late-endosome at 4 hours after doxorubicin exposure. EGFP-ATP7B mainly colocalized with doxorubicin.ATP7B has six metal binding sites (MBSs) in the N-terminal cytoplasmic region. To investigate the role of the MBSs of ATP7B in doxorubicin resistance, we used three mutant ATP7B (Cu0, Cu6 and M6C/S) expressing cells. Cu0 has no MBSs, Cu6 has only the sixth MBS and M6C/S carries CXXC to SXXS mutation in the sixth MBS. Cu6 expressing cells were less resistance to the anticancer agents than wild type ATP7B expressing cells, and had doxorubicin sequestration in the late-endosome. Cu0- and M6C/S-expressing cells were sensitive to doxorubicin. In these cells, doxorubicin did not relocalize to the late-endosome. EGFP-M6C/S mainly localized to the trans-Golgi network (TGN) even in the presence of copper. Thus the cysteine residues in the sixth MBS of ATP7B are essential for MDR phenotype.Finally, we found that ammonium chloride and tamoxifen suppressed late endosomal sequestration of doxorubicin, thereby attenuating drug resistance. These results suggest that the sequestration depends on the acidity of the vesicles partly.We here demonstrate that ATP7B confers MDR by facilitating nuclear drug efflux and late endosomal drug sequestration. PMID:26988911

  8. ATP7B expression confers multidrug resistance through drug sequestration

    PubMed Central

    Moinuddin, F M; Shinsato, Yoshinari; Komatsu, Masaharu; Mitsuo, Ryoichi; Minami, Kentaro; Yamamoto, Masatatsu; Kawahara, Kohich; Hirano, Hirofumi; Arita, Kazunori; Furukawa, Tatsuhiko

    2016-01-01

    We previously reported that ATP7B is involved in cisplatin resistance and ATP7A confers multidrug resistance (MDR) in cancer cells. In this study, we show that ATP7B expressing cells also are resistant to doxorubicin, SN-38, etoposide, and paclitaxel as well as cisplatin. In ATP7B expressing cells, doxorubicin relocated from the nuclei to the late-endosome at 4 hours after doxorubicin exposure. EGFP-ATP7B mainly colocalized with doxorubicin. ATP7B has six metal binding sites (MBSs) in the N-terminal cytoplasmic region. To investigate the role of the MBSs of ATP7B in doxorubicin resistance, we used three mutant ATP7B (Cu0, Cu6 and M6C/S) expressing cells. Cu0 has no MBSs, Cu6 has only the sixth MBS and M6C/S carries CXXC to SXXS mutation in the sixth MBS. Cu6 expressing cells were less resistance to the anticancer agents than wild type ATP7B expressing cells, and had doxorubicin sequestration in the late-endosome. Cu0- and M6C/S-expressing cells were sensitive to doxorubicin. In these cells, doxorubicin did not relocalize to the late-endosome. EGFP-M6C/S mainly localized to the trans-Golgi network (TGN) even in the presence of copper. Thus the cysteine residues in the sixth MBS of ATP7B are essential for MDR phenotype. Finally, we found that ammonium chloride and tamoxifen suppressed late endosomal sequestration of doxorubicin, thereby attenuating drug resistance. These results suggest that the sequestration depends on the acidity of the vesicles partly. We here demonstrate that ATP7B confers MDR by facilitating nuclear drug efflux and late endosomal drug sequestration. PMID:26988911

  9. Acquisition of a single EZH2 D1 domain mutation confers acquired resistance to EZH2-targeted inhibitors

    PubMed Central

    Baker, Theresa; Nerle, Sujata; Pritchard, Justin; Zhao, Boyang; Rivera, Victor M.

    2015-01-01

    Although targeted therapies have revolutionized cancer treatment, overcoming acquired resistance remains a major clinical challenge. EZH2 inhibitors (EZH2i), EPZ-6438 and GSK126, are currently in the early stages of clinical evaluation and the first encouraging signs of efficacy have recently emerged in the clinic. To anticipate mechanisms of resistance to EZH2i, we used a forward genetic platform combining a mutagenesis screen with next generation sequencing technology and identified a hotspot of secondary mutations in the EZH2 D1 domain (Y111 and I109). Y111D mutation within the WT or A677G EZH2 allele conferred robust resistance to both EPZ-6438 and GSK126, but it only drove a partial resistance within the Y641F allele. EZH2 mutants required histone methyltransferase (HMT) catalytic activity and the polycomb repressive complex 2 (PRC2) components, SUZ12 and EED, to drive drug resistance. Furthermore, D1 domain mutations not only blocked the ability of EZH2i to bind to WT and A677G mutant, but also abrogated drug binding to the Y641F mutant. These data provide the first cellular validation of the mechanistic model underpinning the oncogenic function of WT and mutant EZH2. Importantly, our findings suggest that acquired-resistance to EZH2i may arise in WT and mutant EZH2 patients through a single mutation that remains targetable by second generation EZH2i. PMID:26360609

  10. Identification of QTLs conferring resistance to downy mildews of maize in Asia.

    PubMed

    George, M L C; Prasanna, B M; Rathore, R S; Setty, T A S; Kasim, F; Azrai, M; Vasal, S; Balla, O; Hautea, D; Canama, A; Regalado, E; Vargas, M; Khairallah, M; Jeffers, D; Hoisington, D

    2003-08-01

    Downy mildew is one of the most destructive diseases of maize in subtropical and tropical regions in Asia. As a prerequisite for improving downy mildew resistance in maize, we analyzed quantitative trait loci (QTLs) involved in resistance to the important downy mildew pathogens--Peronosclerospora sorghi (sorghum downy mildew) and P. heteropogoni (Rajasthan downy mildew) in India, P. maydis (Java downy mildew) in Indonesia, P. zeae in Thailand and P. philippinensis in the Philippines--using a recombinant inbred line population derived from a cross between Ki3 (downy mildew resistant) and CML139 (susceptible). Resistance was evaluated as percentage disease incidence in replicated field trials at five downy mildew 'hotspots' in the four countries. Heritability estimates of individual environments ranged from 0.58 to 0.75 with an across environment heritability of 0.50. Composite interval mapping was applied for QTL detection using a previously constructed restriction fragment length polymorphism linkage map. The investigation resulted in the identification of six genomic regions on chromosomes 1, 2, 6, 7 and 10 involved in the resistance to the downy mildews under study, explaining, in total, 26-57% of the phenotypic variance for disease response. Most QTL alleles conferring resistance to the downy mildews were from Ki3. All QTLs showed significant QTL x environment interactions, suggesting that the expression of the QTL may be environment-dependent. A strong QTL on chromosome 6 was stable across environments, significantly affecting disease resistance at the five locations in four Asian countries. Simple-sequence repeat markers tightly linked to this QTL were identified for potential use in marker-assisted selection.

  11. QTL conferring fusarium crown rot resistance in the elite bread wheat variety EGA Wylie.

    PubMed

    Zheng, Zhi; Kilian, Andrzej; Yan, Guijun; Liu, Chunji

    2014-01-01

    Fusarium crown rot (FCR) is one of the most damaging cereal diseases in semi-arid regions worldwide. The genetics of FCR resistance in the bread wheat (Triticum eastivum L.) variety EGA Wylie, the most resistant commercial variety available, was studied by QTL mapping. Three populations of recombinant inbred lines were developed with this elite variety as the resistant parent. Four QTL conferring FCR resistance were detected and resistance alleles of all of them were derived from the resistant parent EGA Wylie. One of these loci was located on the short arm of chromosome 5D (designated as Qcrs.cpi-5D). This QTL explains up to 31.1% of the phenotypic variance with an LOD value of 9.6. The second locus was located on the long arm of chromosome 2D (designated as Qcrs.cpi-2D) and explained up to 20.2% of the phenotypic variance with an LOD value of 4.5. Significant effects of both Qcrs.cpi-5D and Qcrs.cpi-2D were detected in each of the three populations assessed. Another two QTL (designated as Qcrs.cpi-4B.1 and Qcrs.cpi-4B.2, respectively) were located on the short arm of chromosome 4B. These two QTL explained up to 16.9% and 18.8% of phenotypic variance, respectively. However, significant effects of Qcrs.cpi-4B.1 and Qcrs.cpi-4B.2 were not detected when the effects of plant height was accounted for by covariance analysis. The elite characteristics of this commercial variety should facilitate the incorporation of the resistance loci it contains into breeding programs.

  12. QTL conferring fusarium crown rot resistance in the elite bread wheat variety EGA Wylie.

    PubMed

    Zheng, Zhi; Kilian, Andrzej; Yan, Guijun; Liu, Chunji

    2014-01-01

    Fusarium crown rot (FCR) is one of the most damaging cereal diseases in semi-arid regions worldwide. The genetics of FCR resistance in the bread wheat (Triticum eastivum L.) variety EGA Wylie, the most resistant commercial variety available, was studied by QTL mapping. Three populations of recombinant inbred lines were developed with this elite variety as the resistant parent. Four QTL conferring FCR resistance were detected and resistance alleles of all of them were derived from the resistant parent EGA Wylie. One of these loci was located on the short arm of chromosome 5D (designated as Qcrs.cpi-5D). This QTL explains up to 31.1% of the phenotypic variance with an LOD value of 9.6. The second locus was located on the long arm of chromosome 2D (designated as Qcrs.cpi-2D) and explained up to 20.2% of the phenotypic variance with an LOD value of 4.5. Significant effects of both Qcrs.cpi-5D and Qcrs.cpi-2D were detected in each of the three populations assessed. Another two QTL (designated as Qcrs.cpi-4B.1 and Qcrs.cpi-4B.2, respectively) were located on the short arm of chromosome 4B. These two QTL explained up to 16.9% and 18.8% of phenotypic variance, respectively. However, significant effects of Qcrs.cpi-4B.1 and Qcrs.cpi-4B.2 were not detected when the effects of plant height was accounted for by covariance analysis. The elite characteristics of this commercial variety should facilitate the incorporation of the resistance loci it contains into breeding programs. PMID:24776887

  13. Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the ß-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in ...

  14. Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although insecticide resistance is a widespread problem for most insect pests, frequently the assessment of resistance occurs over a limited geographic range. Herein we report the first widespread survey of insecticide resistance ever undertaken for the house fly, Musca domestica, a major pest of a...

  15. Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.

    PubMed

    Mokrousov, Igor; Otten, Tatiana; Vyshnevskiy, Boris; Narvskaya, Olga

    2003-07-01

    We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB. PMID:12821473

  16. Food supply confers calcifiers resistance to ocean acidification.

    PubMed

    Ramajo, Laura; Pérez-León, Elia; Hendriks, Iris E; Marbà, Núria; Krause-Jensen, Dorte; Sejr, Mikael K; Blicher, Martin E; Lagos, Nelson A; Olsen, Ylva S; Duarte, Carlos M

    2016-01-01

    Invasion of ocean surface waters by anthropogenic CO2 emitted to the atmosphere is expected to reduce surface seawater pH to 7.8 by the end of this century compromising marine calcifiers. A broad range of biological and mineralogical mechanisms allow marine calcifiers to cope with ocean acidification, however these mechanisms are energetically demanding which affect other biological processes (trade-offs) with important implications for the resilience of the organisms against stressful conditions. Hence, food availability may play a critical role in determining the resistance of calcifiers to OA. Here we show, based on a meta-analysis of existing experimental results assessing the role of food supply in the response of organisms to OA, that food supply consistently confers calcifiers resistance to ocean acidification. PMID:26778520

  17. Food supply confers calcifiers resistance to ocean acidification

    PubMed Central

    Ramajo, Laura; Pérez-León, Elia; Hendriks, Iris E.; Marbà, Núria; Krause-Jensen, Dorte; Sejr, Mikael K.; Blicher, Martin E.; Lagos, Nelson A.; Olsen, Ylva S.; Duarte, Carlos M.

    2016-01-01

    Invasion of ocean surface waters by anthropogenic CO2 emitted to the atmosphere is expected to reduce surface seawater pH to 7.8 by the end of this century compromising marine calcifiers. A broad range of biological and mineralogical mechanisms allow marine calcifiers to cope with ocean acidification, however these mechanisms are energetically demanding which affect other biological processes (trade-offs) with important implications for the resilience of the organisms against stressful conditions. Hence, food availability may play a critical role in determining the resistance of calcifiers to OA. Here we show, based on a meta-analysis of existing experimental results assessing the role of food supply in the response of organisms to OA, that food supply consistently confers calcifiers resistance to ocean acidification. PMID:26778520

  18. Food supply confers calcifiers resistance to ocean acidification.

    PubMed

    Ramajo, Laura; Pérez-León, Elia; Hendriks, Iris E; Marbà, Núria; Krause-Jensen, Dorte; Sejr, Mikael K; Blicher, Martin E; Lagos, Nelson A; Olsen, Ylva S; Duarte, Carlos M

    2016-01-18

    Invasion of ocean surface waters by anthropogenic CO2 emitted to the atmosphere is expected to reduce surface seawater pH to 7.8 by the end of this century compromising marine calcifiers. A broad range of biological and mineralogical mechanisms allow marine calcifiers to cope with ocean acidification, however these mechanisms are energetically demanding which affect other biological processes (trade-offs) with important implications for the resilience of the organisms against stressful conditions. Hence, food availability may play a critical role in determining the resistance of calcifiers to OA. Here we show, based on a meta-analysis of existing experimental results assessing the role of food supply in the response of organisms to OA, that food supply consistently confers calcifiers resistance to ocean acidification.

  19. Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic.

    PubMed

    Larimer, Curtis; Islam, Mohammad Shyful; Ojha, Anil; Nettleship, Ian

    2014-08-01

    Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc(2)155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO3 and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO3. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc(2)155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO4 and ZnSO4 than the mc(2)155 strain.

  20. Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic.

    PubMed

    Larimer, Curtis; Islam, Mohammad Shyful; Ojha, Anil; Nettleship, Ian

    2014-08-01

    Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc(2)155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO3 and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO3. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc(2)155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO4 and ZnSO4 than the mc(2)155 strain. PMID:24989695

  1. Prion protein genotype survey confirms low frequency of scrapie-resistant K222 allele in British goat herds.

    PubMed

    Goldmann, W; Marier, E; Stewart, P; Konold, T; Street, S; Langeveld, J; Windl, O; Ortiz-Pelaez, A

    2016-02-13

    Scrapie in goats is a transmissible, fatal prion disease, which is endemic in the British goat population. The recent success in defining caprine PRNP gene variants that provide resistance to experimental and natural classical scrapie has prompted the authors to conduct a survey of PRNP genotypes in 10 goat breeds and 52 herds to find goats with the resistant K222 allele. They report here the frequencies in 1236 tested animals of the resistance-associated K222 and several other alleles by breed and herd. Eight animals were found to be heterozygous QK222 goats (0.64 per cent genotype frequency, 95 per cent CI 0.28 to 1.27 per cent) but no homozygous KK222 goats were detected. The K222 allele was found in Saanen, Toggenburg and Anglo-Nubian goats. The fact that only a few goats with the K222 allele have been identified does not preclude the possibility to design and implement successful breeding programmes at national level.

  2. Prion protein genotype survey confirms low frequency of scrapie-resistant K222 allele in British goat herds

    PubMed Central

    Goldmann, W.; Stewart, P.; Konold, T.; Street, S.; Langeveld, J.; Windl, O.; Ortiz-Pelaez, A.

    2016-01-01

    Scrapie in goats is a transmissible, fatal prion disease, which is endemic in the British goat population. The recent success in defining caprine PRNP gene variants that provide resistance to experimental and natural classical scrapie has prompted the authors to conduct a survey of PRNP genotypes in 10 goat breeds and 52 herds to find goats with the resistant K222 allele. They report here the frequencies in 1236 tested animals of the resistance-associated K222 and several other alleles by breed and herd. Eight animals were found to be heterozygous QK222 goats (0.64 per cent genotype frequency, 95 per cent CI 0.28 to 1.27 per cent) but no homozygous KK222 goats were detected. The K222 allele was found in Saanen, Toggenburg and Anglo-Nubian goats. The fact that only a few goats with the K222 allele have been identified does not preclude the possibility to design and implement successful breeding programmes at national level. PMID:26755614

  3. Knockdown Resistance Allele Frequencies in North American Head Louse (Anoplura: Pediculidae) Populations

    PubMed Central

    Yoon, Kyong Sup; Previte, Domenic J.; Hodgdon, Hilliary E.; Poole, Bryan C.; Kwon, Deok Ho; El-Ghar, Gamal E. Abo; Lee, Si Hyeock; Clark, J. Marshall

    2014-01-01

    The study examines the extent and frequency of a knockdown-type resistance allele (kdr type) in North American populations of human head lice. Lice were collected from 32 locations in Canada and the United States. DNA was extracted from individual lice and used to determine their zygosity using the serial invasive signal amplification technique to detect the kdr-type T917I (TI) mutation, which is most responsible for nerve insensitivity that results in the kdr phenotype and permethrin resistance. Previously sampled sites were resampled to determine if the frequency of the TI mutation was changing. The TI frequency was also reevaluated using a quantitative sequencing method on pooled DNA samples from selected sites to validate this population genotyping method. Genotyping substantiated that TI occurs at high levels in North American lice (88.4%). Overall, the TI frequency in U.S. lice was 84.4% from 1999 to 2009, increased to 99.6% from 2007 to 2009, and was 97.1% in Canadian lice in 2008. Genotyping results using the serial invasive signal amplification reaction (99.54%) and quantitative sequencing (99.45%) techniques were highly correlated. Thus, the frequencies of TI in North American head louse populations were found to be uniformly high, which may be due to the high selection pressure from the intensive and widespread use of the pyrethrins- or pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins- or permethrin-based products in Canada and the United States. Alternative approaches to treatment of head lice infestations are critically needed. PMID:24724296

  4. Allele mining in the pepper gene pool provided new complementation effects between pvr2-eIF4E and pvr6-eIF(iso)4E alleles for resistance to pepper veinal mottle virus.

    PubMed

    Rubio, Manuel; Nicolaï, Maryse; Caranta, Carole; Palloix, Alain

    2009-11-01

    Molecular cloning of recessive resistance genes to potyviruses in a large range of host species identified the eukaryotic translation initiation factor 4E (eIF4E) as an essential determinant in the outcome of potyvirus infection. Resistance results from a few amino acid changes in the eIF4E protein encoded by the recessive resistance allele that disrupt the direct interaction with the potyviral protein VPg. In plants, several loci encode two protein subfamilies, eIF4E and eIF(iso)4E. While most eIF4E-mediated resistance to potyviruses depends on mutations in a single eIF4E protein, simultaneous mutations in eIF4E (corresponding to the pvr2 locus) and eIF(iso)4E (corresponding to the pvr6 locus) are required to prevent pepper veinal mottle virus (PVMV) infection in pepper. We used this model to look for additional alleles at the pvr2-eIF4E locus that result in resistance when combined with the pvr6-eIF(iso)4E resistant allele. Among the 12 pvr2-eIF4E resistance alleles sequenced in the pepper gene pool, three were shown to have a complementary effect with pvr6-eIF(iso)4E for resistance. Two amino acid changes were exclusively shared by these three alleles and were systematically associated with a second amino acid change, suggesting that these substitutions are associated with resistance expression. The availability of new resistant allele combinations increases the possibility for the durable deployment of resistance against this pepper virus which is prevalent in Africa.

  5. Rifampin resistance, Beijing-W clade-single nucleotide polymorphism cluster group 2 phylogeny, and the Rv2629 191-C allele in Mycobacterium tuberculosis strains.

    PubMed

    Chakravorty, Soumitesh; Aladegbami, Bola; Motiwala, Alifiya S; Dai, Yang; Safi, Hassan; Brimacombe, Michael; Helb, Danica; Alland, David

    2008-08-01

    Rifampin resistance is a key prognostic marker for treatment success in tuberculosis patients. Recently, Wang et al. demonstrated that Rv2629 A191C mutations were present in 99.1% of rifampin-resistant and 0% of rifampin-susceptible clinical Mycobacterium tuberculosis isolates and that overexpression of the Rv2629 191C allele in Mycobacterium smegmatis produced an eightfold increase in rifampin resistance. These results suggested that Rv2629 could be a cause of rifampin resistance and a valuable target for rifampin resistance detection assays. We developed a molecular-beacon assay to study the association between Rv2629 191 alleles and rifampin resistance in 246 geographically and phylogenetically diverse clinical M. tuberculosis isolates. The 191C allele was present in 30/98 (30.6%) rifampin-resistant isolates and 25/148 (16.9%) rifampin-susceptible isolates and was more common in isolates from Asia. Phylogenetic analysis demonstrated complete overlap between the 191C allele and single nucleotide polymorphism cluster group 2 (SCG-2), a phylogenetic lineage that corresponds to the Beijing-W clade of M. tuberculosis. All 55 (100%) 191C isolates were SCG-2, while none of the 191 191A isolates were SCG-2 (P < 0.001). No association was found between the 191C allele and rifampin resistance in an analysis that included the SCG type (P = 1.0). Also, in contrast to the findings of Wang et al., we found that overexpression of either Rv2629 191 allele in M. smegmatis did not produce an increase in rifampin resistance. We conclude that the Rv2629 191C allele is not associated with rifampin resistance and that the allele cannot be used as a molecular target to detect rifampin resistance. The allele appears to be an excellent marker for the Beijing-W clade/SCG-2 phylogenetic group.

  6. Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum.

    PubMed

    Yeam, Inhwa; Kang, Byoung-Cheorl; Lindeman, Wouter; Frantz, James D; Faber, Nanne; Jahn, Molly M

    2005-12-01

    Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 (1), and pvr1 (2). These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.

  7. Allele characterization of genes required for rpg4-mediated wheat stem rust resistance identifies Rpg5 as the R gene.

    PubMed

    Arora, D; Gross, T; Brueggeman, R

    2013-11-01

    A highly virulent form of the wheat stem rust pathogen Puccinia graminis f. sp. tritici race TTKSK is virulent on both wheat and barley, presenting a major threat to world food security. The recessive and temperature-sensitive rpg4 gene is the only effective source of resistance identified in barley (Hordeum vulgare) against P. graminis f. sp. tritici race TTKSK. Efforts to position clone rpg4 localized resistance to a small interval on barley chromosome 5HL, tightly linked to the rye stem rust (P. graminis f. sp. secalis) resistance (R) gene Rpg5. High-resolution genetic analysis and post-transcriptional gene silencing of the genes at the rpg4/Rpg5 locus determined that three tightly linked genes (Rpg5, HvRga1, and HvAdf3) are required together for rpg4-mediated wheat stem rust resistance. Alleles of the three genes were analyzed from a diverse set of 14 domesticated barley lines (H. vulgare) and 8 wild barley accessions (H. vulgare subsp. spontaneum) to characterize diversity that may determine incompatibility (resistance). The analysis determined that HvAdf3 and HvRga1 code for predicted functional proteins that do not appear to contain polymorphisms determining the compatible (susceptible) interactions with the wheat stem rust pathogen and were expressed at the transcriptional level from both resistant and susceptible barley lines. The HvAdf3 alleles shared 100% amino acid identity among all 22 genotypes examined. The P. graminis f. sp. tritici race QCCJ-susceptible barley lines with HvRga1 alleles containing the limited amino acid substitutions unique to the susceptible varieties also contained predicted nonfunctional rpg5 alleles. Thus, susceptibility in these lines is likely due to the nonfunctional RPG5 proteins. The Rpg5 allele analysis determined that 9 of the 13 P. graminis f. sp. tritici race QCCJ-susceptible barley lines contain alleles that either code for predicted truncated proteins as the result of a single nucleotide substitution, resulting in a

  8. Allelic variation in the dihydrofolate reductase gene at amino acid position 95 contributes to antifolate resistance in Chinese hamster cells.

    PubMed

    Yu, M; Melera, P W

    1993-12-15

    The Chinese hamster lung cell line DC-3F contains two polymorphic dihydrofolate reductase (DHFR) alleles that are defined by an Asp-Asn amino acid sequence difference at position 95 in protein. Previously, we reported that the antifolate-resistant subline DC-3F/A3 overexpressed a Leu22-->Phe mutant of the Asp95 (21k) allele and that this was the basis of its resistance to methotrexate (MTX) and methasquin [P. W. Melera, J. P. Davide, C. A. Hession, and K. W. Scotto, Mol. Cell. Biol., 4: 38-48, 1984]. We now show that another independently selected antifolate-resistant subline of DC-3F, DC-3F8/A55, in addition to being severely compromised in its ability to accumulate MTX, overexpresses a Leu22-->Phe mutant form of the Asn95 (20k) allele. Characterization of purified DHFR from these cells showed that the enzyme displayed a 6-fold higher Kd for MTX (3.92 +/- 0.17 pM) than the wild type (0.58 +/- 0.10 pM), thus explaining its lowered sensitivity to drug. Unexpectedly, however, this value was 4-fold lower than that displayed by the DC-3F/A3 enzyme even though both contain the same (Leu22-->Phe) mutation and differ only at position 95. Indeed, we have also shown that the 21k and 20k wild type enzymes, both containing Leu at position 22, in fact differ by 3-fold (1.58 +/- 0.08 and 0.58 +/- 0.10 pM, respectively) in their Kd's for MTX. This demonstrates that the amino acid at position 95 has an effect on the ability of DHFR to bind MTX. On the other hand, these allelic variants are indistinguishable from each other in their catalytic properties and in their respective Kd's for dihydrofolate. Taken together, these characteristics are consistent with the observation that it is the wild type 21k allele which is preferentially overexpressed at a frequency of 3:1 in MTX-resistant Chinese hamster lung sublines derived by long-term selection in MTX. The results of these studies are novel in that they establish a role for allelic variation in the DHFR gene as a contributor to

  9. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

    PubMed

    Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

    2015-01-23

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.

  10. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

    PubMed

    Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

    2015-01-23

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  11. Intestinal trefoil factor confers colonic epithelial resistance to apoptosis.

    PubMed

    Taupin, D R; Kinoshita, K; Podolsky, D K

    2000-01-18

    Intestinal trefoil factor (ITF) is an essential regulator of colonic epithelial restitution, the rapid migration of colonocytes over mucosal wounds. High levels of ITF are frequently present in colorectal cancers and derived cell lines. Mucosal restitution requires the detachment of epithelium from substrate, which would be expected to induce apoptosis. However, mice deficient in ITF showed an increase in colonocyte apoptosis unaccompanied by changes in expression of receptor-related (TNFR/Fas) or stress-related (Bcl-family) cell death regulators. An ITF-expressing colonic (HT-ITF1) cell line was resistant to apoptosis induced by serum starvation and ceramide. Exogenous ITF also protected another human colonic carcinoma-derived cell line (HCT116) and a nontransformed rat intestinal epithelial cell line (IEC-6) from apoptosis. This effect was abrogated by wortmannin and tyrphostin A25, indicating the potential involvement of phosphatidylinositol 3-kinase and epidermal growth factor (EGF) receptor activation. Expression of phosphorylated Akt, which lies downstream of phosphatidylinositol 3-kinase activation, was elevated in this HT-29-ITF line. p53-dependent cell death in the AGS human gastric cancer cell line after etoposide was similarly inhibited by transient expression of ITF but not a C-terminal truncation mutant of ITF, and it required functional phosphatidylinositol 3-kinase and EGF receptor. These findings support a central role for ITF in the maintenance of intestinal mucosal continuity, and conversely demonstrate the potential for ITF expression to confer resistance of colorectal tumors to therapy.

  12. Resistance to the Novel Fungicide Pyrimorph in Phytophthora capsici: Risk Assessment and Detection of Point Mutations in CesA3 That Confer Resistance

    PubMed Central

    Pang, Zhili; Shao, Jingpeng; Chen, Lei; Lu, Xiaohong; Hu, Jian; Qin, Zhaohai; Liu, Xili

    2013-01-01

    Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC50 value of 1.4261 (±0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1×10−4. The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC50 values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations. PMID:23431382

  13. Resistance to the novel fungicide pyrimorph in Phytophthora capsici: risk assessment and detection of point mutations in CesA3 that confer resistance.

    PubMed

    Pang, Zhili; Shao, Jingpeng; Chen, Lei; Lu, Xiaohong; Hu, Jian; Qin, Zhaohai; Liu, Xili

    2013-01-01

    Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC(50) value of 1.4261 (± 0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1 × 10(-4). The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC(50) values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations.

  14. The 2-repeat allele of the MAOA gene confers an increased risk for shooting and stabbing behaviors.

    PubMed

    Beaver, Kevin M; Barnes, J C; Boutwell, Brian B

    2014-09-01

    There has been a great deal of research examining the link between a polymorphism in the promoter region of the MAOA gene and antisocial phenotypes. The results of these studies have consistently revealed that low activity MAOA alleles are related to antisocial behaviors for males who were maltreated as children. Recently, though, some evidence has emerged indicating that a rare allele of the MAOA gene-that is, the 2-repeat allele-may have effects on violence that are independent of the environment. The current study builds on this research and examines the association between the 2-repeat allele and shooting and stabbing behaviors in a sample of males drawn from the National Longitudinal Study of Adolescent Health. Analyses revealed that African-American males who carry the 2-repeat allele are significantly more likely than all other genotypes to engage in shooting and stabbing behaviors and to report having multiple shooting and stabbing victims. The limitations of the study are discussed and suggestions for future research are offered.

  15. Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

    PubMed Central

    Lewis, Jennifer D.; Wu, Ronald

    2010-01-01

    Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a. PMID:20368970

  16. Temporal trends in prevalence of Plasmodium falciparum drug resistance alleles over two decades of changing antimalarial policy in coastal Kenya.

    PubMed

    Okombo, John; Kamau, Alice W; Marsh, Kevin; Sutherland, Colin J; Ochola-Oyier, Lynette Isabella

    2014-12-01

    Molecular surveillance of drug resistance markers through time provides crucial information on genomic adaptations, especially in parasite populations exposed to changing drug pressures. To assess temporal trends of established genotypes associated with tolerance to clinically important antimalarials used in Kenya over the last two decades, we sequenced a region of the pfcrt locus encompassing codons 72-76 of the Plasmodium falciparum chloroquine resistance transporter, full-length pfmdr1 - encoding multi-drug resistance protein, P-glycoprotein homolog (Pgh1) and pfdhfr encoding dihydrofolate reductase, in 485 archived Plasmodium falciparum positive blood samples collected in coastal Kenya at four different time points between 1995 and 2013. Microsatellite loci were also analyzed to compare the genetic backgrounds of parasite populations circulating before and after the withdrawal of chloroquine and sulfadoxine/pyrimethamine. Our results reveal a significant increase in the prevalence of the pfcrt K76 wild-type allele between 1995 and 2013 from 38% to 81.7% (p < 0.0001). In contrast, we noted a significant decline in wild-type pfdhfr S108 allele (p < 0.0001) culminating in complete absence of this allele in 2013. We also observed a significant increase in the prevalence of the wild-type pfmdr1 N86/Y184/D1246 haplotype from 14.6% in 1995 to 66.0% in 2013 (p < 0.0001) and a corresponding decline of the mutant pfmdr1 86Y/184Y/1246Y allele from 36.4% to 0% in 19 years (p < 0.0001). We also show extensive genetic heterogeneity among the chloroquine-sensitive parasites before and after the withdrawal of the drug in contrast to a selective sweep around the triple mutant pfdhfr allele, leading to a mono-allelic population at this locus. These findings highlight the importance of continual surveillance and characterization of parasite genotypes as indicators of the therapeutic efficacy of antimalarials, particularly in the context of changes in malaria treatment

  17. Identification of multiple interacting alleles conferring low glycerol and high ethanol yield in Saccharomyces cerevisiae ethanolic fermentation

    PubMed Central

    2013-01-01

    Background Genetic engineering of industrial microorganisms often suffers from undesirable side effects on essential functions. Reverse engineering is an alternative strategy to improve multifactorial traits like low glycerol/high ethanol yield in yeast fermentation. Previous rational engineering of this trait always affected essential functions like growth and stress tolerance. We have screened Saccharomyces cerevisiae biodiversity for specific alleles causing lower glycerol/higher ethanol yield, assuming higher compatibility with normal cellular functionality. Previous work identified ssk1E330N…K356N as causative allele in strain CBS6412, which displayed the lowest glycerol/ethanol ratio. Results We have now identified a unique segregant, 26B, that shows similar low glycerol/high ethanol production as the superior parent, but lacks the ssk1E330N…K356N allele. Using segregants from the backcross of 26B with the inferior parent strain, we applied pooled-segregant whole-genome sequence analysis and identified three minor quantitative trait loci (QTLs) linked to low glycerol/high ethanol production. Within these QTLs, we identified three novel alleles of known regulatory and structural genes of glycerol metabolism, smp1R110Q,P269Q, hot1P107S,H274Y and gpd1L164P as causative genes. All three genes separately caused a significant drop in the glycerol/ethanol production ratio, while gpd1L164P appeared to be epistatically suppressed by other alleles in the superior parent. The order of potency in reducing the glycerol/ethanol ratio of the three alleles was: gpd1L164P > hot1P107S,H274Y ≥ smp1R110Q,P269Q. Conclusions Our results show that natural yeast strains harbor multiple specific alleles of genes controlling essential functions, that are apparently compatible with survival in the natural environment. These newly identified alleles can be used as gene tools for engineering industrial yeast strains with multiple subtle changes, minimizing the risk of

  18. Academic Conferences: Representative and Resistant Sites for Higher Education Research

    ERIC Educational Resources Information Center

    Henderson, Emily F.

    2015-01-01

    The overarching argument made in this article is twofold. Firstly, academic conferences are posited as sites for higher education research. Secondly, the well-recognised emotional and social processes of conferences are used to make space at the boundaries of higher education research for psychosocial analysis. The article theorises conferences in…

  19. Recent Rapid Rise of a Permethrin Knock Down Resistance Allele in Aedes aegypti in México

    PubMed Central

    García, Gustavo Ponce; Flores, Adriana E.; Fernández-Salas, Ildefonso; Saavedra-Rodríguez, Karla; Reyes-Solis, Guadalupe; Lozano-Fuentes, Saul; Guillermo Bond, J.; Casas-Martínez, Mauricio; Ramsey, Janine M.; García-Rejón, Julián; Domínguez-Galera, Marco; Ranson, Hilary; Hemingway, Janet; Eisen, Lars; Black, William C.

    2009-01-01

    Background Aedes aegypti, the ‘yellow fever mosquito’, is the primary vector to humans of dengue and yellow fever flaviviruses (DENV, YFV), and is a known vector of the chikungunya alphavirus (CV). Because vaccines are not yet available for DENV or CV or are inadequately distributed in developing countries (YFV), management of Ae. aegypti remains the primary option to prevent and control outbreaks of the diseases caused by these arboviruses. Permethrin is one of the most widely used active ingredients in insecticides for suppression of adult Ae. aegypti. In 2007, we documented a replacement mutation in codon 1,016 of the voltage-gated sodium channel gene (para) of Ae. aegypti that encodes an isoleucine rather than a valine and confers resistance to permethrin. Ile1,016 segregates as a recessive allele conferring knockdown resistance to homozygous mosquitoes at 5–10 µg of permethrin in bottle bioassays. Methods and Findings A total of 81 field collections containing 3,951 Ae. aegypti were made throughout México from 1996 to 2009. These mosquitoes were analyzed for the frequency of the Ile1,016 mutation using a melting-curve PCR assay. Dramatic increases in frequencies of Ile1,016 were recorded from the late 1990's to 2006–2009 in several states including Nuevo León in the north, Veracruz on the central Atlantic coast, and Yucatán, Quintana Roo and Chiapas in the south. From 1996 to 2000, the overall frequency of Ile1,016 was 0.04% (95% confidence interval (CI95) = 0.12%; n = 1,359 mosquitoes examined). The earliest detection of Ile1,016 was in Nuevo Laredo on the U.S. border in 1997. By 2003–2004 the overall frequency of Ile1,016 had increased ∼100-fold to 2.7% (±0.80% CI95; n = 808). When checked again in 2006, the frequency had increased slightly to 3.9% (±1.15% CI95; n = 473). This was followed in 2007–2009 by a sudden jump in Ile1,016 frequency to 33.2% (±1.99% CI95; n = 1,074 mosquitoes). There was spatial heterogeneity

  20. Characterization of Klebsiella sp. strain 10982, a colonizer of humans that contains novel antibiotic resistance alleles and exhibits genetic similarities to plant and clinical Klebsiella isolates.

    PubMed

    Hazen, Tracy H; Zhao, LiCheng; Sahl, Jason W; Robinson, Gwen; Harris, Anthony D; Rasko, David A; Johnson, J Kristie

    2014-01-01

    A unique Klebsiella species strain, 10982, was cultured from a perianal swab specimen obtained from a patient in the University of Maryland Medical Center intensive care unit. Klebsiella sp. 10982 possesses a large IncA/C multidrug resistance plasmid encoding a novel FOX AmpC β-lactamase designated FOX-10. A novel variant of the LEN β-lactamase was also identified. Genome sequencing and bioinformatic analysis demonstrated that this isolate contains genes associated with nitrogen fixation, allantoin metabolism, and citrate fermentation. These three gene regions are typically present in either Klebsiella pneumoniae clinical isolates or Klebsiella nitrogen-fixing endophytes but usually not in the same organism. Phylogenomic analysis of Klebsiella sp. 10982 and sequenced Klebsiella genomes demonstrated that Klebsiella sp. 10982 is present on a branch that is located intermediate between the genomes of nitrogen-fixing endophytes and K. pneumoniae clinical isolates. Metabolic features identified in the genome of Klebsiella sp. 10982 distinguish this isolate from other Klebsiella clinical isolates. These features include the nitrogen fixation (nif) gene cluster, which is typically present in endophytic Klebsiella isolates and is absent from Klebsiella clinical isolates. Additionally, the Klebsiella sp. 10982 genome contains genes associated with allantoin metabolism, which have been detected primarily in K. pneumoniae isolates from liver abscesses. Comparative genomic analysis of Klebsiella sp. 10982 demonstrated that this organism has acquired genes conferring new metabolic strategies and novel antibiotic resistance alleles, both of which may enhance its ability to colonize the human body.

  1. High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigera to Bt Soybean in Brazil

    PubMed Central

    Bacalhau, Fabiana B.; Amado, Douglas; Carvalho, Renato A.; Martinelli, Samuel; Head, Graham P.; Omoto, Celso

    2016-01-01

    The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL−1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil. PMID:27532632

  2. High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigers to Bt Soybean in Brazil

    PubMed Central

    Bacalhau, Fabiana B.; Amado, Douglas; Carvalho, Renato A.; Martinelli, Samuel; Head, Graham P.; Omoto, Celso

    2016-01-01

    The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL−1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil. PMID:27532632

  3. High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigers to Bt Soybean in Brazil.

    PubMed

    Dourado, Patrick M; Bacalhau, Fabiana B; Amado, Douglas; Carvalho, Renato A; Martinelli, Samuel; Head, Graham P; Omoto, Celso

    2016-01-01

    The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL-1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil. PMID:27532632

  4. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    PubMed Central

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  5. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

    PubMed

    Tay, Wee Tek; Mahon, Rod J; Heckel, David G; Walsh, Thomas K; Downes, Sharon; James, William J; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K; Gordon, Karl H J

    2015-11-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  6. Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population.

    PubMed

    Chevalier, Frédéric D; Le Clec'h, Winka; Eng, Nina; Rugel, Anastasia R; Assis, Rafael Ramiro de; Oliveira, Guilherme; Holloway, Stephen P; Cao, Xiaohang; Hart, P John; LoVerde, Philip T; Anderson, Timothy J C

    2016-06-01

    Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles. PMID:27073078

  7. A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin▿ †

    PubMed Central

    Vecchione, James J.; Sello, Jason K.

    2009-01-01

    Indolmycin, a potential antibacterial drug, competitively inhibits bacterial tryptophanyl-tRNA synthetases. An effort to identify indolmycin resistance genes led to the discovery of a gene encoding an indolmycin-resistant isoform of tryptophanyl-tRNA synthetase. Overexpression of this gene in an indolmycin-sensitive strain increased the indolmycin MIC 60-fold. Its transcription and distribution in various bacterial genera were assessed. The level of resistance conferred by this gene was compared to that of a known indolmycin resistance gene and to those of genes with resistance-conferring point mutations. PMID:19546369

  8. Allele-specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin-resistant isolates.

    PubMed

    Kim, H Y; Lee, S Y; Kim, B J; Kook, Y H

    2016-01-01

    On the basis of the structural differences of erm, we used a duplex polymerase chain reaction (PCR) to differentiate Mycobacterium abscessus subsp. abscessus and subsp. massiliense isolates and to detect the point mutations of 23S rRNA gene that confer a high level of resistance to clarithromycin. Subsp. massiliense strains occupying almost half of the clinical isolates can be simply identified, and their clarithromycin susceptibility can be rapidly determined. PMID:27514964

  9. Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance

    PubMed Central

    LaMonte, Gregory; Lim, Michelle Yi-Xiu; Wree, Melanie; Reimer, Christin; Nachon, Marie; Corey, Victoria; Gedeck, Peter; Plouffe, David; Du, Alan; Figueroa, Nelissa; Yeung, Bryan; Winzeler, Elizabeth A.

    2016-01-01

    ABSTRACT Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites. PMID:27381290

  10. Quantitative Sequencing for the Determination of Kdr-type Resistance Allele (V419L, L925I, I936F) Frequencies in Common Bed Bug (Hemiptera: Cimicidae) Populations Collected from Israel.

    PubMed

    Palenchar, Daniel J; Gellatly, Kyle J; Yoon, Kyong Sup; Mumcuoglu, Kosta Y; Shalom, Uri; Clark, J Marshall

    2015-09-01

    Human bed bug infestations have dramatically increased worldwide since the mid-1990s. A similar phenomenon was also observed in Israel since 2005, when infestations were reported from all over the country. Two single nucleotide polymorphisms (V419L and L925I) in the bed bug voltage-sensitive sodium channel confer kdr-type resistance to pyrethroids. Using quantitative sequencing (QS), the resistance allele frequencies of Israeli bed bug populations from across the country were determined. Genomic DNA was extracted from samples of 12 populations of bed bugs collected from Israel and DNA fragments containing the V419L or L925I and I936F mutations sites were PCR amplified. The PCR products were analyzed by QS and the nucleotide signal ratios calculated and used to predict the resistance allele frequencies of the unknown populations. Results of the genetic analysis show that resistant nucleotide signals are highly correlated to resistance allele frequencies for both mutations. Ten of the 12 tested populations had 100% of the L925I mutation and 0% of the V419L mutation. One population was heterogeneous for the L925I mutation and had 0% of the V419L mutation and another population was heterogeneous for the V419L mutation and had 100% of the L925I mutation. I936F occurred only at low levels. These results indicate that bed bugs in Israel are genetically resistant to pyrethroids. Thus, pyrethroids should only be used for bed bug management with caution using effective application and careful monitoring procedures. Additionally, new and novel-acting insecticides and nonchemical means of controlling bed bugs should be explored. PMID:26336243

  11. Quantitative Sequencing for the Determination of Kdr-type Resistance Allele (V419L, L925I, I936F) Frequencies in Common Bed Bug (Hemiptera: Cimicidae) Populations Collected from Israel.

    PubMed

    Palenchar, Daniel J; Gellatly, Kyle J; Yoon, Kyong Sup; Mumcuoglu, Kosta Y; Shalom, Uri; Clark, J Marshall

    2015-09-01

    Human bed bug infestations have dramatically increased worldwide since the mid-1990s. A similar phenomenon was also observed in Israel since 2005, when infestations were reported from all over the country. Two single nucleotide polymorphisms (V419L and L925I) in the bed bug voltage-sensitive sodium channel confer kdr-type resistance to pyrethroids. Using quantitative sequencing (QS), the resistance allele frequencies of Israeli bed bug populations from across the country were determined. Genomic DNA was extracted from samples of 12 populations of bed bugs collected from Israel and DNA fragments containing the V419L or L925I and I936F mutations sites were PCR amplified. The PCR products were analyzed by QS and the nucleotide signal ratios calculated and used to predict the resistance allele frequencies of the unknown populations. Results of the genetic analysis show that resistant nucleotide signals are highly correlated to resistance allele frequencies for both mutations. Ten of the 12 tested populations had 100% of the L925I mutation and 0% of the V419L mutation. One population was heterogeneous for the L925I mutation and had 0% of the V419L mutation and another population was heterogeneous for the V419L mutation and had 100% of the L925I mutation. I936F occurred only at low levels. These results indicate that bed bugs in Israel are genetically resistant to pyrethroids. Thus, pyrethroids should only be used for bed bug management with caution using effective application and careful monitoring procedures. Additionally, new and novel-acting insecticides and nonchemical means of controlling bed bugs should be explored.

  12. NRF2 Mutation Confers Malignant Potential and Resistance to Chemoradiation Therapy in Advanced Esophageal Squamous Cancer1

    PubMed Central

    Shibata, Tatsuhiro; Kokubu, Akiko; Saito, Shigeru; Narisawa-Saito, Mako; Sasaki, Hiroki; Aoyagi, Kazuhiko; Yoshimatsu, Yuki; Tachimori, Yuji; Kushima, Ryoji; Kiyono, Tohru; Yamamoto, Masayuki

    2011-01-01

    Esophageal squamous cancer (ESC) is one of the most aggressive tumors of the gastrointestinal tract. A combination of chemotherapy and radiation therapy (CRT) has improved the clinical outcome, but the molecular background determining the effectiveness of therapy remains unknown. NRF2 is a master transcriptional regulator of stress adaptation, and gain of-function mutation of NRF2 in cancer confers resistance to stressors including anticancer therapy. Direct resequencing analysis revealed that Nrf2 gain-of-function mutation occurred recurrently (18/82, 22%) in advanced ESC tumors and ESC cell lines (3/10). The presence of Nrf2 mutation was associated with tumor recurrence and poor prognosis. Short hairpin RNA-mediated down-regulation of NRF2 in ESC cells that harbor only mutated Nrf2 allele revealed that themutant NRF2 conferred increased cell proliferation, attachment-independent survival, and resistance to 5-fluorouracil and γ-irradiation. Based on the Nrf2 mutation status, gene expression signatures associated with NRF2 mutation were extracted from ESC cell lines, and their potential utility for monitoring and prognosis was examined in a cohort of 33 pre-CRT cases of ESC. The molecular signatures of NRF2 mutation were significantly predictive and prognostic for CRT response. In conclusion, recurrent NRF2 mutation confers malignant potential and resistance to therapy in advanced ESC, resulting in a poorer outcome. Molecular signatures of NRF2 mutation can be applied as predictive markers of response to CRT, and efficient inhibition of aberrant NRF2 activation could be a promising approach in combination with CRT. PMID:21969819

  13. Distribution of Voltage-Gated Sodium Channel (Nav) Alleles among the Aedes aegypti Populations In Central Java Province and Its Association with Resistance to Pyrethroid Insecticides.

    PubMed

    Sayono, Sayono; Hidayati, Anggie Puspa Nur; Fahri, Sukmal; Sumanto, Didik; Dharmana, Edi; Hadisaputro, Suharyo; Asih, Puji Budi Setia; Syafruddin, Din

    2016-01-01

    The emergence of insecticide resistant Aedes aegypti mosquitoes has hampered dengue control efforts. WHO susceptibility tests, using several pyrethroid compounds, were conducted on Ae. aegypti larvae that were collected and raised to adulthood from Semarang, Surakarta, Kudus and Jepara in Java. The AaNaV gene fragment encompassing kdr polymorphic sites from both susceptible and resistant mosquitoes was amplified, and polymorphisms were associated with the resistant phenotype. The insecticide susceptibility tests demonstrated Ae, aegypti resistance to the pyrethroids, with mortality rates ranging from 1.6%-15.2%. Three non-synonymous polymorphisms (S989P, V1016G and F1534C) and one synonymous polymorphism (codon 982) were detected in the AaNaV gene. Eight AaNaV alleles were observed in specimens from Central Java. Allele 3 (SGF) and allele 7 (PGF) represent the most common alleles found and demonstrated strong associations with resistance to pyrethroids (OR = 2.75, CI: 0.97-7.8 and OR = 7.37, CI: 2.4-22.5, respectively). This is the first report of 8 Ae. aegypti AaNaV alleles, and it indicates the development of resistance in Ae. aegypti in response to pyrethroid insecticide-based selective pressure. These findings strongly suggest the need for an appropriate integrated use of insecticides in the region. The 989P, 1016G and 1534C polymorphisms in the AaNaV gene are potentially valuable molecular markers for pyrethroid insecticide resistance monitoring.

  14. An independent occurrence of the chimeric P450 enzyme CYP337B3 of Helicoverpa armigera confers cypermethrin resistance in Pakistan.

    PubMed

    Rasool, Akhtar; Joußen, Nicole; Lorenz, Sybille; Ellinger, Renate; Schneider, Bernd; Khan, Sher Afzal; Ashfaq, Muhammad; Heckel, David G

    2014-10-01

    The increasing resistance level of insect pest species is a major concern to agriculture worldwide. The cotton bollworm, Helicoverpa armigera, is one of the most important pest species due to being highly polyphagous, geographically widespread, and resistant towards many chemical classes of insecticides. We previously described the mechanism of fenvalerate resistance in Australian populations conferred by the chimeric cytochrome P450 monooxygenase CYP337B3, which arose by unequal crossing-over between CYP337B1 and CYP337B2. Here, we show that this mechanism is also present in the cypermethrin-resistant FSD strain from Pakistan. The Pakistani and the Australian CYP337B3 alleles differ by 18 synonymous and three nonsynonymous SNPs and additionally in the length and sequence of the intron. Nevertheless, the activity of both CYP337B3 proteins is comparable. We demonstrate that CYP337B3 is capable of metabolizing cypermethrin (trans- and especially cis-isomers) to the main metabolite 4'-hydroxycypermethrin, which exhibits no intrinsic toxicity towards susceptible larvae. In a bioassay, CYP337B3 confers a 7-fold resistance towards cypermethrin in FSD larvae compared to susceptible larvae from the Australian TWB strain lacking CYP337B3. Linkage analysis shows that presence of CYP337B3 accounts for most of the cypermethrin resistance in the FSD strain; up-regulation of other P450s in FSD plays no detectable role in resistance. The presence or absence of CYP337B3 can be easily detected by a simple PCR screen, providing a powerful tool to rapidly distinguish resistant from susceptible individuals in the field and to determine the geographical distribution of this resistance gene. Our results suggest that CYP337B3 evolved twice independently by unequal crossing-over between CYP337B2 and two different CYP337B1 alleles.

  15. The arabidopsis TIR-NB-LRR gene RAC1 confers resistance to Albugo candida (white rust) and is dependent on EDS1 but not PAD4.

    PubMed

    Borhan, Mohammad H; Holub, Eric B; Beynon, Jim L; Rozwadowski, Kevin; Rimmer, S Roger

    2004-07-01

    Resistance to Albugo candida isolate Acem1 is conferred by a dominant gene, RAC1, in accession Ksk-1 of Arabidopsis thaliana. This gene was isolated by positional cloning and is a member of the Drosophila toll and mammalian interleukin-1 receptor (TIR) nucleotide-binding site leucine-rich repeat (NB-LRR) class of plant resistance genes. Strong identity of the TIR and NB domains was observed between the predicted proteins encoded by the Ksk-1 allele and the allele from an Acem1-susceptible accession Columbia (Col) (99 and 98%, respectively). However, major differences between the two predicted proteins occur within the LRR domain and mainly are confined to the beta-strand/beta-turn structure of the LRR. Both proteins contain 14 imperfect repeats. RAC1-mediated resistance was analyzed further using mutations in defense regulation, including: pad4-1, eds1-1, and NahG, in the presence of the RAC1 allele from Ksk-1. White rust resistance was completely abolished by eds1-1 but was not affected by either pad4-1 or NahG.

  16. Foliar resistance to fall armyworm in corn germplasm lines that confer resistance to root- and ear-feeding insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A holistic approach to developing new corn germplasm that confers multiple insect resistance in various plant tissues at different growth stages was examined. Eight corn germplasm lines were examined for their foliage resistance to fall armyworm [Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noc...

  17. Quantitative resistance affects the speed of frequency increase but not the diversity of the virulence alleles overcoming a major resistance gene to Leptosphaeria maculans in oilseed rape.

    PubMed

    Delourme, R; Bousset, L; Ermel, M; Duffé, P; Besnard, A L; Marquer, B; Fudal, I; Linglin, J; Chadœuf, J; Brun, H

    2014-10-01

    Quantitative resistance mediated by multiple genetic factors has been shown to increase the potential for durability of major resistance genes. This was demonstrated in the Leptosphaeria maculans/Brassica napus pathosystem in a 5year recurrent selection field experiment on lines harboring the qualitative resistance gene Rlm6 combined or not with quantitative resistance. The quantitative resistance limited the size of the virulent isolate population. In this study we continued this recurrent selection experiment in the same way to examine whether the pathogen population could adapt and render the major gene ineffective in the longer term. The cultivars Eurol, with a susceptible background, and Darmor, with quantitative resistance, were used. We confirmed that the combination of qualitative and quantitative resistance is an effective approach for controlling the pathogen epidemics over time. This combination did not prevent isolates virulent against the major gene from amplifying in the long term but the quantitative resistance significantly delayed for 5years the loss of effectiveness of the qualitative resistance and disease severity was maintained at a low level on the genotype with both types of resistance after the fungus population had adapted to the major gene. We also showed that diversity of AvrLm6 virulence alleles was comparable in isolates recovered after the recurrent selection on lines carrying either the major gene alone or in combination with quantitative resistance: a single repeat-induced point mutation and deletion events were observed in both situations. Breeding varieties which combine qualitative and quantitative resistance can effectively contribute to disease control by increasing the potential for durability of major resistance genes.

  18. An African ancestry-specific allele of CTLA4 confers protection against rheumatoid arthritis in African Americans.

    PubMed

    Kelley, James M; Hughes, Laura B; Faggard, Jeffrey D; Danila, Maria I; Crawford, Monica H; Edberg, Yuanqing; Padilla, Miguel A; Tiwari, Hemant K; Westfall, Andrew O; Alarcón, Graciela S; Conn, Doyt L; Jonas, Beth L; Callahan, Leigh F; Smith, Edwin A; Brasington, Richard D; Allison, David B; Kimberly, Robert P; Moreland, Larry W; Edberg, Jeffrey C; Bridges, S Louis

    2009-03-01

    Cytotoxic T-lymphocyte associated protein 4 (CTLA4) is a negative regulator of T-cell proliferation. Polymorphisms in CTLA4 have been inconsistently associated with susceptibility to rheumatoid arthritis (RA) in populations of European ancestry but have not been examined in African Americans. The prevalence of RA in most populations of European and Asian ancestry is approximately 1.0%; RA is purportedly less common in black Africans, with little known about its prevalence in African Americans. We sought to determine if CTLA4 polymorphisms are associated with RA in African Americans. We performed a 2-stage analysis of 12 haplotype tagging single nucleotide polymorphisms (SNPs) across CTLA4 in a total of 505 African American RA patients and 712 African American controls using Illumina and TaqMan platforms. The minor allele (G) of the rs231778 SNP was 0.054 in RA patients, compared to 0.209 in controls (4.462 x 10(-26), Fisher's exact). The presence of the G allele was associated with a substantially reduced odds ratio (OR) of having RA (AG+GG genotypes vs. AA genotype, OR 0.19, 95% CI: 0.13-0.26, p = 2.4 x 10(-28), Fisher's exact), suggesting a protective effect. This SNP is polymorphic in the African population (minor allele frequency [MAF] 0.09 in the Yoruba population), but is very rare in other groups (MAF = 0.002 in 530 Caucasians genotyped for this study). Markers associated with RA in populations of European ancestry (rs3087243 [+60C/T] and rs231775 [+49A/G]) were not replicated in African Americans. We found no confounding of association for rs231778 after stratifying for the HLA-DRB1 shared epitope, presence of anti-cyclic citrullinated peptide antibody, or degree of admixture from the European population. An African ancestry-specific genetic variant of CTLA4 appears to be associated with protection from RA in African Americans. This finding may explain, in part, the relatively low prevalence of RA in black African populations.

  19. Nuclear and cytoplasmic genome components of Solanum tuberosum + S. chacoense somatic hybrids and three SSR alleles related to bacterial wilt resistance.

    PubMed

    Chen, Lin; Guo, Xianpu; Xie, Conghua; He, Li; Cai, Xingkui; Tian, Lingli; Song, Botao; Liu, Jun

    2013-07-01

    The somatic hybrids were derived previously from protoplast fusion between Solanum tuberosum and S. chacoense to gain the bacterial wilt resistance from the wild species. The genome components analysis in the present research was to clarify the nuclear and cytoplasmic composition of the hybrids, to explore the molecular markers associated with the resistance, and provide information for better use of these hybrids in potato breeding. One hundred and eight nuclear SSR markers and five cytoplasmic specific primers polymorphic between the fusion parents were used to detect the genome components of 44 somatic hybrids. The bacterial wilt resistance was assessed thrice by inoculating the in vitro plants with a bacterial suspension of race 1. The disease index, relative disease index, and resistance level were assigned to each hybrid, which were further analyzed in relation to the molecular markers for elucidating the potential genetic base of the resistance. All of the 317 parental unique nuclear SSR alleles appeared in the somatic hybrids with some variations in the number of bands detected. Nearly 80 % of the hybrids randomly showed the chloroplast pattern of one parent, and most of the hybrids exhibited a fused mitochondrial DNA pattern. One hundred and nine specific SSR alleles of S. chacoense were analyzed for their relationship with the disease index of the hybrids, and three alleles were identified to be significantly associated with the resistance. Selection for the resistant SSR alleles of S. chacoense may increase the possibility of producing resistant pedigrees.

  20. A new soybean rust resistance allele from PI 423972 at the Rpp4 locus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phakopsora pachyrhizi is a fungal pathogen and the cause of Asian soybean rust (SBR). P. pachyrhizi invaded the continental United States in 2004 and has since been a threat to the soybean industry. There are six described loci that harbor resistance to P. pachyrhizi (Rpp) genes. The resistance of P...

  1. A novel phakopsora pachyrhizi resistance allele (rpp) contributed by PI 567068A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean rust (SBR) caused by the obligate, fungal pathogen Phakopsora pachyrhizi is an economic threat to soybean production, especially in the Americas. Host plant resistance is an important management strategy for SBR. The most recently described resistance to P. pachyrhizi (Rpp) gene is Rpp6 co...

  2. A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis.

    PubMed Central

    Orth, A B; Rzhetskaya, M; Pell, E J; Tien, M

    1995-01-01

    A mutant of Ustilago maydis (VR43) with single-gene resistance to the dicarboximide fungicide vinclozolin was previously isolated and characterized. A genomic library was constructed, and an 8.7-kb resistance-conferring fragment was isolated by sib selection. Sequencing this fragment, we identified an 1,218-bp open reading frame, which, if disrupted by deletion, no longer confers resistance. Analyses of the data in GenBank demonstrated a high degree of homology between the product of the 1,218-bp open reading frame, referred to as the adr-1 gene, and Ser (Thr) protein kinases. PMID:7793954

  3. Alloreactive T Cells to Identify Risk HLA Alleles for Retransplantation After Acute Accelerated Steroid-Resistant Rejection.

    PubMed

    Leyking, S; Wolf, M; Mihm, J; Schaefer, M; Bohle, R M; Fliser, D; Sester, M; Sester, U

    2015-10-01

    The risk of rejection by cellular alloreactivity to the transplant donor is not routinely assessed. Here we analyzed alloreactive T cells in kidney transplant recipients and report how their detection may have helped to prevent rejection of a second kidney graft in a patient with a history of acute accelerated steroid-resistant nonhumoral rejection. Alloreactive CD4 and CD8 T cells were quantified using a flow-cytometric mixed lymphocyte reaction assay based on interferon-γ induction. A group of 16 nonrejecting transplant recipients did not show any alloreactive T-cell immunity to their respective donors, whereas alloreactivity to third-party controls was detectable. In the patient with rejection, HLA-specific antibodies were not detectable before and shortly after rejection, but after transplantation the patient showed exceptionally high frequencies of alloreactive T cells against 2 of 11 HLA-typed controls (0.604% and 0.791% alloreactive CD4 T cells and 0.792% and 0.978% alloreactive CD8 T cells) who shared HLA alleles (HLA-A*24, -B*44, -C*02, -DQB1*5) with the kidney donor. These HLA alleles were subsequently excluded for allocation of a second graft. No alloreactive T cells were observed toward the second kidney donor, and this transplantation was performed successfully. Thus, shared HLA alleles between the donor and third-party controls may suggest that alloreactive T cells had contributed to rejection of the first graft. The rejecting patient highlights that determination of cellular alloreactivity before transplantation may be applied to identify unacceptable mismatches and to reduce the risk for acute cellular rejection episodes. PMID:26518945

  4. Characterization of Vibrio fluvialis qnrVC5 Gene in Native and Heterologous Hosts: Synergy of qnrVC5 with other Determinants in Conferring Quinolone Resistance

    PubMed Central

    Vinothkumar, Kittappa; Kumar, G. N.; Bhardwaj, Ashima K.

    2016-01-01

    Resistance of various pathogens toward quinolones has emerged as a serious threat to combat infections. Analysis of plethora of genes and resistance mechanisms associated with quinolone resistance reveals chromosome-borne and transferable determinants. qnr genes have been found to be responsible for transferable quinolone resistance. In the present work, a new allele qnrVC5 earlier reported in Vibrio fluvialis from this laboratory was characterized in detail for its sequence, genetic context and propensity to decrease the susceptibility for quinolones. The study has revealed persistence of qnrVC5 in clinical isolates of V. fluvialis from Kolkata region through the years 2002–2006. qnrVC5 existed in the form of a gene cassette with the open reading frame being flanked by an upstream promoter and a downstream V. cholerae repeat region suggestive of its superintegron origin. Sequence analysis of different qnrVC alleles showed that qnrVC5 was closely related to qnrVC2 and qnrVC4 and these alleles were associated with V. cholerae repeats. In contrast, qnrVC1, qnrVC3, and qnrVC6 belonging to another group were associated with V. parahaemolyticus repeats. The gene manifested its activity in native V. fluvialis host as well as in Escherichia coli transformants harboring it by elevating the MIC toward various quinolones by twofold to eightfold. In combination with other quinolone resistance factors such as topoisomerase mutations and aac(6’)-Ib-cr gene, qnrVC5 gene product contributed toward higher quinolone resistance displayed by V. fluvialis isolates. Silencing of the gene using antisense peptide nucleic acid sensitized the V. fluvialis parent isolates toward ciprofloxacin. Recombinant QnrVC5 vividly demonstrated its role in conferring quinolone resistance. qnrVC5 gene, its synergistic effect and global dissemination should be perceived as a menace for quinolone-based therapies. PMID:26913027

  5. Leaf rust resistance in wheat line RL6062 is an allele at the Lr3 locus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RL6062, a 'Thatcher' backcross line of wheat (Triticum aestivum L.) has resistance to leaf rust (caused by Puccinia triticina) derived from a line of common wheat from Iran. RL6062 has an intermediate infection type (IT) of small to moderate uredinia surrounded by distinct chlorosis. RL6062 was cros...

  6. Allelic analysis of sheath blight resistance with association mapping in rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sheath blight is one of the most devastating diseases world-wide in rice. For the first time, we adopted association mapping to identify quantitative trait loci for sheath blight resistance from the USDA rice mini-core collection. The phenotyping was conducted with a newly developed micro-chamber me...

  7. HLA-DRB1-DQB1 Haplotypes Confer Susceptibility and Resistance to Multiple Sclerosis in Sardinia

    PubMed Central

    Cocco, Eleonora; Sardu, Claudia; Pieroni, Enrico; Valentini, Maria; Murru, Raffaele; Costa, Gianna; Tranquilli, Stefania; Frau, Jessica; Coghe, Giancarlo; Carboni, Nicola; Floris, Matteo; Contu, Paolo; Marrosu, Maria Giovanna

    2012-01-01

    :02, *06:01 alleles. Conclusions These findings show that the association of specific, independent DRB1*-DQB1* haplotypes confers susceptibility or resistance to MS in the MS-prone Sardinian population. The data also supports a functional role for specific residues of the DRB1 and DQB1 proteins in predisposing patients to MS. PMID:22509268

  8. Characterization of Lr46, a gene conferring partial resistance to wheat leaf rust.

    PubMed

    Martínez, F; Niks, R E; Singh, R P; Rubiales, D

    2001-01-01

    Components of resistance conferred by the Lr46 gene, reported as causing "slow rusting" resistance to leaf rust in wheat, were studied and compared with the effects of Lr34 and genes for quantitative resistance in cv. Akabozu. Lr34 is a gene that confers non-hypersensitive type of resistance. The effect of Lr46 resembles that of Lr34 and other wheats reported with partial resistance. At macroscopic level, Lr46 produced a longer latency period than observed on the susceptible recurrent parent Lalbahadur, and a reduction of the infection frequency not associated with hypersensitivity. Microscopically, Lr46 increased the percentage of early aborted infection units not associated with host cell necrosis and decreased the colony size. The effect of Lr46 is comparable to that of Lr34 in adult plant stage, but in seedling stage its effect is weaker than that of Lr34.

  9. Distribution of Voltage-Gated Sodium Channel (Nav) Alleles among the Aedes aegypti Populations In Central Java Province and Its Association with Resistance to Pyrethroid Insecticides

    PubMed Central

    Sayono, Sayono; Hidayati, Anggie Puspa Nur; Fahri, Sukmal; Sumanto, Didik; Dharmana, Edi; Hadisaputro, Suharyo; Asih, Puji Budi Setia; Syafruddin, Din

    2016-01-01

    The emergence of insecticide resistant Aedes aegypti mosquitoes has hampered dengue control efforts. WHO susceptibility tests, using several pyrethroid compounds, were conducted on Ae. aegypti larvae that were collected and raised to adulthood from Semarang, Surakarta, Kudus and Jepara in Java. The AaNaV gene fragment encompassing kdr polymorphic sites from both susceptible and resistant mosquitoes was amplified, and polymorphisms were associated with the resistant phenotype. The insecticide susceptibility tests demonstrated Ae, aegypti resistance to the pyrethroids, with mortality rates ranging from 1.6%–15.2%. Three non-synonymous polymorphisms (S989P, V1016G and F1534C) and one synonymous polymorphism (codon 982) were detected in the AaNaV gene. Eight AaNaV alleles were observed in specimens from Central Java. Allele 3 (SGF) and allele 7 (PGF) represent the most common alleles found and demonstrated strong associations with resistance to pyrethroids (OR = 2.75, CI: 0.97–7.8 and OR = 7.37, CI: 2.4–22.5, respectively). This is the first report of 8 Ae. aegypti AaNaV alleles, and it indicates the development of resistance in Ae. aegypti in response to pyrethroid insecticide-based selective pressure. These findings strongly suggest the need for an appropriate integrated use of insecticides in the region. The 989P, 1016G and 1534C polymorphisms in the AaNaV gene are potentially valuable molecular markers for pyrethroid insecticide resistance monitoring. PMID:26939002

  10. Gene amplification confers glyphosate resistance in Amaranthus palmeri

    PubMed Central

    Gaines, Todd A.; Zhang, Wenli; Wang, Dafu; Bukun, Bekir; Chisholm, Stephen T.; Shaner, Dale L.; Nissen, Scott J.; Patzoldt, William L.; Tranel, Patrick J.; Culpepper, A. Stanley; Grey, Timothy L.; Webster, Theodore M.; Vencill, William K.; Sammons, R. Douglas; Jiang, Jiming; Preston, Christopher; Leach, Jan E.; Westra, Philip

    2009-01-01

    The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology. PMID:20018685

  11. A pigeonpea gene confers resistance to Asian soybean rust in soybean.

    PubMed

    Kawashima, Cintia G; Guimarães, Gustavo Augusto; Nogueira, Sônia Regina; MacLean, Dan; Cook, Doug R; Steuernagel, Burkhard; Baek, Jongmin; Bouyioukos, Costas; Melo, Bernardo do V A; Tristão, Gustavo; de Oliveira, Jamile Camargos; Rauscher, Gilda; Mittal, Shipra; Panichelli, Lisa; Bacot, Karen; Johnson, Ebony; Iyer, Geeta; Tabor, Girma; Wulff, Brande B H; Ward, Eric; Rairdan, Gregory J; Broglie, Karen E; Wu, Gusui; van Esse, H Peter; Jones, Jonathan D G; Brommonschenkel, Sérgio H

    2016-06-01

    Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement. PMID:27111723

  12. Allele Mining in Barley Genetic Resources Reveals Genes of Race-Non-Specific Powdery Mildew Resistance

    PubMed Central

    Spies, Annika; Korzun, Viktor; Bayles, Rosemary; Rajaraman, Jeyaraman; Himmelbach, Axel; Hedley, Pete E.; Schweizer, Patrick

    2012-01-01

    Race-non-specific, or quantitative, pathogen resistance is of high importance to plant breeders due to its expected durability. However, it is usually controlled by multiple quantitative trait loci (QTL) and therefore difficult to handle in practice. Knowing the genes that underlie race-non-specific resistance (NR) would allow its exploitation in a more targeted manner. Here, we performed an association-genetic study in a customized worldwide collection of spring barley accessions for candidate genes of race-NR to the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) and combined data with results from QTL mapping as well as functional-genomics approaches. This led to the identification of 11 associated genes with converging evidence for an important role in race-NR in the presence of the Mlo gene for basal susceptibility. Outstanding in this respect was the gene encoding the transcription factor WRKY2. The results suggest that unlocking plant genetic resources and integrating functional-genomic with genetic approaches can accelerate the discovery of genes underlying race-NR in barley and other crop plants. PMID:22629270

  13. Development of allele-specific PCR and RT-PCR assays for clustered resistance genes using a potato late blight resistance transgene as a model.

    PubMed

    Millett, B P; Bradeen, J M

    2007-02-01

    Members of the NBS-LRR gene family impart resistance to a wide variety of pathogens and are often found clustered within a plant genome. This clustering of homologous sequences can complicate PCR-based characterizations, especially the study of transgenes. We have developed allele-specific PCR and RT-PCR assays for the potato late blight resistance gene RB. Our assay utilizes two approaches toward primer design, allowing discrimination between the RB transgene and both the endogenous RB gene and numerous RB homeologs. First, a reverse primer was designed to take advantage of an indel present in the RB transgene but absent in rb susceptibility alleles, enhancing specificity for the transgene, though not fully discriminating against RB homeologs. Second, a forward primer was designed according to the principles of mismatch amplification mutation assay (MAMA) PCR, targeting SNPs introduced during the cloning of RB. Together, the indel reverse primer and the MAMA forward primer provide an assay that is highly specific for the RB transgene, being capable of distinguishing the transgene from all RB endogenous gene copies and from all RB paralogs in a diverse collection of wild and cultivated potato genotypes. These primers have been successfully multiplexed with primers of an internal control. The multiplexed assay is useful for both PCR and RT-PCR applications. Double MAMA-PCR, in which both PCR primers target separate transgene-specific SNPs, was also tested and shown to be equally specific for the RB transgene. We propose extending the use of MAMA for the characterization of resistance transgenes. PMID:17177064

  14. Allele mining in the gene pool of wild Solanum species for homologues of late blight resistance gene RB/Rpi-blb1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Solanum bulbocastanum comprising a CC-NBS-LRR gene RB/Rpi-blb1 confers broad-spectrum resistance to Phytophthora infestans and is currently employed in potato breeding for durable late blight (LB) resistance. Genomes of several Solanum species were reported to contain RB homologues with confirmed b...

  15. A Novel Membrane Protein, VanJ, Conferring Resistance to Teicoplanin

    PubMed Central

    Novotna, Gabriela; Hill, Chris; Vincent, Karen; Liu, Chang

    2012-01-01

    Bacterial resistance to the glycopeptide antibiotic teicoplanin shows some important differences from the closely related compound vancomycin. They are currently poorly understood but may reflect significant differences in the mode of action of each antibiotic. Streptomyces coelicolor possesses a vanRSJKHAX gene cluster that when expressed confers resistance to both vancomycin and teicoplanin. The resistance to vancomycin is mediated by the enzymes encoded by vanKHAX, but not by vanJ. vanHAX effect a reprogramming of peptidoglycan biosynthesis, which is considered to be generic, conferring resistance to all glycopeptide antibiotics. Here, we show that vanKHAX are not in fact required for teicoplanin resistance in S. coelicolor, which instead is mediated solely by vanJ. vanJ is shown to encode a membrane protein oriented with its C-terminal active site exposed to the extracytoplasmic space. VanJ also confers resistance to the teicoplanin-like antibiotics ristocetin and A47934 and to a broad range of semisynthetic teicoplanin derivatives, but not generally to antibiotics or semisynthetic derivatives with vancomycin-like structures. vanJ homologues are found ubiquitously in streptomycetes and include staP from the Streptomyces toyocaensis A47934 biosynthetic gene cluster. While overexpression of staP also conferred resistance to teicoplanin, similar expression of other vanJ homologues (SCO2255, SCO7017, and SAV5946) did not. The vanJ and staP orthologues, therefore, appear to represent a subset of a larger protein family whose members have acquired specialist roles in antibiotic resistance. Future characterization of the divergent enzymatic activity within this new family will contribute to defining the molecular mechanisms important for teicoplanin activity and resistance. PMID:22232274

  16. Physiological, biochemical and molecular characterization of an induced mutation conferring imidazolinone resistance in wheat.

    PubMed

    Jimenez, Francisco; Rojano-Delgado, Antonia M; Fernández, Pablo Tomas; Rodríguez-Suárez, Cristina; Atienza, Sergio G; De Prado, Rafael

    2016-09-01

    The Clearfield(®) wheat cultivars possessing imidazolinone (IMI)-resistant traits provide an efficient option for controlling weeds. The imazamox-resistant cultivar Pantera (Clearfield(®) ) was compared with a susceptible cultivar (Gazul). Target and non-target mechanisms of resistance were studied to characterize the resistance of Pantera and to identify the importance of each mechanism involved in this resistance. Pantera is resistant to imazamox as was determined in previous experiments. The molecular study confirmed that it carries a mutation Ser-Asn627 conferring resistance to imazamox in two out of three acetolactate synthase (ALS) genes (imi1 and imi2), located in wheat on chromosomes 6B and 6D, respectively. However, the last gene (imi3) located on chromosome 6A does not carry any mutation conferring resistance. As a result, photosynthetic activity and chlorophyll content were reduced after imazamox treatment. Detoxification was higher in the resistant biotype as shown by metabolomic study while imazamox translocation was higher in the susceptible cultivar. Interestingly, imazamox metabolism was higher at higher doses of herbicide, which suggests that the detoxification process is an inducible mechanism in which the upregulation of key gene coding for detoxification enzymes could play an important role. Thus, the identification of cultivars with a higher detoxification potential would allow the development of more resistant varieties. PMID:26991509

  17. Assessing the Risk That Phytophthora melonis Can Develop a Point Mutation (V1109L) in CesA3 Conferring Resistance to Carboxylic Acid Amide Fungicides

    PubMed Central

    Chen, Lei; Zhu, Shusheng; Lu, Xiaohong; Pang, Zhili; Cai, Meng; Liu, Xili

    2012-01-01

    The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC50 values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10−7 and 1×10−6, respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI  =  mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC50 values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations. PMID:22848705

  18. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay.

    PubMed

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John; Gao, Feng; Yang, Chunfu

    2016-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  19. Conference on Fire Resistant Materials (FIREMEN): A compilation of presentations and papers

    NASA Technical Reports Server (NTRS)

    Kourtides, D. A. (Editor)

    1978-01-01

    The proceedings of the NASA Fire Resistant Materials Engineering (FIREMEN) Program held at Ames Research Center on April, 13, 14, 1978 are reported. The purpose of the conference was to discuss the results of NASA in the field of aircraft fire safety and fire resistant materials. The program components include the following: (1) large-scale testing; (2) fire toxicology; (3) polymeric materials; and (4) bibliography related and/or generated from the program.

  20. The ars operon of Escherichia coli confers arsenical and antimonial resistance.

    PubMed Central

    Carlin, A; Shi, W; Dey, S; Rosen, B P

    1995-01-01

    The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli. When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate. Expression of the ars genes was inducible by arsenite. By Southern hybridization, the operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis. PMID:7860609

  1. Conference on Fire Resistant Materials: A compilation of presentations and papers

    NASA Technical Reports Server (NTRS)

    Kourtides, D. A. (Editor); Johnson, G. A. (Editor)

    1979-01-01

    The proceedings of the NASA IRE Resistant Materials Engineering (FIREMEN) Program held at Boeing Commercial Airplane Company, Seattle, Washington, on March 1-2, 1979 are reported. The conference was to discuss the results of research by the National Aeronautics and Space Administration in the field of aircraft fire safety and fire-resistant materials. The program topics include the following: (1) large-scale testing; (2) fire toxicology; (3) polymeric materials; and (4) fire modeling.

  2. Molecular survey of turfgrass species for mutations conferring resistance to ACCase inhibiting herbicides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The control of grassy weeds in turfgrass is often problematic due to lack of herbicide selectivity. Seven different naturally occurring mutation sites have been reported to confer resistance to Acetyl coenzyme A carboxylase inhibiting herbicides. One or more of these mutation sites may hold potentia...

  3. Inheritance and linkage map positions of genes conferring resistance to stemphylium blight in lentil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stemphylium blight (caused by Stemphylium botryosum Wallr.) is one of the major diseases of lentil (Lens culinaris Medik.) in South Asia and North America. The objective of the study was to identify linkage map position of the genes conferring resistance to stemphylium blight and the markers linked ...

  4. Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations

    PubMed Central

    Menozzi, Philippe; Shi, Ming An; Lougarre, Andrée; Tang, Zhen Hua; Fournier, Didier

    2004-01-01

    Background Organophosphate and carbamate insecticides irreversibly inhibit acetylcholinesterase causing death of insects. Resistance-modified acetylcholinesterases(AChEs) have been described in many insect species and sequencing of their genes allowed several point mutations to be described. However, their relative frequency and their cartography had not yet been addressed. Results To analyze the most frequent mutations providing insecticide resistance in Drosophila melanogaster acetylcholinesterase, the Ace gene was cloned and sequenced in several strains harvested from different parts of the world. Sequence comparison revealed four widespread mutations, I161V, G265A, F330Y and G368A. We confirm here that mutations are found either isolated or in combination in the same protein and we show that most natural populations are heterogeneous, composed of a mixture of different alleles. In vitro expression of mutated proteins showed that combining mutations in the same protein has two consequences: it increases resistance level and provides a wide spectrum of resistance. Conclusion The presence of several alleles in natural populations, offering various resistance to carbamate and organophosphate compounds will complicate the establishment of resistance management programs. PMID:15018651

  5. Pyocyanin Production by Pseudomonas aeruginosa Confers Resistance to Ionic Silver

    PubMed Central

    Merrett, Neil D.

    2014-01-01

    Silver in its ionic form (Ag+), but not the bulk metal (Ag0), is toxic to microbial life forms and has been used for many years in the treatment of wound infections. The prevalence of bacterial resistance to silver is considered low due to the nonspecific nature of its toxicity. However, the recent increased use of silver as an antimicrobial agent for medical, consumer, and industrial products has raised concern that widespread silver resistance may emerge. Pseudomonas aeruginosa is a common pathogen that produces pyocyanin, a redox toxin and a reductant for molecular oxygen and ferric (Fe3+) ions. The objective of this study was to determine whether pyocyanin reduces Ag+ to Ag0, which may contribute to silver resistance due to lower bioavailability of the cation. Using surface plasmon resonance spectroscopy and scanning electron microscopy, pyocyanin was confirmed to be a reductant for Ag+, forming Ag0 nanoparticles and reducing the bioavailability of free Ag+ by >95% within minutes. Similarly, a pyocyanin-producing strain of P. aeruginosa (PA14) reduced Ag+ but not a pyocyanin-deficient (ΔphzM) strain of the bacterium. Challenge of each strain with Ag+ (as AgNO3) gave MICs of 20 and 5 μg/ml for the PA14 and ΔphzM strains, respectively. Removal of pyocyanin from the medium strain PA14 was grown in or its addition to the medium that ΔphzM mutant was grown in gave MICs of 5 and 20 μg/ml, respectively. Clinical isolates demonstrated similar pyocyanin-dependent resistance to Ag+. We conclude that pseudomonal silver resistance exists independently of previously recognized intracellular mechanisms and may be more prevalent than previously considered. PMID:25001302

  6. [Allelic state of the molecular marker for the golden nematode (Globodera rostochiensis) resistance gene H1 among Ukrainian and world cultivars of potato (Solanum tuberosum ssp. tuberosum)].

    PubMed

    Karelov, A V; Pilipenko, L A; Kozub, N A; Bondus, R A; Borzykh, A U; Sozinov, I A; Blium, Ia B; Sozinov, A A

    2013-01-01

    The purpose of our investigation was determination of allelic state of the H1 resistance gene against the pathotypes Ro1 and Ro4 of golden potato cyst nematode (Globodera rostochiensis) among Ukrainian and world potato (Solanum tuberosum ssp. tuberosum) cultivars. The allelic condition of the TG689 marker was determined by PCR with DNA samples isolated from tubers of potato and primers, one pair of which flanks the allele-specific region and the other one was used for the control of DNA quality. Among analyzed 77 potato cultivars the allele of marker associated with the H1-type resistance was found in 74% of Ukrainian and 90% foreign ones although some of those cultivars proved to be susceptible to the golden potato nematode in field. The obtained data confirm the presence of H1-resistance against golden nematode pathotypes Ro1 and Ro4 among the Ukrainian potato cultivars and efficiency of the used marker within the accuracy that has been declared by its authors.

  7. Two genes conferring resistance to Pythium stalk rot in maize inbred line Qi319.

    PubMed

    Song, Feng-Jing; Xiao, Ming-Gang; Duan, Can-Xing; Li, Hong-Jie; Zhu, Zhen-Dong; Liu, Bao-Tao; Sun, Su-Li; Wu, Xiao-Fei; Wang, Xiao-Ming

    2015-08-01

    Stalk rots are destructive diseases in maize around the world, and are most often caused by the pathogen Pythium, Fusarium and other fungi. The most efficient management for controlling stalk rots is to breed resistant cultivars. Pythium stalk rot can cause serious yield loss on maize, and to find the resistance genes from the existing germplasm is the basis to develop Pythium-resistance hybrid lines. In this study, we investigated the genetic resistance to Pythium stalk rot in inbred line Qi319 using F2 and F2:3 population, and found that the resistance to Pythium inflatum in Qi319 was conferred by two independently inherited dominant genes, RpiQI319-1 and RpiQI319-2. Linkage analysis uncovered that the RpiQI319-1 co-segregated with markers bnlg1203, and bnlg2057 on chromosome 1, and that the RpiQI319-2 locus co-segregated with markers umc2069 and bnlg1716 on chromosome 10. The RpiQI319-1 locus was further mapped into a ~500-kb interval flanked by markers SSRZ33 and SSRZ47. These results will facilitate marker-assisted selection of Pythium stalk rot-resistant cultivars in maize breeding. To our knowledge, this is the first report on the resistance to P. inflatum in the inbred line Qi319, and is also the first description of two independently inherited dominant genes conferring the resistance of Pythium stalk rot in maize. PMID:25724693

  8. Two genes conferring resistance to Pythium stalk rot in maize inbred line Qi319.

    PubMed

    Song, Feng-Jing; Xiao, Ming-Gang; Duan, Can-Xing; Li, Hong-Jie; Zhu, Zhen-Dong; Liu, Bao-Tao; Sun, Su-Li; Wu, Xiao-Fei; Wang, Xiao-Ming

    2015-08-01

    Stalk rots are destructive diseases in maize around the world, and are most often caused by the pathogen Pythium, Fusarium and other fungi. The most efficient management for controlling stalk rots is to breed resistant cultivars. Pythium stalk rot can cause serious yield loss on maize, and to find the resistance genes from the existing germplasm is the basis to develop Pythium-resistance hybrid lines. In this study, we investigated the genetic resistance to Pythium stalk rot in inbred line Qi319 using F2 and F2:3 population, and found that the resistance to Pythium inflatum in Qi319 was conferred by two independently inherited dominant genes, RpiQI319-1 and RpiQI319-2. Linkage analysis uncovered that the RpiQI319-1 co-segregated with markers bnlg1203, and bnlg2057 on chromosome 1, and that the RpiQI319-2 locus co-segregated with markers umc2069 and bnlg1716 on chromosome 10. The RpiQI319-1 locus was further mapped into a ~500-kb interval flanked by markers SSRZ33 and SSRZ47. These results will facilitate marker-assisted selection of Pythium stalk rot-resistant cultivars in maize breeding. To our knowledge, this is the first report on the resistance to P. inflatum in the inbred line Qi319, and is also the first description of two independently inherited dominant genes conferring the resistance of Pythium stalk rot in maize.

  9. Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

    PubMed Central

    Stephens, J C; Reich, D E; Goldstein, D B; Shin, H D; Smith, M W; Carrington, M; Winkler, C; Huttley, G A; Allikmets, R; Schriml, L; Gerrard, B; Malasky, M; Ramos, M D; Morlot, S; Tzetis, M; Oddoux, C; di Giovine, F S; Nasioulas, G; Chandler, D; Aseev, M; Hanson, M; Kalaydjieva, L; Glavac, D; Gasparini, P; Kanavakis, E; Claustres, M; Kambouris, M; Ostrer, H; Duff, G; Baranov, V; Sibul, H; Metspalu, A; Goldman, D; Martin, N; Duffy, D; Schmidtke, J; Estivill, X; O'Brien, S J; Dean, M

    1998-01-01

    The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations. PMID:9585595

  10. Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

    PubMed Central

    Grabowicz, Marcin

    2015-01-01

    Gram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens. PMID:26596941

  11. Apoptosis and Tumor Resistance Conferred by Par-4

    PubMed Central

    Zhao, Yanming; Rangnekar, Vivek M.

    2009-01-01

    Par-4 is a tumor suppressor protein with a pro-apoptotic function. Epigenetic silencing of Par-4 is seen in diverse tumors, and Par-4 knockout mice develop spontaneous tumors in various tissues. Endogenous Par-4 is essential for sensitization of cells to diverse apoptotic stimuli, whereas ectopic expression of Par-4 can selectively induce apoptosis in cancer cells. The cancer-specific pro-apoptotic action of Par-4 resides in its centrally located SAC domain. This chapter reviews a novel mouse model with ubiquitous expression of the SAC domain. These SAC transgenic mice display normal development and life span, and, most importantly, are resistant to spontaneous, as well as oncogene-induced, autochthonous tumors. The tumor resistant phenotype and undetectable toxicity of SAC in vivo suggests the SAC domain possesses tremendous therapeutic potential. PMID:18836307

  12. The 2NS Translocation from Aegilops ventricosa Confers Resistance to the Triticum Pathotype of Magnaporthe oryzae

    PubMed Central

    Cruz, C.D.; Peterson, G.L.; Bockus, W.W.; Kankanala, P.; Dubcovsky, J.; Jordan, K.W.; Akhunov, E.; Chumley, F.; Baldelomar, F.D.; Valent, B.

    2016-01-01

    Wheat blast is a serious disease caused by the fungus Magnaporthe oryzae (Triticum pathotype) (MoT). The objective of this study was to determine the effect of the 2NS translocation from Aegilops ventricosa (Zhuk.) Chennav on wheat head and leaf blast resistance. Disease phenotyping experiments were conducted in growth chamber, greenhouse, and field environments. Among 418 cultivars of wheat (Triticum aestivum L.), those with 2NS had 50.4 to 72.3% less head blast than those without 2NS when inoculated with an older MoT isolate under growth chamber conditions. When inoculated with recently collected isolates, cultivars with 2NS had 64.0 to 80.5% less head blast. Under greenhouse conditions when lines were inoculated with an older MoT isolate, those with 2NS had a significant head blast reduction. With newer isolates, not all lines with 2NS showed a significant reduction in head blast, suggesting that the genetic background and/or environment may influence the expression of any resistance conferred by 2NS. However, when near-isogenic lines (NILs) with and without 2NS were planted in the field, there was strong evidence that 2NS conferred resistance to head blast. Results from foliar inoculations suggest that the resistance to head infection that is imparted by the 2NS translocation does not confer resistance to foliar disease. In conclusion, the 2NS translocation was associated with significant reductions in head blast in both spring and winter wheat. PMID:27814405

  13. Targeting PCDH20 gene by microRNA-122 confers 5-FU resistance in hepatic carcinoma.

    PubMed

    Wang, Wei; Liu, Wen Bin; Huang, Da Bing; Jia, Wei; Ji, Chu Shu; Hu, Bing

    2016-01-01

    Drug resistance is one of the main hurdles for the successful treatment of hepatic carcinoma. However, the detailed mechanisms underlying resistance remain largely unknown and therapeutic approaches are limited. In the present study, we show that miR-122 confers resistance to 5-fluorouracil induced hepatocellular carcinoma cell apoptosis in vitro and reduces the potency of 5-fluorouracil in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicate that miR-122 modulates drug resistance through down-regulation of expression of PCDH20, which belongs to the protocadherin gene family and negatively regulates Akt activation. Knockdown of PCDH20 expression increases Akt phosphorylation, which leads to elevated mTOR activity and enhanced 5-fluorouracil resistance; whereas rescue of PCDH20 expression in miR-122-expressing cells decreases Akt and mTOR phosphorylation, re-sensitizing hepatocellular carcinoma cell to 5-fluorouracil induced apoptosis. Moreover, a specific and potent Akt inhibitor reverses miR-122-conferred 5-fluorouracil resistance. These findings indicate that the miR-122/PCDH20/Akt/mTOR signaling axis has an important role in mediating response to chemotherapy in human hepatocellular carcinoma. A major implication of our study is that inhibition of miR-122 or restoration of PCDH20 expression may have significant therapeutic potential to overcome drug resistance in hepatocellular carcinoma and that the combined use of an Akt inhibitor with 5-fluorouracil may increase efficacy in liver cancer treatment. PMID:27648358

  14. Targeting PCDH20 gene by microRNA-122 confers 5-FU resistance in hepatic carcinoma

    PubMed Central

    Wang, Wei; Liu, Wen Bin; Huang, Da Bing; Jia, Wei; Ji, Chu Shu; Hu, Bing

    2016-01-01

    Drug resistance is one of the main hurdles for the successful treatment of hepatic carcinoma. However, the detailed mechanisms underlying resistance remain largely unknown and therapeutic approaches are limited. In the present study, we show that miR-122 confers resistance to 5-fluorouracil induced hepatocellular carcinoma cell apoptosis in vitro and reduces the potency of 5-fluorouracil in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicate that miR-122 modulates drug resistance through down-regulation of expression of PCDH20, which belongs to the protocadherin gene family and negatively regulates Akt activation. Knockdown of PCDH20 expression increases Akt phosphorylation, which leads to elevated mTOR activity and enhanced 5-fluorouracil resistance; whereas rescue of PCDH20 expression in miR-122-expressing cells decreases Akt and mTOR phosphorylation, re-sensitizing hepatocellular carcinoma cell to 5-fluorouracil induced apoptosis. Moreover, a specific and potent Akt inhibitor reverses miR-122-conferred 5-fluorouracil resistance. These findings indicate that the miR-122/PCDH20/Akt/mTOR signaling axis has an important role in mediating response to chemotherapy in human hepatocellular carcinoma. A major implication of our study is that inhibition of miR-122 or restoration of PCDH20 expression may have significant therapeutic potential to overcome drug resistance in hepatocellular carcinoma and that the combined use of an Akt inhibitor with 5-fluorouracil may increase efficacy in liver cancer treatment.

  15. Mutations in the Pneumocystis jirovecii DHPS gene confer cross-resistance to sulfa drugs.

    PubMed

    Iliades, Peter; Meshnick, Steven R; Macreadie, Ian G

    2005-02-01

    Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP) and results in a high degree of mortality in immunocompromised individuals. The drug of choice for PCP is typically sulfamethoxazole (SMX) or dapsone in conjunction with trimethoprim. Drug treatment failure and sulfa drug resistance have been implicated epidemiologically with point mutations in dihydropteroate synthase (DHPS) of P. jirovecii. P. jirovecii cannot be cultured in vitro; however, heterologous complementation of the P. jirovecii trifunctional folic acid synthesis (PjFAS) genes with an E. coli DHPS-disrupted strain was recently achieved. This enabled the evaluation of SMX resistance conferred by DHPS mutations. In this study, we sought to determine whether DHPS mutations conferred sulfa drug cross-resistance to 15 commonly available sulfa drugs. It was established that the presence of amino acid substitutions (T(517)A or P(519)S) in the DHPS domain of PjFAS led to cross-resistance against most sulfa drugs evaluated. The presence of both mutations led to increased sulfa drug resistance, suggesting cooperativity and the incremental evolution of sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine [SCP] and sulfamethoxypyridazine [SMP]) that had a higher inhibitory potential than SMX were identified. In addition, SCP, SMP, and sulfadiazine (SDZ) were found to be capable of inhibiting the clinically observed drug-resistant mutants. We propose that SCP, SMP, and SDZ should be considered for clinical evaluation against PCP or for future development of novel sulfa drug compounds.

  16. Mutations in the Pneumocystis jirovecii DHPS Gene Confer Cross-Resistance to Sulfa Drugs

    PubMed Central

    Iliades, Peter; Meshnick, Steven R.; Macreadie, Ian G.

    2005-01-01

    Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP) and results in a high degree of mortality in immunocompromised individuals. The drug of choice for PCP is typically sulfamethoxazole (SMX) or dapsone in conjunction with trimethoprim. Drug treatment failure and sulfa drug resistance have been implicated epidemiologically with point mutations in dihydropteroate synthase (DHPS) of P. jirovecii. P. jirovecii cannot be cultured in vitro; however, heterologous complementation of the P. jirovecii trifunctional folic acid synthesis (PjFAS) genes with an E. coli DHPS-disrupted strain was recently achieved. This enabled the evaluation of SMX resistance conferred by DHPS mutations. In this study, we sought to determine whether DHPS mutations conferred sulfa drug cross-resistance to 15 commonly available sulfa drugs. It was established that the presence of amino acid substitutions (T517A or P519S) in the DHPS domain of PjFAS led to cross-resistance against most sulfa drugs evaluated. The presence of both mutations led to increased sulfa drug resistance, suggesting cooperativity and the incremental evolution of sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine [SCP] and sulfamethoxypyridazine [SMP]) that had a higher inhibitory potential than SMX were identified. In addition, SCP, SMP, and sulfadiazine (SDZ) were found to be capable of inhibiting the clinically observed drug-resistant mutants. We propose that SCP, SMP, and SDZ should be considered for clinical evaluation against PCP or for future development of novel sulfa drug compounds. PMID:15673759

  17. Targeting PCDH20 gene by microRNA-122 confers 5-FU resistance in hepatic carcinoma

    PubMed Central

    Wang, Wei; Liu, Wen Bin; Huang, Da Bing; Jia, Wei; Ji, Chu Shu; Hu, Bing

    2016-01-01

    Drug resistance is one of the main hurdles for the successful treatment of hepatic carcinoma. However, the detailed mechanisms underlying resistance remain largely unknown and therapeutic approaches are limited. In the present study, we show that miR-122 confers resistance to 5-fluorouracil induced hepatocellular carcinoma cell apoptosis in vitro and reduces the potency of 5-fluorouracil in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicate that miR-122 modulates drug resistance through down-regulation of expression of PCDH20, which belongs to the protocadherin gene family and negatively regulates Akt activation. Knockdown of PCDH20 expression increases Akt phosphorylation, which leads to elevated mTOR activity and enhanced 5-fluorouracil resistance; whereas rescue of PCDH20 expression in miR-122-expressing cells decreases Akt and mTOR phosphorylation, re-sensitizing hepatocellular carcinoma cell to 5-fluorouracil induced apoptosis. Moreover, a specific and potent Akt inhibitor reverses miR-122-conferred 5-fluorouracil resistance. These findings indicate that the miR-122/PCDH20/Akt/mTOR signaling axis has an important role in mediating response to chemotherapy in human hepatocellular carcinoma. A major implication of our study is that inhibition of miR-122 or restoration of PCDH20 expression may have significant therapeutic potential to overcome drug resistance in hepatocellular carcinoma and that the combined use of an Akt inhibitor with 5-fluorouracil may increase efficacy in liver cancer treatment. PMID:27648358

  18. Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival.

    PubMed

    Zaragoza, Oscar; Chrisman, Cara J; Castelli, Maria Victoria; Frases, Susana; Cuenca-Estrella, Manuel; Rodríguez-Tudela, Juan Luis; Casadevall, Arturo

    2008-10-01

    Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.

  19. Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival

    PubMed Central

    Zaragoza, Oscar; Chrisman, Cara J.; Castelli, Maria Victoria; Frases, Susana; Cuenca-Estrella, Manuel; Rodríguez-Tudela, Juan Luis; Casadevall, Arturo

    2015-01-01

    Summary Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H2O2 that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H2O2. Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to β-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells. PMID:18554313

  20. AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

    PubMed Central

    Blair, Jessica M. A.; Bavro, Vassiliy N.; Ricci, Vito; Modi, Niraj; Cacciotto, Pierpaolo; Kleinekathӧfer, Ulrich; Ruggerone, Paolo; Vargiu, Attilio V.; Baylay, Alison J.; Smith, Helen E.; Brandon, Yvonne; Galloway, David; Piddock, Laura J. V.

    2015-01-01

    The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies. PMID:25737552

  1. A transposable element insertion confers xenobiotic resistance in Drosophila.

    PubMed

    Mateo, Lidia; Ullastres, Anna; González, Josefa

    2014-08-01

    The increase in availability of whole genome sequences makes it possible to search for evidence of adaptation at an unprecedented scale. Despite recent progress, our understanding of the adaptive process is still very limited due to the difficulties in linking adaptive mutations to their phenotypic effects. In this study, we integrated different levels of biological information to pinpoint the ecologically relevant fitness effects and the underlying molecular and biochemical mechanisms of a putatively adaptive TE insertion in Drosophila melanogaster: the pogo transposon FBti0019627. We showed that other than being incorporated into Kmn1 transcript, FBti0019627 insertion also affects the polyadenylation signal choice of CG11699 gene. Consequently, only the short 3'UTR transcript of CG11699 gene is produced and the expression level of this gene is higher in flies with the insertion. Our results indicated that increased CG11699 expression leads to xenobiotic stress resistance through increased ALDH-III activity: flies with FBti0019627 insertion showed increased survival rate in response to benzaldehyde, a natural xenobiotic, and to carbofuran, a synthetic insecticide. Although differences in survival rate between flies with and without the insertion were not always significant, when they were, they were consistent with FBti0019627 mediating resistance to xenobiotics. Taken together, our results provide a plausible explanation for the increase in frequency of FBti0019627 in natural populations of D. melanogaster and add to the limited number of examples in which a natural genetic mutation has been linked to its ecologically relevant phenotype. Furthermore, the widespread distribution of TEs across the tree of life and conservation of stress response pathways across organisms make our results relevant not only for Drosophila, but for other organisms as well.

  2. A Transposable Element Insertion Confers Xenobiotic Resistance in Drosophila

    PubMed Central

    Mateo, Lidia; Ullastres, Anna; González, Josefa

    2014-01-01

    The increase in availability of whole genome sequences makes it possible to search for evidence of adaptation at an unprecedented scale. Despite recent progress, our understanding of the adaptive process is still very limited due to the difficulties in linking adaptive mutations to their phenotypic effects. In this study, we integrated different levels of biological information to pinpoint the ecologically relevant fitness effects and the underlying molecular and biochemical mechanisms of a putatively adaptive TE insertion in Drosophila melanogaster: the pogo transposon FBti0019627. We showed that other than being incorporated into Kmn1 transcript, FBti0019627 insertion also affects the polyadenylation signal choice of CG11699 gene. Consequently, only the short 3′UTR transcript of CG11699 gene is produced and the expression level of this gene is higher in flies with the insertion. Our results indicated that increased CG11699 expression leads to xenobiotic stress resistance through increased ALDH-III activity: flies with FBti0019627 insertion showed increased survival rate in response to benzaldehyde, a natural xenobiotic, and to carbofuran, a synthetic insecticide. Although differences in survival rate between flies with and without the insertion were not always significant, when they were, they were consistent with FBti0019627 mediating resistance to xenobiotics. Taken together, our results provide a plausible explanation for the increase in frequency of FBti0019627 in natural populations of D. melanogaster and add to the limited number of examples in which a natural genetic mutation has been linked to its ecologically relevant phenotype. Furthermore, the widespread distribution of TEs across the tree of life and conservation of stress response pathways across organisms make our results relevant not only for Drosophila, but for other organisms as well. PMID:25122208

  3. Bacterial glyphosate resistance conferred by overexpression of an E. coli membrane efflux transporter.

    PubMed

    Staub, Jeffrey M; Brand, Leslie; Tran, Minhtien; Kong, Yifei; Rogers, Stephen G

    2012-04-01

    Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate-resistant crops is due to expression of a variant form of a bacterial 5-enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. To provide insight into glyphosate resistance mechanisms and identify a potential glyphosate transporter, we screened Escherichia coli genomic DNA for alternate sources of glyphosate resistance genes. Our search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. We report here that an alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants.

  4. Novel channel enzyme fusion proteins confer arsenate resistance.

    PubMed

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-12-17

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion.

  5. Novel channel enzyme fusion proteins confer arsenate resistance.

    PubMed

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-12-17

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion. PMID:20947511

  6. Novel Channel Enzyme Fusion Proteins Confer Arsenate Resistance*

    PubMed Central

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-01-01

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H2AsVO4−/HAsVO42−) to arsenite (AsIII(OH)3) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion. PMID:20947511

  7. Mutations in an Atypical TIR-NB-LRR-LIM Resistance Protein Confer Autoimmunity

    PubMed Central

    Bi, Dongling; Johnson, Kaeli C. M.; Zhu, Zhaohai; Huang, Yan; Chen, Fang; Zhang, Yuelin; Li, Xin

    2011-01-01

    In order to defend against microbial infection, plants employ a complex immune system that relies partly on resistance (R) proteins that initiate intricate signaling cascades upon pathogen detection. The resistance signaling network utilized by plants is only partially characterized. A genetic screen conducted to identify novel defense regulators involved in this network resulted in the isolation of the snc6-1D mutant. Positional cloning revealed that this mutant contained a molecular lesion in the chilling sensitive 3 (CHS3) gene, thus the allele was renamed chs3-2D. CHS3 encodes a TIR-NB-LRR R protein that contains a C-terminal zinc-binding LIM (Lin-11, Isl-1, Mec-3) domain. Although this protein has been previously implicated in cold stress and defense response, the role of the LIM domain in modulating protein activity is unclear. The chs3-2D allele contains a G to A point mutation causing a C1340 to Y1340 substitution close to the LIM domain. It encodes a dominant gain-of-function mutation. The chs3-2D mutant is severely stunted and displays curled leaf morphology. Additionally, it constitutively expresses PATHOGENESIS-RELATED (PR) genes, accumulates salicylic acid, and shows enhanced resistance to the virulent oomycete isolate Hyaloperonospora arabidopsidis (H.a.) Noco2. Subcellular localization assays using GFP fusion constructs indicate that both CHS3 and chs3-2D localize to the nucleus. A third chs3 mutant allele, chs3-3D, was identified in an unrelated genetic screen in our lab. This allele contains a C to T point mutation resulting in an M1017 to V1017 substitution in the LRR–LIM linker region. Additionally, a chs3-2D suppressor screen identified two revertant alleles containing secondary mutations that abolish the mutant morphology. Analysis of the locations of these molecular lesions provides support for the hypothesis that the LIM domain represses CHS3 R-like protein activity. This repression may occur through either autoinhibition or binding of a

  8. Rapid assay of A2058T-mutated 23S rRNA allelic profiles associated with high-level macrolide resistance in Moraxella catarrhalis.

    PubMed

    Saito, Ryoichi; Kasai, Ayako; Ogihara, Shinji; Yamada, Kageto; Tao, Kazuyuki

    2015-09-01

    We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.

  9. C239S Mutation in the β-Tubulin of Phytophthora sojae Confers Resistance to Zoxamide

    PubMed Central

    Cai, Meng; Miao, Jianqiang; Song, Xi; Lin, Dong; Bi, Yang; Chen, Lei; Liu, Xili; Tyler, Brett M.

    2016-01-01

    Zoxamide is the sole β-tubulin inhibitor registered for the control of oomycete pathogens. The current study investigated the activity of zoxamide against Phytophthora sojae and baseline sensitivity was established with a mean EC50 of 0.048 μg/ml. The data is critical for monitoring changes in zoxamide-sensitivity in the field. Three stable resistant mutants with a high resistance level were obtained by selection on zoxamide amended media. Although the development of resistance occurred at a low frequency, there were no apparent fitness penalty in the acquired mutants in terms of growth rate, sporulation, germination and pathogenicity. Based on the biological profiles and low mutagenesis rate, the resistance risk of P. sojae to zoxamide can be estimated as low to medium. Further investigation revealed all the zoxamide-resistant mutants had a point mutation of C239S in their β-tubulin. Zoxamide also exhibited high activity against most species from the genus Pythium in which only Pythium aphanidermatum was found naturally resistant to zoxamide and harboring the natural point mutation S239 in the β-tubulin. Back-transformation in P. sojae with the mutated allele (S239) confirmed the C239S mutation can induce resistance to zoxamide, and the resistance level was positively related to the expression level of the mutated gene. In contrast, the overexpression of the wild type gene was unable to cause zoxamide resistance. It is the first report on the resistance molecular mechanism of zoxamide in oomycetes. Based on our study, C239 is supposed to be a key target site of zoxamide, which distinguishes zoxamide from benzimidazoles and accounts for its low resistance risk. The result can provide advice on the design of new β-tubulin inhibitors in future. PMID:27242773

  10. C239S Mutation in the β-Tubulin of Phytophthora sojae Confers Resistance to Zoxamide.

    PubMed

    Cai, Meng; Miao, Jianqiang; Song, Xi; Lin, Dong; Bi, Yang; Chen, Lei; Liu, Xili; Tyler, Brett M

    2016-01-01

    Zoxamide is the sole β-tubulin inhibitor registered for the control of oomycete pathogens. The current study investigated the activity of zoxamide against Phytophthora sojae and baseline sensitivity was established with a mean EC50 of 0.048 μg/ml. The data is critical for monitoring changes in zoxamide-sensitivity in the field. Three stable resistant mutants with a high resistance level were obtained by selection on zoxamide amended media. Although the development of resistance occurred at a low frequency, there were no apparent fitness penalty in the acquired mutants in terms of growth rate, sporulation, germination and pathogenicity. Based on the biological profiles and low mutagenesis rate, the resistance risk of P. sojae to zoxamide can be estimated as low to medium. Further investigation revealed all the zoxamide-resistant mutants had a point mutation of C239S in their β-tubulin. Zoxamide also exhibited high activity against most species from the genus Pythium in which only Pythium aphanidermatum was found naturally resistant to zoxamide and harboring the natural point mutation S239 in the β-tubulin. Back-transformation in P. sojae with the mutated allele (S239) confirmed the C239S mutation can induce resistance to zoxamide, and the resistance level was positively related to the expression level of the mutated gene. In contrast, the overexpression of the wild type gene was unable to cause zoxamide resistance. It is the first report on the resistance molecular mechanism of zoxamide in oomycetes. Based on our study, C239 is supposed to be a key target site of zoxamide, which distinguishes zoxamide from benzimidazoles and accounts for its low resistance risk. The result can provide advice on the design of new β-tubulin inhibitors in future. PMID:27242773

  11. Genetic analysis of new 16S rRNA mutations conferring aminoglycoside resistance in Mycobacterium abscessus

    PubMed Central

    Nessar, Rachid; Reyrat, Jean Marc; Murray, Alan; Gicquel, Brigitte

    2011-01-01

    Objectives We studied the development and fitness cost of 2-deoxystreptamine aminoglycoside resistance of Mycobacterium abscessus. Methods Spontaneous 2-deoxystreptamine aminoglycoside-resistant mutants were selected and the frequency of their appearance was determined. The 3′ part of the rrs gene was sequenced to characterize mutations. Additionally, we determined the MICs of aminoglycoside drugs for the different mutants obtained. The dominance/recessivity traits of the different mutations were examined and we explored the potential cost conferred by the mutations selected in vitro on the fitness of these isolates compared with the wild-type strain. Results The in vitro mutation rate for 2-deoxystreptamine aminoglycoside resistance was ∼10−7 mutations/cell division. In addition to the known rrs A→G substitution at position 1408 (Escherichia coli numbering), which confers kanamycin resistance (KanR), three new substitutions in rrs were identified in M. abscessus KanR mutants, i.e. T→A at 1406, C→T at 1409 and G→T at 1491. Heterodiploids carrying genomic mutations T→A at 1406 and A→G at 1408 with the wild-type rrs gene carried by the pNBV1 vector showed a resistant phenotype. In contrast, heterodiploids carrying genomic mutations C→T at 1409 and G→T at 1491 with the wild-type rrs gene carried by the pNBV1 vector had a susceptible phenotype. No burden on fitness was observed for the different mutations. Conclusion Mutations in the rrs gene that confer high-level 2-deoxystreptamine aminoglycoside resistance on M. abscessus differ in their dominance/recessivity traits and have no biological cost under our experimental conditions. PMID:21652621

  12. Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1.

    PubMed

    Humphry, Matt; Reinstädler, Anja; Ivanov, Sergey; Bisseling, Ton; Panstruga, Ralph

    2011-12-01

    Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.

  13. Vitamin B3 confers resistance to sulfa drugs in Saccharomyces cerevisiae.

    PubMed

    Kornfeld, Olga; Nichols, Brian P

    2005-10-01

    Sulfa drugs are ubiquitous antibiotics used to treat bacterial infections and diseases caused by eukaryotes, such as Pneumocystis carinii, the leading cause of pneumonia (PCP) in HIV patients. A daily regimen of sulfonamides and multivitamins including vitamin B3 is also recommended for persons with HIV. We show that exogenous vitamin B3 (nicotinate) confers resistance to sulfa drugs in Saccharomyces cerevisiae, a model for P. carinii. We propose a model of metabolic rerouting in which increased nicotinate leads to increased intracellular concentration of p-aminobenzoate, thus leading to sulfonamide resistance.

  14. Diversified mcr-1-Harbouring Plasmid Reservoirs Confer Resistance to Colistin in Human Gut Microbiota

    PubMed Central

    Ye, Huiyan; Li, Yihui; Li, Zhencui; Gao, Rongsui; Zhang, Han; Wen, Ronghui; Gao, George F.; Hu, Qinghua

    2016-01-01

    ABSTRACT Colistin is an ultimate line of refuge against multidrug-resistant Gram-negative pathogens. Very recently, the emergence of plasmid-mediated mcr-1 colistin resistance has become a great challenge to global public health, raising the possibility that dissemination of the mcr-1 gene is underestimated and diversified. Here, we report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients. Structural and functional analyses determined that the colistin resistance is conferred purely by the single mcr-1 gene. Genetic and sequence mapping revealed that mcr-1-harbouring plasmid reservoirs are present in diversity. Together, the data represent the first evidence of diversity in mcr-1-harbouring plasmid reservoirs of human gut microbiota. PMID:27048797

  15. Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance.

    PubMed

    Rallos, Lynn Esther E; Baudoin, Anton B

    2016-01-01

    Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970

  16. Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance

    PubMed Central

    Rallos, Lynn Esther E.; Baudoin, Anton B.

    2016-01-01

    Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970

  17. APOL1 Risk Alleles are Associated with More Severe Arteriosclerosis in Renal Resistance Vessels with Aging and Hypertension

    PubMed Central

    Hughson, Michael D; Hoy, Wendy E; Mott, Susan A; Puelles, Victor G; Bertram, John F; Winkler, Cheryl L; Kopp, Jeffrey B

    2016-01-01

    The increased risk of end-stage kidney disease (ESKD) among hypertensive African Americans is partly related to APOL1 allele variants. Hypertension-associated arterionephrosclerosis consists of arteriosclerosis, glomerulosclerosis, and cortical fibrosis. The initial glomerulosclerosis, attributed to preglomerular arteriosclerosis and ischemia, consists of focal global glomerulosclerosis (FGGS), but in biopsy studies, focal segmental glomerulosclerosis (FSGS) is found with progression to ESKD, particularly in African Americans. This is a study of arterionephrosclerosis in successfully APOL1 genotyped autopsy kidney tissue of 159 African Americans (61 no risk alleles, 68 one risk allele, 30 two risk alleles) and 135 whites aged 18–89 years from a general population with no clinical renal disease. Glomerulosclerosis was nearly exclusively FGGS with only three subjects having FSGS-like lesions that were unrelated to APOL1 risk status. For both races, in multivariable analysis, the dependent variables of arteriosclerosis, glomerulosclerosis, and cortical fibrosis were all significantly related to the independent variables of older age (P < 0.001) and hypertension (P < 0.001). A relationship between APOL1 genotype and arteriosclerosis was apparent only after 35 years of age when, for any level of elevated blood pressure, more severe arteriosclerosis was found in the interlobular arteries of 14 subjects with two APOL1 risk alleles when compared to African Americans with none (n = 37, P = 0.02) or one risk alleles (n = 35, P = 0.02). With the limitation of the small number of subjects contributing to the positive results, the findings imply that APOL1 risk alleles recessively augment small vessel arteriosclerosis in conjunction with age and hypertension. FSGS was not a significant finding, indicating that in the early stages of arterionephrosclerosis, the primary pathologic influence of APOL1 genotype is vascular rather than glomerular. PMID:27610422

  18. Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa.

    PubMed

    Ueno, Hiroki; Matsumoto, Etsuo; Aruga, Daisuke; Kitagawa, Satoshi; Matsumura, Hideo; Hayashida, Nobuaki

    2012-12-01

    Clubroot disease is one of the major diseases affecting Brassicaceae crops, and a number of these crops grown commercially, such as Chinese cabbage (Brassica rapa L. ssp. pekinensis), are known to be highly susceptible to clubroot disease. To provide protection from this disease, plant breeders have introduced genes for resistance to clubroot from the European turnip into susceptible lines. The CRa gene confers specific resistance to the clubroot pathogen Plasmodiophora brassicae isolate M85. Fine mapping of the CRa locus using synteny to the Arabidopsis thaliana genome and partial genome sequences of B. rapa revealed a candidate gene encoding a TIR-NBS-LRR protein. Several structural differences in this candidate gene were found between susceptible and resistant lines, and CRa expression was observed only in the resistant line. Four mutant lines lacking clubroot resistance were obtained by the UV irradiation of pollen from a resistant line, and all of these mutant lines carried independent mutations in the candidate TIR-NBS-LRR gene. This genetic and molecular evidence strongly suggests that the identified gene is CRa. This is the first report on the molecular characterization of a clubroot Resistance gene in Brassicaceae and of the disease resistance gene in B. rapa.

  19. Plant eR Genes That Encode Photorespiratory Enzymes Confer Resistance against Disease

    PubMed Central

    Taler, Dvir; Galperin, Marjana; Benjamin, Ido; Cohen, Yigal; Kenigsbuch, David

    2004-01-01

    Downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis is a devastating foliar disease of cucurbits worldwide. We previously demonstrated that the wild melon line PI 124111F (PI) is highly resistant to all pathotypes of P. cubensis. That resistance was controlled genetically by two partially dominant, complementary loci. Here, we show that unlike other plant disease resistance genes, which confer an ability to resist infection by pathogens expressing corresponding avirulence genes, the resistance of PI to P. cubensis is controlled by enhanced expression of the enzymatic resistance (eR) genes At1 and At2. These constitutively expressed genes encode the photorespiratory peroxisomal enzyme proteins glyoxylate aminotransferases. The low expression of At1 and At2 in susceptible melon lines is regulated mainly at the transcriptional level. This regulation is independent of infection with the pathogen. Transgenic melon plants overexpressing either of these eR genes displayed enhanced activity of glyoxylate aminotransferases and remarkable resistance against P. cubensis. The cloned eR genes provide a new resource for developing downy mildew–resistant melon varieties. PMID:14688292

  20. A new finely mapped Oryza australiensis-derived QTL in rice confers resistance to brown planthopper.

    PubMed

    Hu, Jie; Xiao, Cong; Cheng, Ming-Xing; Gao, Guan-Jun; Zhang, Qing-Lu; He, Yu-Qing

    2015-04-25

    Brown planthopper (BPH) is the most destructive pest of rice in Asia. The BPH resistance in the introgression line IR65482-17-511-5-7 (IR65482-17) is derived from the wild rice species Oryza australiensis. An F2:3 population from a cross between Zhenshan 97 (ZS97) and IR65482-17 was used to map three quantitative trait loci (QTLs) for seedling resistance and feeding rate to BPH. The loci were distributed on chromosomes 2, 4 and 12. The QTL qBph4.2 on chromosome 4 had the largest effect, and contributed 36-44% of the phenotypic variance with a LOD score of 19-29. To validate the effect of qBph4.2, two near-isogenic lines (NILs) containing the qBph4.2 locus in the backgrounds of ZS97 and 9311 were developed by marker-assisted backcrossing (MABC). BPH bioassays showed that lines homozygous for the IR65482-17 allele (NIL+) of qBph4.2 tented to have significantly higher seedling resistance to BPH than those homozygous for the ZS97 or 9311 alleles (NIL-). Resistance was associated with a lower feeding rate by the insect. qBph4.2 was delimited to a ~300 kb (0.04 cM) region flanked by markers RM261 and S1, and co-segregating with XC4-27. This study will facilitate map-based cloning and marker-assisted selection of the gene, and permits further studies of gene function and resistance mechanisms in rice: BPH interaction.

  1. Postnatal establishment of allelic Gαs silencing as a plausible explanation for delayed onset of parathyroid hormone-resistance due to heterozygous Gαs disruption

    PubMed Central

    Turan, Serap; Fernandez-Rebollo, Eduardo; Aydin, Cumhur; Zoto, Teuta; Reyes, Monica; Bounoutas, George; Chen, Min; Weinstein, Lee S.; Erben, Reinhold G.; Marshansky, Vladimir; Bastepe, Murat

    2013-01-01

    Pseudohypoparathyroidism type-Ia (PHP-Ia), characterized by renal proximal tubular resistance to parathyroid hormone (PTH), results from maternal mutations of GNAS that lead to loss of Gαs activity. Gαs expression is paternally silenced in the renal proximal tubule, and this genomic event is critical for the development of PTH-resistance, as patients display impaired hormone action only if the mutation is inherited maternally. The primary clinical finding of PHP-Ia is hypocalcemia, which can lead to various neuromuscular defects including seizures. PHP-Ia patients frequently do not present with hypocalcemia until after infancy, but it has remained uncertain whether PTH-resistance occurs in a delayed fashion. Analyzing reported cases of PHP-Ia with documented GNAS mutations and mice heterozygous for disruption of Gnas, we herein determined that the manifestation of PTH-resistance caused by the maternal loss of Gαs, i.e. hypocalcemia and elevated serum PTH, occurs after early postnatal life. To investigate whether this delay could reflect gradual development of paternal Gαs silencing, we then analyzed renal proximal tubules isolated by laser capture microdissection from mice with either maternal or paternal disruption of Gnas. Our results revealed that, whereas expression of Gαs mRNA in this tissue is predominantly from the maternal Gnas allele at weaning (three-weeks postnatal) and in adulthood, the contributions of the maternal and paternal Gnas alleles to Gαs mRNA expression are equal at postnatal day 3. In contrast, we found that paternal Gαs expression is already markedly repressed in brown adipose tissue at birth. Thus, the mechanisms silencing the paternal Gαs allele in renal proximal tubules are not operational during early postnatal development, and this finding correlates well with the latency of PTH-resistance in patients with PHP-Ia. PMID:23956044

  2. Bactobolin Resistance Is Conferred by Mutations in the L2 Ribosomal Protein

    PubMed Central

    Chandler, Josephine R.; Truong, Thao T.; Silva, Patricia M.; Seyedsayamdost, Mohammad R.; Carr, Gavin; Radey, Matthew; Jacobs, Michael A.; Sims, Elizabeth H.; Clardy, Jon; Greenberg, E. Peter

    2012-01-01

    ABSTRACT Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). PMID:23249812

  3. Double mutations in eIF4E and eIFiso4E confer recessive resistance to Chilli veinal mottle virus in pepper.

    PubMed

    Hwang, Jeena; Li, Jinjie; Liu, Wing-Yee; An, Song-Ji; Cho, Hwajin; Her, Nam Han; Yeam, Inhwa; Kim, Dosun; Kang, Byoung-Cheorl

    2009-03-31

    To evaluate the involvement of translation initiation factors eIF4E and eIFiso4E in Chilli veinai mottle virus (ChiVMV) infection in pepper, we conducted a genetic analysis using a segregating population derived from a cross between Capsicum annuum 'Dempsey' containing an eIF4E mutation (pvr1(2)) and C. annuum 'Perennial' containing an eIFiso4E mutation (pvr6). C. annuum 'Dempsey' was susceptible and C. annuum 'Perennial' was resistant to ChiVMV. All F(1) plants showed resistance, and F(2) individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five F(2) and 329 F(3) plants of 17 families were genotyped with pvr1(2) and pvr6 allele-specific markers, and the genotype data were compared with observed resistance to viral infection. All plants containing homozygous genotypes of both pvr1(2) and pvr6 were resistant to ChiVMV, demonstrating that simultaneous mutations in eIF4E and eIFiso4E confer resistance to ChiVMV in pepper. Genotype analysis of F2 plants revealed that all plants containing homozygous genotypes of both pvr1(2) and pvr6 showed resistance to ChiVMV. In protein-protein interaction experiments, ChiVMV viral genome-linked protein (VPg) interacted with both eIF4E and eIFiso4E. Silencing of eIF4E and eIFiso4E in the VIGS experiment showed reduction in ChiVMV accumulation. These results demonstrated that ChiVMV can use both eIF4E and eIFiso4E for replication, making simultaneous mutations in eIF4E and eIFiso4E necessary to prevent ChiVMV infection in pepper.

  4. FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

    PubMed Central

    Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

    2014-01-01

    Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

  5. Analysis of acetohydroxyacid synthase1 gene in chickpea conferring resistance to imazamox herbicide.

    PubMed

    Jain, Parul; Tar'an, Bunyamin

    2014-11-01

    Chickpea (Cicer arietinum L.) production in the Canadian prairies is challenging due to a lack of effective weed management mainly because of poor competition ability of the crop and limited registered herbicide options. Chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified. A point mutation in the acetohydroxyacid synthase1 (AHAS1) gene at C581 to T581, resulting in an amino acid substitution from Ala194 to Val194 (position 205, standardized to arabidopsis), confers the resistance to imazamox in chickpea. However, the molecular mechanism leading to the resistance is not fully understood. In many plant species, contrasting transcription levels of AHAS gene has been implicated in the resistant and susceptible genotypes in response to IMI. The objectives of this research were to compare the AHAS gene expression and AHAS enzyme activity in resistant and susceptible chickpea cultivars in response to imazamox herbicide treatment. Results from RT-qPCR indicated that there is no significant change in the transcript levels of AHAS1 between the susceptible and the resistant genotypes in response to imazamox treatment. Protein hydrophobic cluster analysis, protein-ligand docking analysis, and AHAS enzyme activity assay all indicated that the resistance to imazamox in chickpea is due to the alteration of interaction of the AHAS1 enzyme with the imazamox herbicide.

  6. Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

    PubMed

    Statt, Sarah; Ruan, Jhen-Wei; Hung, Li-Yin; Chang, Ching-Yun; Huang, Chih-Ting; Lim, Jae Hyang; Li, Jian-Dong; Wu, Reen; Kao, Cheng-Yuan

    2015-11-01

    Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

  7. The Nematode Resistance Allele at the rhg1 Locus Alters the Proteome and Primary Metabolism of Soybean Roots1[C][W][OA

    PubMed Central

    Afzal, Ahmed J.; Natarajan, Aparna; Saini, Navinder; Iqbal, M. Javed; Geisler, Matt; El Shemy, Hany A.; Mungur, Rajsree; Willmitzer, Lothar; Lightfoot, David A.

    2009-01-01

    Heterodera glycines, the soybean cyst nematode (SCN), causes the most damaging chronic disease of soybean (Glycine max). Host resistance requires the resistance allele at rhg1. Resistance destroys the giant cells created in the plant's roots by the nematodes about 24 to 48 h after commencement of feeding. In addition, 4 to 8 d later, a systemic acquired resistance develops that discourages later infestations. The molecular mechanisms that control the rhg1-mediated resistance response appear to be multigenic and complex, as judged by transcript abundance changes, even in near isogenic lines (NILs). This study aimed to focus on key posttranscriptional changes by identifying proteins and metabolites that were increased in abundance in both resistant and susceptible NILs. Comparisons were made among NILs 10 d after SCN infestation and without SCN infestation. Two-dimensional gel electrophoresis resolved more than 1,000 protein spots on each gel. Only 30 protein spots with a significant (P < 0.05) difference in abundance of 1.5-fold or more were found among the four treatments. The proteins in these spots were picked, trypsin digested, and analyzed using quadrupole time-of-flight tandem mass spectrometry. Protein identifications could be made for 24 of the 30 spots. Four spots contained two proteins, so that 28 distinct proteins were identified. The proteins were grouped into six functional categories. Metabolite analysis by gas chromatography-mass spectrometry identified 131 metabolites, among which 58 were altered by one or more treatment; 28 were involved in primary metabolism. Taken together, the data showed that 17 pathways were altered by the rhg1 alleles. Pathways altered were associated with systemic acquired resistance-like responses, including xenobiotic, phytoalexin, ascorbate, and inositol metabolism, as well as primary metabolisms like amino acid synthesis and glycolysis. The pathways impacted by the rhg1 allelic state and SCN infestation agreed with

  8. Introgression of resistance-conferring ALS mutations in herbicide-resistant weedy rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Weedy red rice (Oryza sativa) competes aggressively with rice, reducing yields and grain quality. Clearfield™ rice, a nontransgenic, herbicide-resistant (HR) rice introduced in 2002 to control weedy rice, has resulted in some ALS-resistant weedy rice apparently due to gene flow. Studies were conduct...

  9. Polar flagella rotation in Vibrio parahaemolyticus confers resistance to bacteriophage infection.

    PubMed

    Zhang, Hui; Li, Lu; Zhao, Zhe; Peng, Daxin; Zhou, Xiaohui

    2016-01-01

    Bacteriophage has been recognized as a novel approach to treat bacterial infectious diseases. However, phage resistance may reduce the efficacy of phage therapy. Here, we described a mechanism of bacterial resistance to phage infections. In Gram-negative enteric pathogen Vibrio parahaemolyticus, we found that polar flagella can reduce the phage infectivity. Deletion of polar flagella, but not the lateral flagella, can dramatically promote the adsorption of phage to the bacteria and enhances the phage infectivity to V. parahaemolyticus, indicating that polar flagella play an inhibitory role in the phage infection. Notably, it is the rotation, not the physical presence, of polar flagella that inhibits the phage infection of V. parahaemolyticus. Strikingly, phage dramatically reduces the virulence of V. parahaemolyticus only when polar flagella were absent both in vitro and in vivo. These results indicated that polar flagella rotation is a previously unidentified mechanism that confers bacteriophage resistance.

  10. Polar flagella rotation in Vibrio parahaemolyticus confers resistance to bacteriophage infection

    PubMed Central

    Zhang, Hui; Li, Lu; Zhao, Zhe; Peng, Daxin; Zhou, Xiaohui

    2016-01-01

    Bacteriophage has been recognized as a novel approach to treat bacterial infectious diseases. However, phage resistance may reduce the efficacy of phage therapy. Here, we described a mechanism of bacterial resistance to phage infections. In Gram-negative enteric pathogen Vibrio parahaemolyticus, we found that polar flagella can reduce the phage infectivity. Deletion of polar flagella, but not the lateral flagella, can dramatically promote the adsorption of phage to the bacteria and enhances the phage infectivity to V. parahaemolyticus, indicating that polar flagella play an inhibitory role in the phage infection. Notably, it is the rotation, not the physical presence, of polar flagella that inhibits the phage infection of V. parahaemolyticus. Strikingly, phage dramatically reduces the virulence of V. parahaemolyticus only when polar flagella were absent both in vitro and in vivo. These results indicated that polar flagella rotation is a previously unidentified mechanism that confers bacteriophage resistance. PMID:27189325

  11. MRP1 expression in CTCs confers resistance to irinotecan-based chemotherapy in metastatic colorectal cancer.

    PubMed

    Abdallah, Emne Ali; Fanelli, Marcello Ferretti; Souza E Silva, Virgílio; Machado Netto, Marcelo Calil; Gasparini Junior, José Luiz; Araújo, Daniel Vilarim; Ocea, Luciana Menezes Mendonça; Buim, Marcilei Eliza Cavicchioli; Tariki, Milena Shizue; Alves, Vanessa da Silva; Piana de Andrade, Victor; Dettino, Aldo Lourenço Abbade; Abdon Lopes de Mello, Celso; Chinen, Ludmilla Thomé Domingos

    2016-08-15

    Circulating tumor cells are important markers of tumor progression and can reflect tumor behavior in metastatic colorectal cancer (mCRC). Identification of proteins that confer resistance to treatment is an important step to predict response and better selection of treatment for patients. Multidrug resistance-associated protein 1 (MRP1) and Multidrug resistance-associated protein 4 (MRP4) play a role in irinotecan-resistance, and Excision Repair Cross-Complementation group 1 (ERCC1) expression can confer resistance to platinum compounds. Here, we included 34 patients with mCRC and most of them received FOLFIRI or FOLFOX chemotherapy (91.1%). CTCs were isolated by ISET(®) Technology and identified in 30 patients (88.2%), with a median of 2.0 CTCs/mL (0-31.0). We analyzed the immunocytochemical expression of MRP1, MRP4 and ERCC1 only in patients who had previously detectable CTCs, accordingly to treatment received (n = 19, 15 and 13 patients, respectively). Among patients treated with irinotecan-based chemotherapy, 4 out of 19 cases with MRP1 positive CTCs showed a worse progression free survival (PFS) in comparison to those with MRP1 negative CTCs (2.1 months vs. 9.1 months; p = 0.003). None of the other proteins studied in CTCs had significant association with PFS. We analyzed also histological sections of primary tumors and metastases by immunohistochemistry, and found no association with clinicopathological characteristics or with PFS. Our results show MRP1 as a potential biomarker of resistance to treatment with irinotecan when found in CTCs from mCRC patients. This is a small proof-of-principle study and these early findings need to be validated in a larger cohort of patients. PMID:26950035

  12. SHEA conference on antimicrobial resistance. Society for Healthcare Epidemiology of America.

    PubMed

    Gerding, D N; Martone, W J

    2000-05-01

    Antimicrobial resistance is an increasing problem in healthcare institutions and in the community. Public concern about resistance is also increasing. The issue is broad and complex and not readily addressed by government, industry, or professional societies alone. On October 29-30, 1998, 19 representatives of various professional societies and governmental agencies met under the auspices of the Society for Healthcare Epidemiology of America (SHEA) at Brook Lodge Conference Center in Augusta, Michigan. The purpose of the meeting was to discuss the current status of antimicrobial resistance in the United States and Canada, including present society and governmental efforts to address the problem. Representatives exchanged experiences through presentations and discussions on the first day, then on the second day held a brainstorming session to address future needs and priorities in addressing the resistance problem. It was agreed that a national coordinated effort was needed. As part of this national effort, representatives called for the creation of a National Coalition on Antibiotic Resistance (NCAR) to combat antibiotic resistance through education, research, prevention, and advocacy. Priorities for NCAR were focused in four areas: (1) education of the public and professionals; (2) support of basic and applied research; (3) provision of an information resource and clearinghouse; and (4) advocacy initiatives. At the recommendation of the SHEA Board, discussions with the National Foundation for Infectious Diseases for the joint development of NCAR have begun.

  13. Activation tagging of ATHB13 in Arabidopsis thaliana confers broad-spectrum disease resistance.

    PubMed

    Gao, Dongli; Appiano, Michela; Huibers, Robin P; Chen, Xi; Loonen, Annelies E H M; Visser, Richard G F; Wolters, Anne-Marie A; Bai, Yuling

    2014-12-01

    Powdery mildew species Oidium neolycopersici (On) can cause serious yield losses in tomato production worldwide. Besides on tomato, On is able to grow and reproduce on Arabidopsis. In this study we screened a collection of activation-tagged Arabidopsis mutants and identified one mutant, 3221, which displayed resistance to On, and in addition showed a reduced stature and serrated leaves. Additional disease tests demonstrated that the 3221 mutant exhibited resistance to downy mildew (Hyaloperonospora arabidopsidis) and green peach aphid (Myzus persicae), but retained susceptibility to bacterial pathogen Pseudomonas syringae pv tomato DC3000. The resistance trait and morphological alteration were mutually linked in 3221. Identification of the activation tag insertion site and microarray analysis revealed that ATHB13, a homeodomain-leucine zipper (HD-Zip) transcription factor, was constitutively overexpressed in 3221. Silencing of ATHB13 in 3221 resulted in the loss of both the morphological alteration and resistance, whereas overexpression of the cloned ATHB13 in Col-0 and Col-eds1-2 backgrounds resulted in morphological alteration and resistance. Microarray analysis further revealed that overexpression of ATHB13 influenced the expression of a large number of genes. Previously, it was reported that ATHB13-overexpressing lines conferred tolerance to abiotic stress. Together with our results, it appears that ATHB13 is involved in the crosstalk between abiotic and biotic stress resistance pathways.

  14. Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

    PubMed Central

    Johnson, Neil; Johnson, Shawn F.; Yao, Wei; Li, Yu-Chen; Choi, Young-Eun; Bernhardy, Andrea J.; Wang, Yifan; Capelletti, Marzia; Sarosiek, Kristopher A.; Moreau, Lisa A.; Chowdhury, Dipanjan; Wickramanayake, Anneka; Harrell, Maria I.; Liu, Joyce F.; D’Andrea, Alan D.; Miron, Alexander; Swisher, Elizabeth M.; Shapiro, Geoffrey I.

    2013-01-01

    Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance. PMID:24085845

  15. ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating HER3

    PubMed Central

    Ebbing, Eva A.; Medema, Jan Paul; Damhofer, Helene; Meijer, Sybren L.; Krishnadath, Kausilia K.; van Berge Henegouwen, Mark I.

    2016-01-01

    Receptor tyrosine kinases of the HER-family are involved in the development and progression of multiple epithelial tumors, and have consequently become widely used targets for new anti-cancer therapies. Trastuzumab, an antibody against HER2, has shown potent growth inhibitory effects on HER2 overexpressing tumors, including gastro-esophageal cancer, however, resistance to this therapy is inevitable. Unfortunately, a paucity of data on the cellular mechanisms of resistance to targeted therapeutic agents exists in esophageal adenocarcinoma. Using primary established HER2-overexpressing cultures and patient-derived xenograft models, we now reveal a novel resistance mechanism to trastuzumab in esophageal cancer: In response to trastuzumab, both HER3 and the metalloprotease ADAM10 are simultaneously upregulated. The proteolytic activity of the latter then releases the HER3 ligand heregulin from the cell surface to activate HER3 and confer resistance to trastuzumab by inducing compensatory growth factor receptor signaling. Blocking either HER3 or ADAM10 effectively reverts the acquired resistance to trastuzumab. Our data thus provide strategies to inhibit this signaling and circumvent resistance to trastuzumab. PMID:26863569

  16. Interfamily transfer of dual NB-LRR genes confers resistance to multiple pathogens.

    PubMed

    Narusaka, Mari; Kubo, Yasuyuki; Hatakeyama, Katsunori; Imamura, Jun; Ezura, Hiroshi; Nanasato, Yoshihiko; Tabei, Yutaka; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

    2013-01-01

    A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens.

  17. The evolutionary ecology of symbiont-conferred resistance to parasitoids in aphids.

    PubMed

    Vorburger, Christoph

    2014-06-01

    Aphids may harbor a wide variety of facultative bacterial endosymbionts. These symbionts are transmitted maternally with high fidelity and they show horizontal transmission as well, albeit at rates too low to enable infectious spread. Such symbionts need to provide a net fitness benefit to their hosts to persist and spread. Several symbionts have achieved this by evolving the ability to protect their hosts against parasitoids. Reviewing empirical work and some models, I explore the evolutionary ecology of symbiont-conferred resistance to parasitoids in order to understand how defensive symbiont frequencies are maintained at the intermediate levels observed in aphid populations. I further show that defensive symbionts alter the reciprocal selection between aphids and parasitoids by augmenting the heritable variation for resistance, by increasing the genetic specificity of the host-parasitoid interaction, and by inducing environment-dependent trade-offs. These effects are conducive to very dynamic, symbiont-mediated coevolution that is driven by frequency-dependent selection. Finally I argue that defensive symbionts represent a problem for biological control of pest aphids, and I propose to mitigate this problem by exploiting the parasitoids' demonstrated ability to rapidly evolve counteradaptations to symbiont-conferred resistance.

  18. Rapid Detection of rpoB Gene Mutations Conferring Rifampin Resistance in Mycobacterium tuberculosis

    PubMed Central

    Ao, Wanyuan; Aldous, Stephen; Woodruff, Evelyn; Hicke, Brian; Rea, Larry; Kreiswirth, Barry

    2012-01-01

    Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute. PMID:22518852

  19. The effects of resistant and sensitive alleles on survivorship, weight, and heterozygosity in the fathead minnow, Pimephales promelas

    SciTech Connect

    Schlueter, M.; Guftman, S.

    1995-12-31

    In three separate acute copper exposures a highly significant (p = 0.0001) negative relationship was detected between fish weight and survivorship. Overall heterozygosity was not related to survivorship. The effects of two single loci IDHP 1* and MDH-2* were shown to be partially responsible for these results. For the IDHP-1* locus allele *a was associated with increased copper survivorship; however, it was also associated with small size. Conversely, the allele *a at the MDH-2* locus, which was present in higher frequencies in larger fish, was associated with decreased survivorship. If the genotype at an individual locus strongly affects survivorship, being a homozygote at that locus may be more significant to survivorship than overall individual heterozygosity. These results suggest that genetic selection for one characteristic may have a negative impact on other characteristics. These results may help explain unusual relationships between weight and other characteristics and survivorship found in other studies.

  20. The Arabidopsis NPR1 gene confers broad-spectrum disease resistance in strawberry.

    PubMed

    Silva, Katchen Julliany P; Brunings, Asha; Peres, Natalia A; Mou, Zhonglin; Folta, Kevin M

    2015-08-01

    Although strawberry is an economically important fruit crop worldwide, production of strawberry is limited by its susceptibility to a wide range of pathogens and the lack of major commercial cultivars with high levels of resistance to multiple pathogens. The objective of this study is to ectopically express the Arabidopsis thaliana NPR1 gene (AtNPR1) in the diploid strawberry Fragaria vesca L. and to test transgenic plants for disease resistance. AtNPR1 is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR) and has been shown to confer resistance to a number of pathogens when overexpressed in Arabidopsis or ectopically expressed in several crop species. We show that ectopic expression of AtNPR1 in strawberry increases resistance to anthracnose, powdery mildew, and angular leaf spot, which are caused by different fungal or bacterial pathogens. The increased resistance is related to the relative expression levels of AtNPR1 in the transgenic plants. In contrast to Arabidopsis plants overexpressing AtNPR1, which grow normally and do not constitutively express defense genes, the strawberry transgenic plants are shorter than non-transformed controls, and most of them fail to produce runners and fruits. Consistently, most of the transgenic lines constitutively express the defense gene FvPR5, suggesting that the SAR activation mechanisms in strawberry and Arabidopsis are different. Nevertheless, our results indicate that overexpression of AtNPR1 holds the potential for generation of broad-spectrum disease resistance in strawberry.

  1. The Arabidopsis NPR1 gene confers broad-spectrum disease resistance in strawberry.

    PubMed

    Silva, Katchen Julliany P; Brunings, Asha; Peres, Natalia A; Mou, Zhonglin; Folta, Kevin M

    2015-08-01

    Although strawberry is an economically important fruit crop worldwide, production of strawberry is limited by its susceptibility to a wide range of pathogens and the lack of major commercial cultivars with high levels of resistance to multiple pathogens. The objective of this study is to ectopically express the Arabidopsis thaliana NPR1 gene (AtNPR1) in the diploid strawberry Fragaria vesca L. and to test transgenic plants for disease resistance. AtNPR1 is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR) and has been shown to confer resistance to a number of pathogens when overexpressed in Arabidopsis or ectopically expressed in several crop species. We show that ectopic expression of AtNPR1 in strawberry increases resistance to anthracnose, powdery mildew, and angular leaf spot, which are caused by different fungal or bacterial pathogens. The increased resistance is related to the relative expression levels of AtNPR1 in the transgenic plants. In contrast to Arabidopsis plants overexpressing AtNPR1, which grow normally and do not constitutively express defense genes, the strawberry transgenic plants are shorter than non-transformed controls, and most of them fail to produce runners and fruits. Consistently, most of the transgenic lines constitutively express the defense gene FvPR5, suggesting that the SAR activation mechanisms in strawberry and Arabidopsis are different. Nevertheless, our results indicate that overexpression of AtNPR1 holds the potential for generation of broad-spectrum disease resistance in strawberry. PMID:25812515

  2. Systematic identification of signaling pathways with potential to confer anticancer drug resistance.

    PubMed

    Martz, Colin A; Ottina, Kathleen A; Singleton, Katherine R; Jasper, Jeff S; Wardell, Suzanne E; Peraza-Penton, Ashley; Anderson, Grace R; Winter, Peter S; Wang, Tim; Alley, Holly M; Kwong, Lawrence N; Cooper, Zachary A; Tetzlaff, Michael; Chen, Pei-Ling; Rathmell, Jeffrey C; Flaherty, Keith T; Wargo, Jennifer A; McDonnell, Donald P; Sabatini, David M; Wood, Kris C

    2014-12-23

    Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts. PMID:25538079

  3. MicroRNAs Suppress NB Domain Genes in Tomato That Confer Resistance to Fusarium oxysporum

    DOE PAGES

    Ouyang, Shouqiang; Park, Gyungsoon; Atamian, Hagop S.; Han, Cliff S.; Stajich, Jason E.; Kaloshian, Isgouhi; Borkovich, Katherine A.

    2014-10-16

    MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site–leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. Here, we explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulatedmore » by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. In conclusion, taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.« less

  4. MicroRNAs Suppress NB Domain Genes in Tomato That Confer Resistance to Fusarium oxysporum

    SciTech Connect

    Ouyang, Shouqiang; Park, Gyungsoon; Atamian, Hagop S.; Han, Cliff S.; Stajich, Jason E.; Kaloshian, Isgouhi; Borkovich, Katherine A.

    2014-10-16

    MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site–leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. Here, we explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. In conclusion, taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

  5. MicroRNAs Suppress NB Domain Genes in Tomato That Confer Resistance to Fusarium oxysporum

    PubMed Central

    Ouyang, Shouqiang; Park, Gyungsoon; Atamian, Hagop S.; Han, Cliff S.; Stajich, Jason E.; Kaloshian, Isgouhi; Borkovich, Katherine A.

    2014-01-01

    MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site–leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs. PMID:25330340

  6. Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements.

    PubMed

    Klima, Cassidy L; Zaheer, Rahat; Cook, Shaun R; Booker, Calvin W; Hendrick, Steve; Alexander, Trevor W; McAllister, Tim A

    2014-02-01

    In this study, we determined the prevalence of bovine respiratory disease (BRD)-associated viral and bacterial pathogens in cattle and characterized the genetic profiles, antimicrobial susceptibilities, and nature of antimicrobial resistance determinants in collected bacteria. Nasopharyngeal swab and lung tissue samples from 68 BRD mortalities in Alberta, Canada (n = 42), Texas (n = 6), and Nebraska (n = 20) were screened using PCR for bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus, bovine herpesvirus 1, parainfluenza type 3 virus, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Excepting bovine herpesvirus 1, all agents were detected. M. haemolytica (91%) and BVDV (69%) were the most prevalent, with cooccurrence in 63% of the cattle. Isolates of M. haemolytica (n = 55), P. multocida (n = 8), and H. somni (n = 10) from lungs were also collected. Among M. haemolytica isolates, a clonal subpopulation (n = 8) was obtained from a Nebraskan feedlot. All three bacterial pathogens exhibited a high rate of antimicrobial resistance, with 45% exhibiting resistance to three or more antimicrobials. M. haemolytica (n = 18), P. multocida (n = 3), and H. somni (n = 3) from Texas and Nebraska possessed integrative conjugative elements (ICE) that conferred resistance for up to seven different antimicrobial classes. ICE were shown to be transferred via conjugation from P. multocida to Escherichia coli and from M. haemolytica and H. somni to P. multocida. ICE-mediated multidrug-resistant profiles of bacterial BRD pathogens could be a major detriment to many of the therapeutic antimicrobial strategies currently used to control BRD.

  7. Genetic analysis of Tn916-like elements conferring tetracycline resistance in clinical isolates of Clostridium difficile.

    PubMed

    Dong, Danfeng; Chen, Xu; Jiang, Cen; Zhang, Lihua; Cai, Gang; Han, Lizhong; Wang, Xuefeng; Mao, Enqiang; Peng, Yibing

    2014-01-01

    As an important clinically relevant pathogen, Clostridium difficile has a high multidrug resistance rate. Conjugative transposons play a vital role in its resistance phenotype. In the present study, 34 tetracycline-resistant clinical isolates of C. difficile were studied to detect tetracycline resistance genes and the presence of transposons. Thirty-two isolates were found to harbour Tn916-like elements carrying the tet(M) resistance gene, of which only one copy existed in the genome by Southern blot analysis. To characterise the genetic organisation of the Tn916-like elements, overlap PCR assays were performed with nine primer pairs, revealing three types of elements designated T1 to T3. The prevalent element T1 lacking PCRA (ORF23 to ORF21) and PCRB (ORF21 to ORF20) products, present in the epidemic ST37 clone, was further analysed by genome walking PCR in the left and right end sequences of the novel Tn916-like element. A gene coding for an FtsK/SpoIIIE family protein was found to replace the ORF24 to ORF21 region in Tn916. Moreover, the element could hardly conjugate between cells by filter mating experiments. These findings suggest that the dissemination of Tn916-like elements in epidemic ST37 strains in China was likely to have been conferred by clonal spread, signifying the importance of future surveillance and characterisation of conjugative transposons.

  8. Pathogens of Bovine Respiratory Disease in North American Feedlots Conferring Multidrug Resistance via Integrative Conjugative Elements

    PubMed Central

    Klima, Cassidy L.; Zaheer, Rahat; Cook, Shaun R.; Booker, Calvin W.; Hendrick, Steve

    2014-01-01

    In this study, we determined the prevalence of bovine respiratory disease (BRD)-associated viral and bacterial pathogens in cattle and characterized the genetic profiles, antimicrobial susceptibilities, and nature of antimicrobial resistance determinants in collected bacteria. Nasopharyngeal swab and lung tissue samples from 68 BRD mortalities in Alberta, Canada (n = 42), Texas (n = 6), and Nebraska (n = 20) were screened using PCR for bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus, bovine herpesvirus 1, parainfluenza type 3 virus, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Excepting bovine herpesvirus 1, all agents were detected. M. haemolytica (91%) and BVDV (69%) were the most prevalent, with cooccurrence in 63% of the cattle. Isolates of M. haemolytica (n = 55), P. multocida (n = 8), and H. somni (n = 10) from lungs were also collected. Among M. haemolytica isolates, a clonal subpopulation (n = 8) was obtained from a Nebraskan feedlot. All three bacterial pathogens exhibited a high rate of antimicrobial resistance, with 45% exhibiting resistance to three or more antimicrobials. M. haemolytica (n = 18), P. multocida (n = 3), and H. somni (n = 3) from Texas and Nebraska possessed integrative conjugative elements (ICE) that conferred resistance for up to seven different antimicrobial classes. ICE were shown to be transferred via conjugation from P. multocida to Escherichia coli and from M. haemolytica and H. somni to P. multocida. ICE-mediated multidrug-resistant profiles of bacterial BRD pathogens could be a major detriment to many of the therapeutic antimicrobial strategies currently used to control BRD. PMID:24478472

  9. Genetic analysis of Tn916-like elements conferring tetracycline resistance in clinical isolates of Clostridium difficile.

    PubMed

    Dong, Danfeng; Chen, Xu; Jiang, Cen; Zhang, Lihua; Cai, Gang; Han, Lizhong; Wang, Xuefeng; Mao, Enqiang; Peng, Yibing

    2014-01-01

    As an important clinically relevant pathogen, Clostridium difficile has a high multidrug resistance rate. Conjugative transposons play a vital role in its resistance phenotype. In the present study, 34 tetracycline-resistant clinical isolates of C. difficile were studied to detect tetracycline resistance genes and the presence of transposons. Thirty-two isolates were found to harbour Tn916-like elements carrying the tet(M) resistance gene, of which only one copy existed in the genome by Southern blot analysis. To characterise the genetic organisation of the Tn916-like elements, overlap PCR assays were performed with nine primer pairs, revealing three types of elements designated T1 to T3. The prevalent element T1 lacking PCRA (ORF23 to ORF21) and PCRB (ORF21 to ORF20) products, present in the epidemic ST37 clone, was further analysed by genome walking PCR in the left and right end sequences of the novel Tn916-like element. A gene coding for an FtsK/SpoIIIE family protein was found to replace the ORF24 to ORF21 region in Tn916. Moreover, the element could hardly conjugate between cells by filter mating experiments. These findings suggest that the dissemination of Tn916-like elements in epidemic ST37 strains in China was likely to have been conferred by clonal spread, signifying the importance of future surveillance and characterisation of conjugative transposons. PMID:24176599

  10. Frequency of spontaneous mutations that confer antibiotic resistance in Chlamydia spp.

    PubMed

    Binet, Rachel; Maurelli, Anthony T

    2005-07-01

    Mutations in rRNA genes (rrn) that confer resistance to ribosomal inhibitors are typically recessive or weakly codominant and have been mostly reported for clinical strains of pathogens possessing only one or two rrn operons, such as Helicobacter pylori and Mycobacterium spp. An analysis of the genome sequences of several members of the Chlamydiaceae revealed that these obligate intracellular bacteria harbor only one or two sets of rRNA genes. To study the contribution of rRNA mutations to the emergence of drug resistance in the Chlamydiaceae, we used the sensitivities of Chlamydia trachomatis L2 (two rrn operons) and Chlamydophila psittaci 6BC (one rrn operon) to the aminoglycoside spectinomycin as a model. Confluent cell monolayers were infected in a plaque assay with about 10(8) wild-type infectious particles and then treated with the antibiotic. After a 2-week incubation time, plaques formed by spontaneous spectinomycin-resistant (Spc(r)) mutants appeared with a frequency of 5 x 10(-5) for C. psittaci 6BC. No Spc(r) mutants were isolated for C. trachomatis L2, although the frequencies of rifampin resistance were in the same range for both strains (i.e., 10(-7)). The risk of emergence of Chlamydia strains resistant to tetracyclines and macrolides, the ribosomal drugs currently used to treat chlamydial infections, is discussed.

  11. Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay

    PubMed Central

    Zhang, Guoqing; Cai, Fangping; Zhou, Zhiyong; DeVos, Joshua; Wagar, Nick; Diallo, Karidia; Zulu, Isaac; Wadonda-Kabondo, Nellie; Stringer, Jeffrey S. A.; Weidle, Paul J.; Ndongmo, Clement B.; Sikazwe, Izukanji; Sarr, Abdoulaye; Kagoli, Matthew; Nkengasong, John

    2013-01-01

    High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5′ end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring. PMID:23985909

  12. Haplotype variation of Glu-D1 locus and the origin of Glu-D1d allele conferring superior end-use qualities in common wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In common wheat (Triticum aestivum, AABBDD), the Glu-D1 locus possesses multiple alleles, with Glu-D1a (coding for 1Dx2 and 1Dy12 subunits) and Glu-D1d (encoding 1Dx5 and 1Dy10 subunits) being intensively used in the genetic improvement of end-use qualities. Here, we studied the molecular variatio...

  13. 4th international conference on tumor progression and therapeutic resistance: meeting report

    PubMed Central

    Prabhu, Varun V; El-Deiry, Wafik S

    2015-01-01

    The fourth international conference on tumor progression and therapeutic resistance organized in association with GTCbio was held in Boston, MA from March 9 to 11, 2014. The meeting attracted a diverse group of experts in the field of cancer biology, therapeutics and medical oncology from academia and industry. The meeting addressed the current challenges in the treatment of cancer including tumor heterogeneity, therapy resistance and metastasis along with the need for improved biomarkers of tumor progression and clinical trial design. Keynote speakers included Clifton Leaf, Editor at Fortune Magazine, Dr. Mina Bissell from the Lawrence Berkeley National Laboratory and Dr. Levi Garraway from the Dana Farber Cancer Institute. The meeting featured cutting edge tools, preclinical models and the latest basic, translational and clinical research findings in the field. PMID:25782066

  14. Draft genome sequences of two Aeromonas salmonicida subsp. salmonicida isolates harboring plasmids conferring antibiotic resistance.

    PubMed

    Vincent, Antony T; Tanaka, Katherine H; Trudel, Melanie V; Frenette, Michel; Derome, Nicolas; Charette, Steve J

    2015-02-01

    The bacterium Aeromonas salmonicida is the etiological agent of furunculosis, a widespread fish disease causing important economic losses to the fish farming industry. Antibiotic treatments in fish farms may be challenging given the existence of multidrug-resistant isolates of this bacterium. Here, we report the draft genome sequences of the 2004-05MF26 and 2009-144K3 isolates, which harbor plasmids conferring antibiotic resistance. Both isolates also carry the large plasmid pAsa5, which is known to encode a type three secretion system (TTSS) and the pAsal1 plasmid which has the aopP gene producing a TTSS effector. These two isolates are good representatives of the plasmid diversity in A. salmonicida subsp. salmonicida. PMID:25724776

  15. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors.

    PubMed

    Ceccon, Monica; Mologni, Luca; Bisson, William; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2013-02-01

    The dual ALK/MET inhibitor crizotinib was recently approved for the treatment of metastatic and late-stage ALK+ NSCLC, and is currently in clinical trial for other ALK-related diseases. As predicted after other tyrosine kinase inhibitors' clinical experience, the first mutations that confer resistance to crizotinib have been described in patients with non-small cell lung cancer (NSCLC) and in one patient inflammatory myofibroblastic tumor (IMT). Here, we focused our attention on the anaplastic large cell lymphoma (ALCL), where the oncogenic fusion protein NPM-ALK, responsible for 70% to 80% of cases, represents an ideal crizotinib target. We selected and characterized 2 human NPM-ALK+ ALCL cell lines, KARPAS-299 and SUP-M2, able to survive and proliferate at different crizotinib concentrations. Sequencing of ALK kinase domain revealed that a single mutation became predominant at high crizotinib doses in each cell line, namely L1196Q and I1171N in Karpas-299 and SUP-M2 cells, respectively. These mutations also conferred resistance to crizotinib in Ba/F3 cells expressing human NPM-ALK. The resistant cell populations, as well as mutated Ba/F3 cells, were characterized for sensitivity to two additional ALK inhibitors: the dual ALK/EGFR inhibitor AP26113 and NVP-TAE684. While L1196Q-positive cell lines were sensitive to both inhibitors, cells carrying I1171N substitution showed cross-resistance to all ALK inhibitors tested. This study provides potentially relevant information for the management of patients with ALCL that may relapse after crizotinib treatment.

  16. The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.

    PubMed

    Leprivier, Gabriel; Remke, Marc; Rotblat, Barak; Dubuc, Adrian; Mateo, Abigail-Rachele F; Kool, Marcel; Agnihotri, Sameer; El-Naggar, Amal; Yu, Bin; Somasekharan, Syam Prakash; Faubert, Brandon; Bridon, Gaëlle; Tognon, Cristina E; Mathers, Joan; Thomas, Ryan; Li, Amy; Barokas, Adi; Kwok, Brian; Bowden, Mary; Smith, Stephanie; Wu, Xiaochong; Korshunov, Andrey; Hielscher, Thomas; Northcott, Paul A; Galpin, Jason D; Ahern, Christopher A; Wang, Ye; McCabe, Martin G; Collins, V Peter; Jones, Russell G; Pollak, Michael; Delattre, Olivier; Gleave, Martin E; Jan, Eric; Pfister, Stefan M; Proud, Christopher G; Derry, W Brent; Taylor, Michael D; Sorensen, Poul H

    2013-05-23

    Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

  17. Tn5406, a New Staphylococcal Transposon Conferring Resistance to Streptogramin A and Related Compounds Including Dalfopristin

    PubMed Central

    Haroche, Julien; Allignet, Jeanine; El Solh, Névine

    2002-01-01

    We characterized a new transposon, Tn5406 (5,467 bp), in a clinical isolate of Staphylococcus aureus (BM3327). It carries a variant of vgaA, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn554. Like Tn554, Tn5406 failed to generate target repeats. In BM3327, the single copy of Tn5406 was inserted into the chromosomal att554 site, which is the preferential insertion site of Tn554. In three other independent S. aureus clinical isolates, Tn5406 was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn5406-carrying plasmids also contain two other genes, vgaB and vatB. The insertion sites of Tn5406 in BM3252 were studied: one copy was in att554, and one copy was in the additional SCCmec element. Amplification experiments revealed circular forms of Tn5406, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described. PMID:12121902

  18. The eEF2 Kinase Confers Resistance to Nutrient Deprivation by Blocking Translation Elongation

    PubMed Central

    Leprivier, Gabriel; Remke, Marc; Rotblat, Barak; Dubuc, Adrian; Mateo, Abigail-Rachele F.; Kool, Marcel; Agnihotri, Sameer; El-Naggar, Amal; Yu, Bin; Somasekharan, Syam Prakash; Faubert, Brandon; Bridon, Gaëlle; Tognon, Cristina E.; Mathers, Joan; Thomas, Ryan; Li, Amy; Barokas, Adi; Kwok, Brian; Bowden, Mary; Smith, Stephanie; Wu, Xiaochong; Korshunov, Andrey; Hielscher, Thomas; Northcott, Paul A.; Galpin, Jason D.; Ahern, Christopher A.; Wang, Ye; McCabe, Martin G.; Collins, V. Peter; Jones, Russell G.; Pollak, Michael; Delattre, Olivier; Gleave, Martin E.; Jan, Eric; Pfister, Stefan M.; Proud, Christopher G.; Derry, W. Brent; Taylor, Michael D.; Sorensen, Poul H.

    2015-01-01

    SUMMARY Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PMID:23706743

  19. Identification of Bone-Derived Factors Conferring De Novo Therapeutic Resistance in Metastatic Prostate Cancer.

    PubMed

    Lee, Yu-Chen; Lin, Song-Chang; Yu, Guoyu; Cheng, Chien-Jui; Liu, Bin; Liu, Hsuan-Chen; Hawke, David H; Parikh, Nila U; Varkaris, Andreas; Corn, Paul; Logothetis, Christopher; Satcher, Robert L; Yu-Lee, Li-Yuan; Gallick, Gary E; Lin, Sue-Hwa

    2015-11-15

    Resistance to currently available targeted therapies significantly hampers the survival of patients with prostate cancer with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a "resistance niche" adjacent to prostate cancer-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed "osteocrines") found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to the newly formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic prostate cancer.

  20. Diversity of Molecular Mechanisms Conferring Carbapenem Resistance to Pseudomonas aeruginosa Isolates from Saudi Arabia.

    PubMed

    Al-Agamy, Mohamed H; Jeannot, Katy; El-Mahdy, Taghrid S; Samaha, Hassan A; Shibl, Atef M; Plésiat, Patrick; Courvalin, Patrice

    2016-01-01

    Background. This study described various molecular and epidemiological characters determining antibiotic resistance patterns in Pseudomonas aeruginosa isolates. Methods. A total of 34 carbapenem-resistant P. aeruginosa clinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE) were performed. Results. All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC) > 256 mg/L). Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrum β-lactamase (VEB-1) (n = 16/34) and oxacillinase (OXA-10) (n = 14/34) were the most prevalent extended-spectrum β-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28) and imipenemase (IMP-7) variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD) expression was seen in nine isolates, and an increase in efflux pump gene (MexAB) expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15) were found among the 34 isolates. The predominant serotype was O:11 (16 isolates), followed by O:15 (nine isolates). PFGE analysis of the 34 carbapenem-resistant P. aeruginosa isolates revealed 14 different pulsotypes. Conclusions. These results revealed diverse mechanisms conferring carbapenem resistance to P. aeruginosa isolates from Saudi Arabia. PMID:27597874

  1. Diversity of Molecular Mechanisms Conferring Carbapenem Resistance to Pseudomonas aeruginosa Isolates from Saudi Arabia

    PubMed Central

    Jeannot, Katy; El-Mahdy, Taghrid S.; Samaha, Hassan A.; Shibl, Atef M.; Plésiat, Patrick; Courvalin, Patrice

    2016-01-01

    Background. This study described various molecular and epidemiological characters determining antibiotic resistance patterns in Pseudomonas aeruginosa isolates. Methods. A total of 34 carbapenem-resistant P. aeruginosa clinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE) were performed. Results. All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC) > 256 mg/L). Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrum β-lactamase (VEB-1) (n = 16/34) and oxacillinase (OXA-10) (n = 14/34) were the most prevalent extended-spectrum β-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28) and imipenemase (IMP-7) variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD) expression was seen in nine isolates, and an increase in efflux pump gene (MexAB) expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15) were found among the 34 isolates. The predominant serotype was O:11 (16 isolates), followed by O:15 (nine isolates). PFGE analysis of the 34 carbapenem-resistant P. aeruginosa isolates revealed 14 different pulsotypes. Conclusions. These results revealed diverse mechanisms conferring carbapenem resistance to P. aeruginosa isolates from Saudi Arabia.

  2. Diversity of Molecular Mechanisms Conferring Carbapenem Resistance to Pseudomonas aeruginosa Isolates from Saudi Arabia

    PubMed Central

    Jeannot, Katy; El-Mahdy, Taghrid S.; Samaha, Hassan A.; Shibl, Atef M.; Plésiat, Patrick; Courvalin, Patrice

    2016-01-01

    Background. This study described various molecular and epidemiological characters determining antibiotic resistance patterns in Pseudomonas aeruginosa isolates. Methods. A total of 34 carbapenem-resistant P. aeruginosa clinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE) were performed. Results. All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC) > 256 mg/L). Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrum β-lactamase (VEB-1) (n = 16/34) and oxacillinase (OXA-10) (n = 14/34) were the most prevalent extended-spectrum β-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28) and imipenemase (IMP-7) variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD) expression was seen in nine isolates, and an increase in efflux pump gene (MexAB) expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15) were found among the 34 isolates. The predominant serotype was O:11 (16 isolates), followed by O:15 (nine isolates). PFGE analysis of the 34 carbapenem-resistant P. aeruginosa isolates revealed 14 different pulsotypes. Conclusions. These results revealed diverse mechanisms conferring carbapenem resistance to P. aeruginosa isolates from Saudi Arabia. PMID:27597874

  3. Apoptosis-related genes confer resistance to Fusarium wilt in transgenic 'Lady Finger' bananas.

    PubMed

    Paul, Jean-Yves; Becker, Douglas K; Dickman, Martin B; Harding, Robert M; Khanna, Harjeet K; Dale, James L

    2011-12-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

  4. Superbugs and Superdrugs-SMi's 12th annual conference--Overcoming resistance. 17-18 March 2010, London, UK.

    PubMed

    Oni, Adekemi

    2010-05-01

    The 12th Annual Superbugs and Superdrugs conference, held in London, included topics covering new therapeutic developments in the field of antimicrobial research. This conference report highlights selected presentations on antimicrobial peptides, addressing bacterial resistance, and new treatments for bacterial infections. Investigational drugs discussed include DPK-060 (DermaGen AB), DAV-132 (Da Volterra), PF-4287881, PNU-100480 and PF-02538084 (all Pfizer Inc), BAL-30072 (Basilea Pharmaceutica International Ltd) and lanbiotics from Novacta.

  5. Combination of essential oils and antibiotics reduce antibiotic resistance in plasmid-conferred multidrug resistant bacteria.

    PubMed

    Yap, Polly Soo Xi; Lim, Swee Hua Erin; Hu, Cai Ping; Yiap, Beow Chin

    2013-06-15

    In this study we investigated the relationship between several selected commercially available essential oils and beta-lactam antibiotics on their antibacterial effect against multidrug resistant bacteria. The antibacterial activity of essential oils and antibiotics was assessed using broth microdilution. The combined effects between essential oils of cinnamon bark, lavender, marjoram, tea tree, peppermint and ampicillin, piperacillin, cefazolin, cefuroxime, carbenicillin, ceftazidime, meropenem, were evaluated by means of the checkerboard method against beta-lactamase-producing Escherichia coli. In the latter assays, fractional inhibitory concentration (FIC) values were calculated to characterize interaction between the combinations. Substantial susceptibility of the bacteria toward natural antibiotics and a considerable reduction in the minimum inhibitory concentrations (MIC) of the antibiotics were noted in some paired combinations of antibiotics and essential oils. Out of 35 antibiotic-essential oil pairs tested, four of them showed synergistic effect (FIC≤0.5) and 31 pairs showed no interaction (FIC>0.5-4.0). The preliminary results obtained highlighted the occurrence of a pronounced synergistic relationship between piperacillin/cinnamon bark oil, piperacillin/lavender oil, piperacillin/peppermint oil as well as meropenem/peppermint oil against two of the three bacteria under study with a FIC index in the range 0.26-0.5. The finding highlighted the potential of peppermint, cinnamon bark and lavender essential oils being as antibiotic resistance modifying agent. Reduced usage of antibiotics could be employed as a treatment strategy to decrease the adverse effects and possibly to reverse the beta-lactam antibiotic resistance.

  6. The Tomato Yellow Leaf Curl Virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases.

    PubMed

    Verlaan, Maarten G; Hutton, Samuel F; Ibrahem, Ragy M; Kormelink, Richard; Visser, Richard G F; Scott, John W; Edwards, Jeremy D; Bai, Yuling

    2013-03-01

    Tomato Yellow Leaf Curl Virus Disease incited by Tomato yellow leaf curl virus (TYLCV) causes huge losses in tomato production worldwide and is caused by different related begomovirus species. Breeding for TYLCV resistance has been based on the introgression of multiple resistance genes originating from several wild tomato species. In this study we have fine-mapped the widely used Solanum chilense-derived Ty-1 and Ty-3 genes by screening nearly 12,000 plants for recombination events and generating recombinant inbred lines. Multiple molecular markers were developed and used in combination with disease tests to fine-map the genes to a small genomic region (approximately 70 kb). Using a Tobacco Rattle Virus-Virus Induced Gene Silencing approach, the resistance gene was identified. It is shown that Ty-1 and Ty-3 are allelic and that they code for a RNA-dependent RNA polymerase (RDR) belonging to the RDRγ type, which has an atypical DFDGD motif in the catalytic domain. In contrast to the RDRα type, characterized by a catalytic DLDGD motif, no clear function has yet been described for the RDRγ type, and thus the Ty-1/Ty-3 gene unveils a completely new class of resistance gene. Although speculative, the resistance mechanism of Ty-1/Ty-3 and its specificity towards TYLCV are discussed in light of the function of the related RDRα class in the amplification of the RNAi response in plants and transcriptional silencing of geminiviruses in plants. PMID:23555305

  7. A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat.

    PubMed

    Moore, John W; Herrera-Foessel, Sybil; Lan, Caixia; Schnippenkoetter, Wendelin; Ayliffe, Michael; Huerta-Espino, Julio; Lillemo, Morten; Viccars, Libby; Milne, Ricky; Periyannan, Sambasivam; Kong, Xiuying; Spielmeyer, Wolfgang; Talbot, Mark; Bariana, Harbans; Patrick, John W; Dodds, Peter; Singh, Ravi; Lagudah, Evans

    2015-12-01

    As there are numerous pathogen species that cause disease and limit yields of crops, such as wheat (Triticum aestivum), single genes that provide resistance to multiple pathogens are valuable in crop improvement. The mechanistic basis of multi-pathogen resistance is largely unknown. Here we use comparative genomics, mutagenesis and transformation to isolate the wheat Lr67 gene, which confers partial resistance to all three wheat rust pathogen species and powdery mildew. The Lr67 resistance gene encodes a predicted hexose transporter (LR67res) that differs from the susceptible form of the same protein (LR67sus) by two amino acids that are conserved in orthologous hexose transporters. Sugar uptake assays show that LR67sus, and related proteins encoded by homeoalleles, function as high-affinity glucose transporters. LR67res exerts a dominant-negative effect through heterodimerization with these functional transporters to reduce glucose uptake. Alterations in hexose transport in infected leaves may explain its ability to reduce the growth of multiple biotrophic pathogen species.

  8. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure.

    PubMed

    Unemo, Magnus; Golparian, Daniel; Nicholas, Robert; Ohnishi, Makoto; Gallay, Anne; Sednaoui, Patrice

    2012-03-01

    Recently, the first Neisseria gonorrhoeae strain (H041) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type 1901 (ST1901) and NG-MAST ST1407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/ml) and ceftriaxone (MIC = 1 to 2 μg/ml) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A501P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management.

  9. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure.

    PubMed

    Unemo, Magnus; Golparian, Daniel; Nicholas, Robert; Ohnishi, Makoto; Gallay, Anne; Sednaoui, Patrice

    2012-03-01

    Recently, the first Neisseria gonorrhoeae strain (H041) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type 1901 (ST1901) and NG-MAST ST1407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/ml) and ceftriaxone (MIC = 1 to 2 μg/ml) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A501P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management. PMID:22155830

  10. Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis.

    PubMed

    Ruhland, Aaron; Leal, Nicole; Kima, Peter E

    2007-01-01

    Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NFkappaB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFkappaB signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.

  11. Overcoming MITF-conferred drug resistance through dual AURKA/MAPK targeting in human melanoma cells

    PubMed Central

    Pathria, G; Garg, B; Borgdorff, V; Garg, K; Wagner, C; Superti-Furga, G; Wagner, S N

    2016-01-01

    MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma. PMID:26962685

  12. Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.

    PubMed Central

    Zimmerman, P. A.; Buckler-White, A.; Alkhatib, G.; Spalding, T.; Kubofcik, J.; Combadiere, C.; Weissman, D.; Cohen, O.; Rubbert, A.; Lam, G.; Vaccarezza, M.; Kennedy, P. E.; Kumaraswami, V.; Giorgi, J. V.; Detels, R.; Hunter, J.; Chopek, M.; Berger, E. A.; Fauci, A. S.; Nutman, T. B.; Murphy, P. M.

    1997-01-01

    BACKGROUND: CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus-1 (HIV-1). Recently, an inactive CCR5 allele (designated here as CCR5-2) was identified that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. The reports conflict on the effect of heterozygous CCR5-2 on HIV-1 susceptibility, and race and risk levels have not yet been fully analyzed. Here we report our independent identification of CCR5-2 and test its effects on HIV-1 pathogenesis in individuals with contrasting clinical outcomes, defined race, and quantified risk. MATERIALS AND METHODS: Mutant CCR5 alleles were sought by directed heteroduplex analysis of genomic DNA from random blood donors. Genotypic frequencies were then determined in (1) random blood donors from North America, Asia, and Africa; (2) HIV-1+ individuals; and (3) highly exposed-seronegative homosexuals with quantified risk. RESULTS: CCR5-2 was the only mutant allele found. It was common in Caucasians, less common in other North American racial groups, and not detected in West Africans or Tamil Indians. Homozygous CCR5-2 frequencies differed reciprocally in highly exposed-seronegative (4.5%, n = 111) and HIV-1-seropositive (0%, n = 614) Caucasians relative to Caucasian random blood donors (0.8%, n = 387). This difference was highly significant (p < 0.0001). By contrast, heterozygous CCR5-2 frequencies did not differ significantly in the same three groups (21.6, 22.6, and 21.7%, respectively). A 55% increase in the frequency of heterozygous CCR5-2 was observed in both of two cohorts of Caucasian homosexual male, long-term nonprogressors compared with other HIV-1+ Caucasian homosexuals (p = 0.006) and compared with Caucasian random blood donors. Moreover, Kaplan-Meier estimates indicated that CCR5-2 heterozygous seroconvertors had a 52.6% lower risk of developing AIDS than homozygous wild-type seroconvertors

  13. The Hippo coactivator YAP1 mediates EGFR overexpression and confers chemo-resistance in esophageal cancer

    PubMed Central

    Song, Shumei; Honjo, Soichiro; Jin, Jiankang; Chang, Shih-Shin; Scott, Ailing W; Chen, Qiongrong; Kalhor, Neda; Correa, Arlene M.; Hofstetter, Wayne L.; Albarracin, Constance T.; Wu, Tsung-Teh; Johnson, Randy L.; Hung, Mien-Chie; Ajani, Jaffer A.

    2015-01-01

    Purpose Esophageal cancer (EC) is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of EC patients. However, clinical trials using EGFR inhibitors have not provided benefit for EC patients. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. Experimental Design In this study, expression of YAP1 and EGFR were examined in EAC resistant tumor tissues vs sensitive tissues by immunohistochemistry. Western blot, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis and in vitro and in vivo functional assays were performed to elucidate the YAP1 mediate EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. Results We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant EC tissues compared to sensitive EC tissues. Further, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-FU and docetaxcel, while knockdown of YAP sensitizes EC cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. Conclusions Our data provide evidence that YAP1 up-regulation of EGFR plays an important role in conferring therapy resistance in EC cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in EC. PMID:25739674

  14. A Balance between Inhibitor Binding and Substrate Processing Confers Influenza Drug Resistance.

    PubMed

    Jiang, Li; Liu, Ping; Bank, Claudia; Renzette, Nicholas; Prachanronarong, Kristina; Yilmaz, Lutfu S; Caffrey, Daniel R; Zeldovich, Konstantin B; Schiffer, Celia A; Kowalik, Timothy F; Jensen, Jeffrey D; Finberg, Robert W; Wang, Jennifer P; Bolon, Daniel N A

    2016-02-13

    The therapeutic benefits of the neuraminidase (NA) inhibitor oseltamivir are dampened by the emergence of drug resistance mutations in influenza A virus (IAV). To investigate the mechanistic features that underlie resistance, we developed an approach to quantify the effects of all possible single-nucleotide substitutions introduced into important regions of NA. We determined the experimental fitness effects of 450 nucleotide mutations encoding positions both surrounding the active site and at more distant sites in an N1 strain of IAV in the presence and absence of oseltamivir. NA mutations previously known to confer oseltamivir resistance in N1 strains, including H275Y and N295S, were adaptive in the presence of drug, indicating that our experimental system captured salient features of real-world selection pressures acting on NA. We identified mutations, including several at position 223, that reduce the apparent affinity for oseltamivir in vitro. Position 223 of NA is located adjacent to a hydrophobic portion of oseltamivir that is chemically distinct from the substrate, making it a hotspot for substitutions that preferentially impact drug binding relative to substrate processing. Furthermore, two NA mutations, K221N and Y276F, each reduce susceptibility to oseltamivir by increasing NA activity without altering drug binding. These results indicate that competitive expansion of IAV in the face of drug pressure is mediated by a balance between inhibitor binding and substrate processing.

  15. Cellular robustness conferred by genetic crosstalk underlies resistance against chemotherapeutic drug doxorubicin in fission yeast.

    PubMed

    Tay, Zoey; Eng, Ru Jun; Sajiki, Kenichi; Lim, Kim Kiat; Tang, Ming Yi; Yanagida, Mitsuhiro; Chen, Ee Sin

    2013-01-01

    Doxorubicin is an anthracycline antibiotic that is among one of the most commonly used chemotherapeutic agents in the clinical setting. The usage of doxorubicin is faced with many problems including severe side effects and chemoresistance. To overcome these challenges, it is important to gain an understanding of the underlying molecular mechanisms with regards to the mode of action of doxorubicin. To facilitate this aim, we identified the genes that are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe. We further demonstrated interplay between factors controlling various aspects of chromosome metabolism, mitochondrial respiration and membrane transport. In the nucleus we observed that the subunits of the Ino80, RSC, and SAGA complexes function in the similar epistatic group that shares significant overlap with the homologous recombination genes. However, these factors generally act in synergistic manner with the chromosome segregation regulator DASH complex proteins, possibly forming two major arms for regulating doxorubicin resistance in the nucleus. Simultaneous disruption of genes function in membrane efflux transport or the mitochondrial respiratory chain integrity in the mutants defective in either Ino80 or HR function resulted in cumulative upregulation of drug-specific growth defects, suggesting a rewiring of pathways that synergize only when the cells is exposed to the cytotoxic stress. Taken together, our work not only identified factors that are required for survival of the cells in the presence of doxorubicin but has further demonstrated that an extensive molecular crosstalk exists between these factors to robustly confer doxorubicin resistance.

  16. Human ABCB1 confers cells resistance to cytotoxic guanidine alkaloids from Pterogyne nitens.

    PubMed

    Satake, Kazuhiro; Tsukamoto, Megumi; Mitani, Yuji; Regasini, Luis Octavio; da Silva Bolzani, Vanderlan; Efferth, Thomas; Nakagawa, Hiroshi

    2015-01-01

    Multidrug resistance (MDR) caused by human ABCB1 (P-glycoprotein/MDR1) is one of the major obstacles in chemotherapy. To understand the mechanism of MDR by ABCB1 and circumvent the MDR, in the present study, we established human ABCB1-expressing cells (Flp-In-293/ABCB1 cells) and examined the cytotoxic effects of four guanidine alkaloids from Pterogyne nitens (galegine, nitensidine A, pterogynidine and pterogynine) using Flp-In-293/Mock and Flp-In-293/ABCB1 cells. The activity of ABCB1 in Flp-In-293/ABCB1 cells were confirmed by typical substrates for ABCB1 (taxol and vinblastine) in MTT assay. Flp-In-293/ABCB1 cells were also resistant to the four guanidine alkaloids as well as taxol and vinblastine compared to Flp-In-293/Mock cells although the four guanidine alkaloids exhibited cytotoxicity against the two Flp-In-293 cells. Furthermore, the four guanidine alkaloids were also found to stimulate the ATPase activity of ABCB1 in ATPase assays. These results suggest that ABCB1 can confer the resistance to the cytotoxic guanidine alkaloids by transporting them.

  17. NUMB inactivation confers resistance to imatinib in chronic myeloid leukemia cells.

    PubMed

    García-Alegría, Eva; Lafita-Navarro, M Carmen; Aguado, Rocío; García-Gutiérrez, Lucia; Sarnataro, Kyle; Ruiz-Herguido, Cristina; Martín, Francisco; Bigas, Anna; Canelles, Matilde; León, Javier

    2016-05-28

    Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase, where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and its derivatives. NUMB is an evolutionary well-conserved protein initially described as a functional antagonist of NOTCH function. NUMB is an endocytic protein associated with receptor internalization, involved in multiple cellular functions. It has been reported that MSI2 protein, a NUMB inhibitor, is upregulated in CML blast crisis, whereas NUMB itself is downregulated. This suggest that NUMB plays a role in the malignant progression of CML. Here we have generated K562 cells (derived from CML in blast crisis) constitutively expressing a dominant negative form of NUMB (dnNUMB). We show that dnNUMB expression confers a high proliferative phenotype to the cells. Importantly, dnNUMB triggers a partial resistance to imatinib in these cells, antagonizing the apoptosis mediated by the drug. Interestingly, imatinib resistance is not linked to p53 status or NOTCH signaling, as K562 lack p53 and imatinib resistance is reproduced in the presence of NOTCH inhibitors. Taken together, our data support the hypothesis that NUMB activation could be a new therapeutic target in CML.

  18. More about the Viking hypothesis of origin of the delta32 mutation in the CCR5 gene conferring resistance to HIV-1 infection.

    PubMed

    Lucotte, Gérard; Dieterlen, Florent

    2003-11-01

    The chemokine receptor CCR5 constitutes the major coreceptor for the HIV-1, because a mutant allele of the CCR5 gene named delta32 was shown to provide to homozygotes a strong resistance against infection. In the present study the frequency of the delta32 allele was collected in 36 European populations and in Cyprus, and the highest allele frequencies were found in Nordic countries. We constructed an allele map of delta32 frequencies in Europe; the map is in accordance to the Vikings hypothesis of the origin of the mutation and his dissemination during the eighth to the tenth centuries.

  19. Is the European spatial distribution of the HIV-1-resistant CCR5-Delta32 allele formed by a breakdown of the pathocenosis due to the historical Roman expansion?

    PubMed

    Faure, Eric; Royer-Carenzi, Manuela

    2008-12-01

    We studied the possible effects of the expansion of ancient Mediterranean civilizations during the five centuries before and after Christ on the European distribution of the mutant allele for the chemokine receptor gene CCR5 which has a 32-bp deletion (CCR5-Delta32). There is a strong evidence for the unitary origin of the CCR5-Delta32 mutation, this it is found principally in Europe and Western Asia, with generally a north-south downhill cline frequency. Homozygous carriers of this mutation show a resistance to HIV-1 infection and a slower progression towards AIDS. However, HIV has clearly emerged too recently to have been the selective force on CCR5. Our analyses showed strong negative correlations in Europe between the allele frequency and two historical parameters, i.e. the first colonization dates by the great ancient Mediterranean civilizations, and the distances from the Northern frontiers of the Roman Empire in its greatest expansion. Moreover, other studies have shown that the deletion frequencies in both German Bronze Age and Swedish Neolithic populations were similar to those found in the corresponding modern populations, and this deletion has been found in ancient DNA of around 7000 years ago, suggesting that in the past, the deletion frequency could have been relatively high in European populations. In addition, in West Nile virus pathogenesis, CCR5 plays an antimicrobial role showing that host genetic factors are highly pathogen-specific. Our results added to all these previous data suggest that the actual European allele frequency distribution might not be due to genes spreading, but to a negative selection resulting in the spread of pathogens principally during Roman expansion. Indeed, as gene flows from colonizers to European native populations were extremely low, the mutational changes might be associated with vulnerability to imported infections. To date, the nature of the parasites remains unknown; however, zoonoses could be incriminated.

  20. Mapping QTL conferring resistance in maize to gray leaf spot disease caused by Cercospora zeina

    PubMed Central

    2014-01-01

    Background Gray leaf spot (GLS) is a globally important foliar disease of maize. Cercospora zeina, one of the two fungal species that cause the disease, is prevalent in southern Africa, China, Brazil and the eastern corn belt of the USA. Identification of QTL for GLS resistance in subtropical germplasm is important to support breeding programmes in developing countries where C. zeina limits production of this staple food crop. Results A maize RIL population (F7:S6) from a cross between CML444 and SC Malawi was field-tested under GLS disease pressure at five field sites over three seasons in KwaZulu-Natal, South Africa. Thirty QTL identified from eleven field trials (environments) were consolidated to seven QTL for GLS resistance based on their expression in at least two environments and location in the same core maize bins. Four GLS resistance alleles were derived from the more resistant parent CML444 (bin 1.10, 4.08, 9.04/9.05, 10.06/10.07), whereas the remainder were from SC Malawi (bin 6.06/6.07, 7.02/7.03, 9.06). QTLs in bin 4.08 and bin 6.06/6.07 were also detected as joint QTLs, each explained more than 11% of the phenotypic variation, and were identified in four and seven environments, respectively. Common markers were used to allocate GLS QTL from eleven previous studies to bins on the IBM2005 map, and GLS QTL “hotspots” were noted. Bin 4.08 and 7.02/7.03 GLS QTL from this study overlapped with hotspots, whereas the bin 6.06/6.07 and bin 9.06 QTLs appeared to be unique. QTL for flowering time (bin 1.07, 4.09) in this population did not correspond to QTL for GLS resistance. Conclusions QTL mapping of a RIL population from the subtropical maize parents CML444 and SC Malawi identified seven QTL for resistance to gray leaf spot disease caused by C. zeina. These QTL together with QTL from eleven studies were allocated to bins on the IBM2005 map to provide a basis for comparison. Hotspots of GLS QTL were identified on chromosomes one, two, four, five and

  1. Diversity of mechanisms conferring resistance to β-lactams among OXA-23-producing Acinetobacter baumannii clones.

    PubMed

    Cardoso, Juliana Provasi; Cayô, Rodrigo; Girardello, Raquel; Gales, Ana Cristina

    2016-05-01

    A total of 31 unrelated OXA-23-producing Acinetobacter baumannii strains isolated from 14 hospitals located in distinct Brazilian regions were evaluated in this study. These isolates were grouped into 12 different sequence types (STs), of which 7 had unique allelic sequences (ST188, ST189, ST190, ST191, ST192, ST228, and ST299). Most isolates belonged to the clonal complex CC79 followed by CC15 and CC1. Only polymyxin B and minocycline showed good activity against the OXA-23-producing A. baumannii clones. The ISAba1 upstream blaOXA-23, blaOXA-51-like, or ampC was found in 100%, 54.8%, and 77.4% of the isolates, respectively. High resistance rates to ceftazidime and cefotaxime were observed among those isolates possessing ISAba1 upstream ampC, in contrast to those isolates that did not carry this configuration. Moreover, a ≥2 Log2 decrease in the MICs of meropenem and ceftazidime was observed in the presence of phenyl-arginine-β-naphthylamide for 80.6% and 54.8% of isolates, respectively. Overexpression of the adeB was observed in 61.3% of isolates, particularly among those isolates belonging to the ST1 (CC1). It was also verified that ompW was down-regulated in all isolates belonging to the ST15 (CC15). On the other hand, carO and omp33-36 genes were overexpressed in 48.4% and 58.1% of the isolates, respectively. In this study, we show that overexpression of AdeABC system could significantly contribute for resistance to meropenem and ceftazidime among OXA-23-producing A. baumannii clones in Brazil, demonstrating the complexity involved in the β-lactam resistance in such isolates. PMID:26971181

  2. Detection, introgression and localization of genes conferring specific resistance to Leptosphaeria maculans from Brassica rapa into B. napus.

    PubMed

    Leflon, M; Brun, H; Eber, F; Delourme, R; Lucas, M O; Vallée, P; Ermel, M; Balesdent, M H; Chèvre, A M

    2007-11-01

    Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of "new" resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.

  3. satG, Conferring Resistance to Streptogramin A, Is Widely Distributed in Enterococcus faecium Strains but Not in Staphylococci

    PubMed Central

    Haroche, Julien; Allignet, Jeanine; Aubert, Sylvie; Van Den Bogaard, Anthony E.; El Solh, Névine

    2000-01-01

    A gene almost identical to satG was isolated from an Enterococcus faecium strain. This gene was transferred to a Staphylococcus aureus recipient strain where it conferred resistance to streptogramin A. satG was found to be widely distributed among E. faecium strains but not detected among staphylococci. PMID:10602747

  4. Molecular mapping and characterization of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, a...

  5. Microsomal epoxide hydrolase (EPHX1), slow (exon 3, 113His) and fast (exon 4, 139Arg) alleles confer susceptibility to squamous cell esophageal cancer

    SciTech Connect

    Jain, Meenu; Tilak, Anup Raj; Upadhyay, Rohit; Kumar, Ashwani; Mittal, Balraj

    2008-07-15

    Genetic polymorphisms in xenobiotic metabolizing enzymes may alter risk of various cancers. Present case-control study evaluated the influence of EPHX1 genetic variations on squamous cell esophageal cancer (ESCC) susceptibility in 107 patients and 320 controls. EPHX1 polymorphic alleles were genotyped by direct sequencing (exon 3, Tyr113His) or PCR-RFLP (exon 4, His139Arg). Patients with exon 3 genotypes (Tyr113His, His113His) and 113His allele were at risk of ESCC (OR{sub Tyr113His} 2.0, 95% CI = 1.2-3.4, p = 0.007; OR{sub His113His} 2.3 95% CI = 1.0-5.2, p = 0.03 and OR{sub His} 1.5, 95% CI = 1.0-2.1, p = 0.01). In contrast, individuals with exon 4, 139Arg allele were at low risk of cancer (OR 0.34, 95% CI = 0.20-0.56, p = 0.001). However, none of haplotype combinations of exon 3 (Tyr113His) and exon 4 (His139Arg) polymorphisms showed modulation of risk for ESCC. Sub-grouping of patients based on anatomical location of tumor predicted that patients with exon 3, His113His and Tyr113His genotypes were at higher risk for developing ESCC tumor at upper and middle third locations (OR 4.4, 95% CI = 1.0-18.5, p = 0.04; OR 2.5, 95% CI = 1.3-5.0, p = 0.005 respectively). The frequency of exon 4, His139Arg genotype was significantly lower in ESCC patients with lower third tumor location as compared to controls (14.8% vs. 36.3%, p = 0.02). In case-only study, gene-environment interaction of EPHX1 genotypes with tobacco, alcohol and occupational exposures did not appear to modulate the cancer susceptibility. In conclusion, exon 3, Tyr113His genotype was associated with higher risk of ESCC particularly at upper and middle-third anatomical locations of tumor. However, His139Arg genotype of exon 4, exhibited low risk for ESCC as well as its clinical characteristics.

  6. Functional analysis of Pid3-A4, an ortholog of rice blast resistance gene Pid3 revealed by allele mining in common wild rice.

    PubMed

    Lv, Qiming; Xu, Xiao; Shang, Junjun; Jiang, Guanghuai; Pang, Zhiqian; Zhou, Zhuangzhi; Wang, Jing; Liu, Ya; Li, Ting; Li, Xiaobing; Xu, Jichen; Cheng, Zhukuan; Zhao, Xianfeng; Li, Shigui; Zhu, Lihuang

    2013-06-01

    The rice blast resistance gene Pid3 encodes a nucleotide-binding-site leucine-rich repeat (NBS-LRR) protein. This gene was cloned from the rice 'Digu' (indica) by performing a genome-wide comparison of the NBS-LRR gene family between two genome-sequenced varieties, '9311' (indica) and 'Nipponbare' (japonica). In this study, we performed functional analysis of Pid3-A4, an ortholog of Pid3 revealed by allele mining in the common wild rice A4 (Oryza rufipogon). The predicted protein encoded by Pid3-A4 shares 99.03% sequence identity with Pid3, with only nine amino-acid substitutions. In wild rice plants, Pid3-A4 is constitutively expressed, and its expression is not induced by Magnaporthe oryzae isolate Zhong-10-8-14 infection. Importantly, in transgenic plants, Pid3-A4, as compared with Pid3, displays a distinct resistance spectrum to a set of M. oryzae isolates, including those that prevail in the rice fields of Sichuan Province. Therefore, Pid3-A4 should be quite useful for the breeding of rice blast resistance, especially in southwestern China.

  7. RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and -susceptible alleles.

    PubMed

    Kim, Kil Hyun; Kang, Yang Jae; Kim, Dong Hyun; Yoon, Min Young; Moon, Jung-Kyung; Kim, Moon Young; Van, Kyujung; Lee, Suk-Ha

    2011-12-01

    Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean. PMID:21987089

  8. Activation of the Met kinase confers acquired drug resistance in FGFR-targeted lung cancer therapy.

    PubMed

    Kim, S-M; Kim, H; Yun, M R; Kang, H N; Pyo, K-H; Park, H J; Lee, J M; Choi, H M; Ellinghaus, P; Ocker, M; Paik, S; Kim, H R; Cho, B C

    2016-01-01

    Aberrant fibroblast growth factor receptor (FGFR) activation/expression is a common feature in lung cancer (LC). In this study, we evaluated the antitumor activity of and the mechanisms underlying acquired resistance to two potent selective FGFR inhibitors, AZD4547 and BAY116387, in LC cell lines. The antitumor activity of AZD4547 and BAY1163877 was screened in 24 LC cell lines, including 5 with FGFR1 amplification. Two cell lines containing FGFR1 amplifications, H1581 and DMS114, were sensitive to FGFR inhibitors (IC50<250 nm). Clones of FGFR1-amplified H1581 cells resistant to AZD4547 or BAY116387 (H1581AR and H1581BR cells, respectively) were established. Receptor tyrosine kinase (RTK) array and immunoblotting analyses showed strong overexpression and activation of Met in H1581AR/BR cells, compared with that in the parental cells. Gene set enrichment analysis against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that cytokine-cytokine receptor interaction pathways were significantly enriched in H1581AR/BR cells, with Met contributing significantly to the core enrichment. Genomic DNA quantitative PCR and fluorescent in situ hybridization analyses showed MET amplification in H1581AR, but not in H1581BR, cells. Met amplification drives acquired resistance to AZD4547 in H1581AR cells by activating ErbB3. Combination treatment with FGFR inhibitors and an anaplastic lymphoma kinase (ALK)/Met inhibitor, crizotinib, or Met-specific short interfering RNA (siRNA) synergistically inhibited cell proliferation in both H1581AR and H1581BR cells. Conversely, ectopic expression of Met in H1581 cells conferred resistance to AZD4547 and BAY1163877. Acquired resistance to FGFR inhibitors not only altered cellular morphology, but also promoted migration and invasion of resistant clones, in part by inducing epithelial-to-mesenchymal transition. Taken together, our data suggest that Met activation is sufficient to bypass dependency on FGFR signaling. Concurrent

  9. Rapid Microarray-Based Identification of Different mecA Alleles in Staphylococci

    PubMed Central

    Müller, Elke; Schwarz, Stefan; Hotzel, Helmut; Ehricht, Ralf

    2012-01-01

    To screen isolates and to identify mecA alleles, published mecA sequences were analyzed, and a microarray for the rapid discrimination of mecA alleles was designed. A GenBank analysis yielded 135 full-length gene sequences annotated as mecA. These sequences clustered into 32 different alleles corresponding to 28 unique amino acid sequences and to 15 distinct hybridization patterns on this microarray. A collection of 78 clinical and veterinary isolates of Staphylococcus spp. was characterized using this assay. Nine of the 15 expected patterns, as well as one as-yet-unknown pattern, were identified. These patterns were detected in various epidemic methicillin-resistant Staphylococcus aureus strains, in S. pseudintermedius, and in coagulase-negative species such as S. epidermidis, S. fleurettii, or S. haemolyticus. There was no correlation between the different mecA hybridization patterns and the SCCmec type. Determination of MICs showed that mecA alleles corresponding to only four of these nine patterns were associated with β-lactam resistance. The mecA alleles that did not confer β-lactam resistance were largely restricted to coagulase-negative staphylococci of animal origin, such as S. sciuri and S. vitulinus. Because of the diversity of sequences and the different impact on β-lactam susceptibility, the existence of different mecA alleles needs to be taken into account when designing diagnostic assays for the detection of mecA. PMID:22890767

  10. Rapid microarray-based identification of different mecA alleles in Staphylococci.

    PubMed

    Monecke, Stefan; Müller, Elke; Schwarz, Stefan; Hotzel, Helmut; Ehricht, Ralf

    2012-11-01

    To screen isolates and to identify mecA alleles, published mecA sequences were analyzed, and a microarray for the rapid discrimination of mecA alleles was designed. A GenBank analysis yielded 135 full-length gene sequences annotated as mecA. These sequences clustered into 32 different alleles corresponding to 28 unique amino acid sequences and to 15 distinct hybridization patterns on this microarray. A collection of 78 clinical and veterinary isolates of Staphylococcus spp. was characterized using this assay. Nine of the 15 expected patterns, as well as one as-yet-unknown pattern, were identified. These patterns were detected in various epidemic methicillin-resistant Staphylococcus aureus strains, in S. pseudintermedius, and in coagulase-negative species such as S. epidermidis, S. fleurettii, or S. haemolyticus. There was no correlation between the different mecA hybridization patterns and the SCCmec type. Determination of MICs showed that mecA alleles corresponding to only four of these nine patterns were associated with β-lactam resistance. The mecA alleles that did not confer β-lactam resistance were largely restricted to coagulase-negative staphylococci of animal origin, such as S. sciuri and S. vitulinus. Because of the diversity of sequences and the different impact on β-lactam susceptibility, the existence of different mecA alleles needs to be taken into account when designing diagnostic assays for the detection of mecA.

  11. Lung-specific loss of the laminin α3 subunit confers resistance to mechanical injury.

    PubMed

    Urich, Daniela; Eisenberg, Jessica L; Hamill, Kevin J; Takawira, Desire; Chiarella, Sergio E; Soberanes, Saul; Gonzalez, Angel; Koentgen, Frank; Manghi, Tomas; Hopkinson, Susan B; Misharin, Alexander V; Perlman, Harris; Mutlu, Gokhan M; Budinger, G R Scott; Jones, Jonathan C R

    2011-09-01

    Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of α3 laminin (Lama3(fl/fl)), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3(fl/fl) mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of α3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the α3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the α3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of α3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of α3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury. PMID:21878500

  12. Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP-1 and p38.

    PubMed

    Escolano, Amelia; Martínez-Martínez, Sara; Alfranca, Arántzazu; Urso, Katia; Izquierdo, Helena M; Delgado, Mario; Martín, Francisco; Sabio, Guadalupe; Sancho, David; Gómez-del Arco, Pablo; Redondo, Juan Miguel

    2014-05-16

    Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti-inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN-targeted macrophages or direct injection of LxVP-encoding lentivirus has anti-inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP-1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN-inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti-inflammatory phenotype of CN-targeted macrophages, and mice with defective p38-activation were resistant to the anti-inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti-inflammatory status.

  13. Crp-dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis.

    PubMed

    Fu, Huihui; Chen, Haijiang; Wang, Jixuan; Zhou, Guangqi; Zhang, Haiyan; Zhang, Lili; Gao, Haichun

    2013-08-01

    Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (cyclic AMP receptor protein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp-independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.

  14. Loss of the RNA polymerase III repressor MAF1 confers obesity resistance

    PubMed Central

    Bonhoure, Nicolas; Byrnes, Ashlee; Moir, Robyn D.; Hodroj, Wassim; Preitner, Frédéric; Praz, Viviane; Marcelin, Genevieve; Chua, Streamson C.; Martinez-Lopez, Nuria; Singh, Rajat; Moullan, Norman; Auwerx, Johan; Willemin, Gilles; Shah, Hardik; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Kurland, Irwin

    2015-01-01

    MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1−/− mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1−/− mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD+, and is associated with obesity resistance. Consistent with this, NAD+ levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. PMID:25934505

  15. Characterization of a Variant of vga(A) Conferring Resistance to Streptogramin A and Related Compounds

    PubMed Central

    Haroche, Julien; Allignet, Jeanine; Buchrieser, Carmen; El Solh, Névine

    2000-01-01

    A variant of the vga(A) gene (1,575 bp), encoding an ATP-binding cassette protein conferring resistance to streptogramin A and related antibiotics, was cloned from the chromosome of a Staphylococcus aureus clinical isolate and sequenced. The sequence of the variant was similar to that of the vga(A) gene (83.2% identity). However, the G+C content of the variant (35.6%) was higher than that of vga(A) (29%) and there was no cross hybridization between vga(A) and the variant at high stringency (≥60°C), the highest temperature at which a signal was detected being 55°C. Unlike previous reports for vga(A) and vga(B), the variant of vga(A) may be present in multiple copies in the genome. These copies are chromosomal in some isolates and both chromosomal and plasmid-borne in others. Nucleotide sequences hybridizing at 65°C with the vga(A) variant were found in all the staphylococcal strains harboring plasmids carrying both vga(B) and vat(B), which also encode resistance to streptogramin A. PMID:10952567

  16. Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death

    PubMed Central

    Narayanan, Kannan Badri; Ali, Manaf; Barclay, Barry J.; Cheng, Qiang (Shawn); D’Abronzo, Leandro; Dornetshuber-Fleiss, Rita; Ghosh, Paramita M.; Gonzalez Guzman, Michael J.; Lee, Tae-Jin; Leung, Po Sing; Li, Lin; Luanpitpong, Suidjit; Ratovitski, Edward; Rojanasakul, Yon; Romano, Maria Fiammetta; Romano, Simona; Sinha, Ranjeet K.; Yedjou, Clement; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G.; Ryan, Elizabeth P.; Colacci, Anna Maria; Hamid, Roslida A.; Mondello, Chiara; Raju, Jayadev; Salem, Hosni K.; Woodrick, Jordan; Scovassi, A.Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Kim, Seo Yun; Bisson, William H.; Lowe, Leroy; Park, Hyun Ho

    2015-01-01

    Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis. PMID:26106145

  17. Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP-1 and p38

    PubMed Central

    Escolano, Amelia; Martínez-Martínez, Sara; Alfranca, Arántzazu; Urso, Katia; Izquierdo, Helena M; Delgado, Mario; Martín, Francisco; Sabio, Guadalupe; Sancho, David; Gómez-del Arco, Pablo; Redondo, Juan Miguel

    2014-01-01

    Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti-inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN-targeted macrophages or direct injection of LxVP-encoding lentivirus has anti-inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP-1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN-inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti-inflammatory phenotype of CN-targeted macrophages, and mice with defective p38-activation were resistant to the anti-inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti-inflammatory status. PMID:24596247

  18. Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

    PubMed

    Bonhoure, Nicolas; Byrnes, Ashlee; Moir, Robyn D; Hodroj, Wassim; Preitner, Frédéric; Praz, Viviane; Marcelin, Genevieve; Chua, Streamson C; Martinez-Lopez, Nuria; Singh, Rajat; Moullan, Norman; Auwerx, Johan; Willemin, Gilles; Shah, Hardik; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Kurland, Irwin; Hernandez, Nouria; Willis, Ian M

    2015-05-01

    MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. PMID:25934505

  19. HDAC6 promotes cell proliferation and confers resistance to temozolomide in glioblastoma.

    PubMed

    Wang, Zhihao; Hu, Pengchao; Tang, Fang; Lian, Haiwei; Chen, Xiong; Zhang, Yingying; He, Xiaohua; Liu, Wanhong; Xie, Conghua

    2016-08-28

    Histone deacetylases are considered to be among the most promising targets in drug development for cancer therapy. Histone deacetylase 6 (HDAC6) is a unique cytoplasmic enzyme that regulates many biological processes involved in tumorigenesis through its deacetylase and ubiquitin-binding activities. Here, we report that HDAC6 is overexpressed in glioblastoma tissues and cell lines. Overexpression of HDAC6 promotes the proliferation and spheroid formation of glioblastoma cells. HDAC6 overexpression confers resistance to temozolomide (TMZ) mediated cell proliferation inhibition and apoptosis induction. Conversely, knockdown of HDAC6 inhibits cell proliferation, impairs spheroid formation and sensitizes glioblastoma cells to TMZ. The inhibition of HDAC6 deacetylase activity by selective inhibitors inhibits the proliferation of glioblastoma cells and induces apoptosis. HDAC6 selective inhibitors can sensitize glioblastoma cells to TMZ. Moreover, we showed that HDAC6 mediated EGFR stabilization might partly account for its oncogenic role in glioblastoma. TMZ resistant glioblastoma cells showed higher expression of HDAC6 and more activation of EGFR. HDAC6 inhibitors decrease EGFR protein levels and impair the activation of the EGFR pathway. Taken together, our results suggest that the inhibition of HDAC6 may be a promising strategy for the treatment of glioblastoma.

  20. HPV-18 confers resistance to TNF-{alpha} in organotypic cultures of human keratinocytes

    SciTech Connect

    Boccardo, Enrique . E-mail: eboccardo@ludwig.org.br; Noya, Francisco; Broker, Thomas R.; Chow, Louise T.; Villa, Luisa L.

    2004-10-25

    The proinflammatory cytokine tumor necrosis factor-alpha (TNF-{alpha}) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-{alpha} antiproliferative effect. The present study analyzes the effects of TNF-{alpha} on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-{alpha}. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-{alpha} cultures transfected with HPV-18 whole genome showed proliferation in all cell strata. Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-{alpha}. Besides, TNF-{alpha} treatment did not alter p16{sup ink4a}, p21{sup cip1}, p27{sup kip1}, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.

  1. Heritable variation in the foliar secondary metabolite sideroxylonal in Eucalyptus confers cross-resistance to herbivores.

    PubMed

    Andrew, Rose L; Wallis, Ian R; Harwood, Chris E; Henson, Michael; Foley, William J

    2007-10-01

    Plants encounter a broad range of natural enemies and defend themselves in diverse ways. The cost of defense can be reduced if a plant secondary metabolite confers resistance to multiple herbivores. However, there are few examples of positively correlated defenses in plants against herbivores of different types. We present evidence that a genetically variable chemical trait that acts as a strong antifeedant to mammalian herbivores of Eucalyptus also deters insect herbivores, suggesting a possible mechanism for cross-resistance. We provide field confirmation that sideroxylonal, an important antifeedant for mammalian herbivores, also determines patterns of damage by Christmas beetles, a specialist insect herbivore of Eucalyptus. In a genetic progeny trial of Eucalyptus tricarpa, we found significant heritabilities of sideroxylonal concentration (0.60), overall insect damage (0.34), and growth traits (0.30-0.53). Population of origin also had a strong effect on each trait. Negative phenotypic correlations were observed between sideroxylonal and damage, and between damage and growth. No relationship was observed between sideroxylonal concentration and any growth trait. Our results suggest that potential for evolution by natural selection of sideroxylonal concentrations is not strongly constrained by growth costs and that both growth and defense traits can be successfully incorporated into breeding programs for plantation trees.

  2. Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

    PubMed Central

    Biadglegne, Fantahun; Tessema, Belay; Rodloff, Arne C.; Sack, Ulrich

    2013-01-01

    Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients. Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType® MTBDRplus and GenoType® MTBDRsl assay. Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay. Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was

  3. Two Non-target Recessive Genes Confer Resistance to the Anti-Oomycete Microtubule Inhibitor Zoxamide in Phytophthora capsici

    PubMed Central

    Cai, Meng; Zhu, Shusheng; Pang, Zhili; Liu, Xili

    2014-01-01

    This study characterized isolates of P. capsici that had developed a novel mechanism of resistance to zoxamide, which altered the minimum inhibition concentration (MIC) but not the EC50. Molecular analysis revealed that the β-tubulin gene of the resistant isolates contained no mutations and was expressed at the same level as in zoxamide-sensitive isolates. This suggested that P. capsici had developed a novel non-target-site-based resistance to zoxamide. Analysis of the segregation ratio of zoxamide-resistance in the sexual progeny of the sensitive isolates PCAS1 and PCAS2 indicated that the resistance to zoxamide was controlled by one or more recessive nuclear genes. Furthermore, the segregation of resistance in the F1, F2, and BC1 progeny was in accordance with the theoretical ratios of the χ2 test (P>0.05), which suggested that the resistance to zoxamide was controlled by two recessive genes, and that resistance to zoxamide occurred when at least one pair of these alleles was homozygous. This implies that the risk of zoxamide-resistance in P. capsici is low to moderate. Nevertheless this potential for resistance should be monitored closely, especially if two compatible mating types co-exist in the same field. PMID:24586697

  4. Analysis of proteins responsive to acetic acid in Acetobacter: molecular mechanisms conferring acetic acid resistance in acetic acid bacteria.

    PubMed

    Nakano, Shigeru; Fukaya, Masahiro

    2008-06-30

    Acetic acid bacteria are used for industrial vinegar production because of their remarkable ability to oxidize ethanol and high resistance to acetic acid. Although several molecular machineries responsible for acetic acid resistance in acetic acid bacteria have been reported, the entire mechanism that confers acetic acid resistance has not been completely understood. One of the promising methods to elucidate the entire mechanism is global analysis of proteins responsive to acetic acid by two-dimensional gel electrophoresis. Recently, two proteins whose production was greatly enhanced by acetic acid in Acetobacter aceti were identified to be aconitase and a putative ABC-transporter, respectively; furthermore, overexpression or disruption of the genes encoding these proteins affected acetic acid resistance in A. aceti, indicating that these proteins are involved in acetic acid resistance. Overexpression of each gene increased acetic acid resistance in Acetobacter, which resulted in an improvement in the productivity of acetic acid fermentation. Taken together, the results of the proteomic analysis and those of previous studies indicate that acetic acid resistance in acetic acid bacteria is conferred by several mechanisms. These findings also provide a clue to breed a strain having high resistance to acetic acid for vinegar fermentation.

  5. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode.

    PubMed

    Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; Zhu, Junwei J; Zhuang, Xiaofeng; Liu, Wusheng; Pantalone, Vincent R; Arelli, Prakash R; Stewart, Charles N; Chen, Feng

    2013-12-01

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

  6. WRR4 encodes a TIR-NB-LRR protein that confers broad-spectrum white rust resistance in Arabidopsis thaliana to four physiological races of Albugo candida.

    PubMed

    Borhan, M Hossein; Gunn, Nick; Cooper, Abigail; Gulden, Sigrun; Tör, Mahmut; Rimmer, S Roger; Holub, Eric B

    2008-06-01

    White blister rust in the Brassicaceae is emerging as a superb model for exploring how plant biodiversity has channeled speciation of biotrophic parasites. The causal agents of white rust across a wide breadth of cruciferous hosts currently are named as variants of a single oomycete species, Albugo candida. The most notable examples include a major group of physiological races that each are economically destructive in a different vegetable or oilseed crop of Brassica juncea (A. candida race 2), B. rapa (race 7), or B. oleracea (race 9); or parasitic on wild crucifers such as Capsella bursa-pastoris (race 4). Arabidopsis thaliana is innately immune to these races of A. candida under natural conditions; however, it commonly hosts its own molecularly distinct subspecies of A. candida (A. candida subsp. arabidopsis). In the laboratory, we have identified several accessions of Arabidopsis thaliana (e.g.,. Ws-3) that can permit varying degrees of rust development following inoculation with A. candida races 2, 4, and 7, whereas race 9 is universally incompatible in Arabidopsis thaliana and nonrusting resistance is the most prevalent outcome of interactions with the other races. Subtle variation in resistance phenotypes is evident, observed initially with an isolate of A. candida race 4, indicating additional genetic variation. Therefore, we used the race 4 isolate for map-based cloning of the first of many expected white rust resistance (WRR) genes. This gene was designated WRR4 and encodes a cytoplasmic toll-interleukin receptor-like nucleotide-binding leucine-rich repeat receptor-like protein that confers a dominant, broad-spectrum white rust resistance in the Arabidopsis thaliana accession Columbia to representative isolates of A. candida races 2, 4, 7, and 9, as verified by transgenic expression of the Columbia allele in Ws-3. The WRR4 protein requires functional expression of the lipase-like protein EDS1 but not the paralogous protein PAD4, and confers full immunity

  7. A Methyl Viologen-Resistant Mutant of Arabidopsis, Which Is Allelic to Ozone-Sensitive rcd1, Is Tolerant to Supplemental Ultraviolet-B Irradiation1

    PubMed Central

    Fujibe, Takahiro; Saji, Hikaru; Arakawa, Keita; Yabe, Naoto; Takeuchi, Yuichi; Yamamoto, Kotaro T.

    2004-01-01

    To better understand the role of active oxygen species (AOS) in acquired resistance to increased levels of ultraviolet (UV)-B irradiation in plants, we isolated an Arabidopsis mutant that is resistant to methyl viologen, and its sensitivity to UV-B was investigated. A complementation test revealed that the obtained mutant was allelic to the ozone-sensitive radical-induced cell death1-1 (rcd1-1). Therefore, this mutant was named rcd1-2. rcd1-2 was recessive and nearly 4-fold more resistant to methyl viologen than wild type. It exhibited a higher tolerance to short-term UV-B supplementation treatments than the wild type: UV-B-induced formation of cyclobutane pyrimidine dimers was reduced by one-half after 24 h of exposure; the decrease in quantum yield of photosystem II was also diminished by 40% after 12 h of treatment. Furthermore, rcd1-2 was tolerant to freezing. Steady-state mRNA levels of plastidic Cu/Zn superoxide dismutase and stromal ascorbate peroxidase were higher in rcd1-2 than in wild type, and the mRNA level of the latter enzyme was enhanced by UV-B exposure more effectively in rcd1-2. UV-B-absorbing compounds were more accumulated in rcd1-2 than in wild type after UV-B exposure for 24 h. These findings suggest that rcd1-2 methyl viologen resistance is due to the enhanced activities of the AOS-scavenging enzymes in chloroplasts and that the acquired tolerance to the short-term UV-B exposure results from a higher accumulation of sunscreen pigments. rcd1 appears to be a mutant that constitutively shows stress responses, leading to accumulation of more pigments and AOS-scavenging enzymes without any stresses. PMID:14657410

  8. A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference.

    PubMed

    Takahashi, Masaki; Hohjoh, Hirohiko

    2014-11-01

    Allele-specific silencing by RNA interference (ASP-RNAi) is an atypical RNAi that is capable of discriminating target alleles from non-target alleles, and may be therapeutically useful for specific inhibition of disease-causing alleles without affecting their corresponding normal alleles. However, it is difficult to design and select small interfering RNA (siRNAs) that confer ASP-RNAi. A major problem is that there are few appropriate measures in determining optimal allele-specific siRNAs. Here we show two novel formulas for calculating a new measure of allele-discrimination, named "ASP-score". The formulas and ASP-score allow for an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs.

  9. Concordance between allele-specific PCR and ultra-deep pyrosequencing for the detection of HIV-1 non-nucleoside reverse transcriptase inhibitor resistance mutations

    PubMed Central

    Hunt, Gillian M; Morris, Lynn; Moorthy, Anitha; Coovadia, Ashraf; Abrams, Elaine J; Strehlau, Renate; Kuhn, Louise; Persaud, Deborah

    2014-01-01

    Recent advances in genotyping technologies have allowed for detection of HIV-1 drug resistance mutations present at low levels. The presence and percentage of Y181C and K103N drug-resistant variants in the blood of 105 subtype C HIV-infected infants who failed single-dose nevirapine prophylaxis for HIV transmission were compared using two highly sensitive genotyping methods, allele-specific PCR (AS-PCR) and ultra-deep pyrosequencing. Significant correlations in detection between both methods were found for both Y181C (correlation coefficients of 0.94 [95% CI 0.91-0.96]) and K103N (0.89 [95% CI 0.84 – 0.92]) mutations. The majority of discordant specimens (3/5 Y181C and 8/11 K103N) had wild-type variants when population sequencing was used, but mutant variants were detectable at very low levels (≤5%) with either assay. This difference is most likely due to stochastic variations in the appearance of mutant variants. Overall, both AS-PCR and ultra-deep pyrosequencing methods have proven to be sensitive and accurate, and may confidently be used where feasible. PMID:25034127

  10. Loss-of-Function Mutations in CsMLO1 Confer Durable Powdery Mildew Resistance in Cucumber (Cucumis sativus L.).

    PubMed

    Nie, Jingtao; Wang, Yunli; He, Huanle; Guo, Chunli; Zhu, Wenying; Pan, Jian; Li, Dandan; Lian, Hongli; Pan, Junsong; Cai, Run

    2015-01-01

    Powdery mildew (PM) is a serious fungal disease of cucumber worldwide. The identification of resistance genes is very important for resistance breeding to ensure cucumber production. Here, natural loss-of-function mutations at an MLO homologous locus, CsMLO1, were found to confer durable PM resistance in cucumber. CsMLO1 encoded a cell membrane protein, was mainly expressed in leaves and cotyledons, and was up-regulated by PM at the early stage of host-pathogen interaction. Ectopic expression of CsMLO1 rescued the phenotype of the PM resistant Atmlo2 Atmlo12 double mutant to PM susceptible in Arabidopsis. Domesticated and wild resistant cucumbers originating from various geographical regions of the world were found to harbor three independent natural mutations that resulted in CsMLO1 loss of function. In addition, between the near-isogenic lines (NILs) of PM resistant and susceptible, S1003 and NIL(Pm5.1), quantitative RT-PCR revealed that there is no difference at expression levels of several genes in the pathways of ethylene, jasmonic acid or salicylic acid. Moreover, the two NILs were used for transcriptome profiling to explore the mechanism underlying the resistance. Several genes correlated with plant cell wall thickening are possibly involved in the PM resistance. This study revealed that loss of function of CsMLO1 conferred durable PM resistance, and that this loss of function is necessary but alone may not be sufficient for PM resistance in cucumber. These findings will facilitate the molecular breeding of PM resistant varieties to control this destructive disease in cucumber.

  11. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. PMID:26464158

  12. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations.

  13. Fine mapping of QTL and genomic prediction using allele-specific expression SNPs demonstrates that the complex trait of genetic resistance to Marek’s disease is predominantly determined by transcriptional regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hypothesis that polymorphisms associated with transcriptional regulation are critical for viral disease resistance was tested by selecting birds using SNPs exhibiting allele-specific expression (ASE) in response to viral challenge. Analysis indicates ASE markers account for 83% of the disease re...

  14. Genome-wide identification and quantification of cis- and trans-regulated genes responding to Marek's disease virus infection via analysis of allele-specific expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Marek’s disease (MD) is a commercially important neoplastic disease of chickens caused by the Marek’s disease virus (MDV), a naturally-occurring oncogenic alphaherpesvirus. We attempted to identify genes conferring MD resistance, by completing a genome-wide screen for allele-specific expr...

  15. Irradiation-resistance conferred by superoxide dismutase: possible adaptive role of a natural polymorphism in Drosophila melanogaster

    SciTech Connect

    Peng, T.X.; Moya, A.; Ayala, F.J.

    1986-02-01

    The toxic effects of ionizing radiation to DNA are thought to be due to the generation of the superoxide radical, 02-. Superoxide dismutase (SOD), which scavenges 02-., has been invoked as a protecting enzyme against ionizing radiation in viruses, bacteria, mammalian cells in culture, and live mice. We now demonstrate that SOD is involved in the resistance of Drosophila melanogaster against irradiation. The protection is greatest when flies carry the S form of the enzyme (which exhibits highest in vitro specific activity), intermediate when they carry the F form of the enzyme, and lowest when they are homozygous for N, an allele that reduces the amount of the enzyme to 3.5% of the normal level. Natural selection experiments show that the fitness of the high-activity S allele is increased in an irradiated population relative to the nonirradiated control. These results point towards a possible adaptive function of the S/F polymorphism found in natural populations of D. melanogaster.

  16. The Brassica napus receptor-like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus.

    PubMed

    Larkan, Nicholas J; Ma, Lisong; Borhan, Mohammad Hossein

    2015-09-01

    Leucine-rich repeat receptor-like proteins (LRR-RLPs) are highly adaptable parts of the signalling apparatus for extracellular detection of plant pathogens. Resistance to blackleg disease of Brassica spp. caused by Leptosphaeria maculans is largely governed by host race-specific R-genes, including the LRR-RLP gene LepR3. The blackleg resistance gene Rlm2 was previously mapped to the same genetic interval as LepR3. In this study, the LepR3 locus of the Rlm2 Brassica napus line 'Glacier DH24287' was cloned, and B. napus transformants were analysed for recovery of the Rlm2 phenotype. Multiple B. napus, B. rapa and B. juncea lines were assessed for sequence variation at the locus. Rlm2 was found to be an allelic variant of the LepR3 LRR-RLP locus, conveying race-specific resistance to L. maculans isolates harbouring AvrLm2. Several defence-related LRR-RLPs have previously been shown to associate with the RLK SOBIR1 to facilitate defence signalling. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation of RLM2-SOBIR1 studies revealed that RLM2 interacts with SOBIR1 of Arabidopsis thaliana when co-expressed in Nicotiana benthamiana. The interaction of RLM2 with AtSOBIR1 is suggestive of a conserved defence signalling pathway between B. napus and its close relative A. thaliana.

  17. A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum.

    PubMed

    Wang, Yong; Duan, Yabing; Wang, Jianxin; Zhou, Mingguo

    2015-09-01

    Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs.

  18. A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum.

    PubMed

    Wang, Yong; Duan, Yabing; Wang, Jianxin; Zhou, Mingguo

    2015-09-01

    Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs. PMID:25441450

  19. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper.

    PubMed

    Rao, K V; Rathore, K S; Hodges, T K; Fu, X; Stoger, E; Sudhakar, D; Williams, S; Christou, P; Bharathi, M; Bown, D P; Powell, K S; Spence, J; Gatehouse, A M; Gatehouse, J A

    1998-08-01

    Snowdrop lectin (Galanthus nivalis agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper (Nilaparvata lugens; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes 'hopper burn', as well as being an important virus vector. To evaluate the potential of the gna gene to confer resistance towards BPH, transgenic rice (Oryza sativa L.) plants were produced, containing the gna gene in constructs where its expression was driven by a phloem-specific promoter (from the rice sucrose synthase RSs1 gene) and by a constitutive promoter (from the maize ubiquitin ubi1 gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self-fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the RSs1 promoter was tissue-specific, as shown by immunohistochemical localization of the protein in the non-lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding. gna is the first transgene to exhibit insecticidal activity towards sap-sucking insects in an important cereal crop plant.

  20. Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism.

    PubMed

    Song, Xiaozhao; Kain, Wendy; Cassidy, Douglas; Wang, Ping

    2015-08-01

    The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism. PMID:26025894

  1. Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

    PubMed Central

    Song, Xiaozhao; Kain, Wendy; Cassidy, Douglas

    2015-01-01

    The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism. PMID:26025894

  2. Mutations in the herpes simplex virus DNA polymerase gene can confer resistance to 9-beta-D-arabinofuranosyladenine.

    PubMed Central

    Coen, D M; Furman, P A; Gelep, P T; Schaffer, P A

    1982-01-01

    Mutants of herpes simplex virus type 1 resistant to the antiviral drug 9-beta-D-arabinofuranosyladenine (araA) have been isolated and characterized. AraA-resistant mutants can be isolated readily and appear at an appreciable frequency in low-passage stocks of wild-type virus. Of 13 newly isolated mutants, at least 11 were also resistant to phosphonoacetic acid (PAA). Of four previously described PAA-resistant mutants, two exhibited substantial araA resistance. The araA resistance phenotype of one of these mutants, PAAr5, has been mapped to the HpaI-B fragment of herpes simplex virus DNA by marker transfer, and araA resistance behaved in marker transfer experiments as if it were closely linked to PAA resistance, a recognized marker for the viral DNA polymerase locus. PAAr5 induced viral DNA polymerase activity which was much less susceptible to inhibition by the triphosphate derivative of araA than was wild-type DNA polymerase. These genetic and biochemical data indicate that the herpes simplex virus DNA polymerase gene is a locus which, when mutated, can confer resistance to araA and thus that the herpes simplex virus DNA polymerase is a target for this antiviral drug. PMID:6284981

  3. The Oxidative Stress Responsive Transcription Factor Pap1 Confers DNA Damage Resistance on Checkpoint-Deficient Fission Yeast Cells

    PubMed Central

    Belfield, Carrie; Queenan, Craig; Rao, Hui; Kitamura, Kenji; Walworth, Nancy C.

    2014-01-01

    Eukaryotic cells invoke mechanisms to promote survival when confronted with cellular stress or damage to the genome. The protein kinase Chk1 is an integral and conserved component of the DNA damage response pathway. Mutation or inhibition of Chk1 results in mitotic death when cells are exposed to DNA damage. Oxidative stress activates a pathway that results in nuclear accumulation of the bZIP transcription factor Pap1. We report the novel finding that fission yeast Pap1 confers resistance to drug- and non-drug-induced DNA damage even when the DNA damage checkpoint is compromised. Multi-copy expression of Pap1 restores growth to chk1-deficient cells exposed to camptothecin or hydroxyurea. Unexpectedly, increased Pap1 expression also promotes survival of chk1-deficient cells with mutations in genes encoding DNA ligase (cdc17) or DNA polymerase δ (cdc6), but not DNA replication initiation mutants. The ability of Pap1 to confer resistance to DNA damage was not specific to chk1 mutants, as it also improved survival of rad1- and rad9-deficient cells in the presence of CPT. To confer resistance to DNA damage Pap1 must localize to the nucleus and be transcriptionally active. PMID:24587136

  4. The Cfr rRNA Methyltransferase Confers Resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A Antibiotics

    PubMed Central

    Long, Katherine S.; Poehlsgaard, Jacob; Kehrenberg, Corinna; Schwarz, Stefan; Vester, Birte

    2006-01-01

    A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones. PMID:16801432

  5. Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco.

    PubMed

    Cox, K D; Layne, D R; Scorza, R; Schnabel, G

    2006-11-01

    Diseases of agricultural crops are caused by pathogens from several higher-order phylogenetic lineages including fungi, straminipila, eubacteria, and metazoa. These pathogens are commonly managed with pesticides due to the lack of broad-spectrum host resistance. Gastrodia anti-fungal protein (GAFP; gastrodianin) may provide a level of broad-spectrum resistance due to its documented anti-fungal activity in vitro and structural similarity to insecticidal lectins. We transformed tobacco (Nicotiana tabacum cv. Wisconsin 38) with GAFP-1 and challenged transformants with agriculturally important plant pathogens from several higher-order lineages including Rhizoctonia solani (fungus), Phytophthora nicotianae (straminipile), Ralstonia solanacearum (eubacterium), and Meloidogyne incognita (metazoan). Quantitative real-time PCR and western blotting analysis indicated that GAFP-1 was transcribed and translated in transgenic lines. When challenged by R. solani and P. nicotianae, GAFP-1 expressing lines had reduced symptom development and improved plant vigor compared to non-transformed and empty vector control lines. These lines also exhibited reduced root galling when challenged by M. incognita. Against R. solanacearum expression of GAFP-1 neither conferred resistance, nor exacerbated disease development. These results indicate that heterologous expression of GAFP-1 can confer enhanced resistance to a diverse set of plant pathogens and may be a good candidate gene for the development of transgenic, root-disease-resistant crops.

  6. The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat.

    PubMed

    Sasaki, Kentaro; Kuwabara, Chikako; Umeki, Natsuki; Fujioka, Mari; Saburi, Wataru; Matsui, Hirokazu; Abe, Fumitaka; Imai, Ryozo

    2016-06-20

    TAD1 (Triticum aestivum defensin 1) is induced during cold acclimation in winter wheat and encodes a plant defensin with antimicrobial activity. In this study, we demonstrated that recombinant TAD1 protein inhibits hyphal growth of the snow mold fungus, Typhula ishikariensis in vitro. Transgenic wheat plants overexpressing TAD1 were created and tested for resistance against T. ishikariensis. Leaf inoculation assays revealed that overexpression of TAD1 confers resistance against the snow mold. In addition, the TAD1-overexpressors showed resistance against Fusarium graminearum, which causes Fusarium head blight, a devastating disease in wheat and barley. These results indicate that TAD1 is a candidate gene to improve resistance against multiple fungal diseases in cereal crops. PMID:27080445

  7. The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat.

    PubMed

    Sasaki, Kentaro; Kuwabara, Chikako; Umeki, Natsuki; Fujioka, Mari; Saburi, Wataru; Matsui, Hirokazu; Abe, Fumitaka; Imai, Ryozo

    2016-06-20

    TAD1 (Triticum aestivum defensin 1) is induced during cold acclimation in winter wheat and encodes a plant defensin with antimicrobial activity. In this study, we demonstrated that recombinant TAD1 protein inhibits hyphal growth of the snow mold fungus, Typhula ishikariensis in vitro. Transgenic wheat plants overexpressing TAD1 were created and tested for resistance against T. ishikariensis. Leaf inoculation assays revealed that overexpression of TAD1 confers resistance against the snow mold. In addition, the TAD1-overexpressors showed resistance against Fusarium graminearum, which causes Fusarium head blight, a devastating disease in wheat and barley. These results indicate that TAD1 is a candidate gene to improve resistance against multiple fungal diseases in cereal crops.

  8. Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

    PubMed

    Yassien, M A M; Elfaky, M A

    2015-11-01

    A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α. PMID:26375447

  9. Genetic resistance to flaviviruses.

    PubMed

    Brinton, Margo A; Perelygin, Andrey A

    2003-01-01

    Resistance to flavivirus-induced disease in mice was first discovered in the 1920s and was subsequently shown to be controlled by the resistant allele of a single dominant autosomal gene. While the majority of current laboratory mouse stains have a homozygous-susceptible phenotype, the resistant allele has been found to segregate in wild mouse populations in many different parts of the world. Resistance is flavivirus specific and extends to both mosquito- and tick-borne flaviviruses. Resistant animals are infected productively by flaviviruses but produce lower virus titers, especially in their brains, as compared to susceptible mice. Decreased virus production is observed in resistant animals even during a lethal infection and the times of disease onset and death are also delayed as compared to susceptible mice. An intact immune response is required to clear flaviviruses from resistant mice. The resistant phenotype is expressed constitutively and does not require interferon induction. The Flv gene was discovered using a positional cloning approach and identified as Oas1b. Susceptible mice produce a truncated Oas1b protein. A C820T transition in the fourth exon of the gene introduced a premature stop codon and was found in all susceptible mouse strains tested. Possible mechanisms by which the product of the resistant allele could confer the resistant phenotype are discussed. PMID:14689691

  10. 3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors.

    PubMed Central

    Larder, B A

    1992-01-01

    Nonnucleoside reverse transcriptase (NNRT) inhibitors (R82913; (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione; Cl-TIBO; and BI-RG-587, nevirapine) were used to select resistant human immunodeficiency virus type 1 (HIV-1) variants by passage in cell cultures of wild-type or 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant strains. Similar to other NNRT inhibitors, Cl-TIBO induced a single mutation (Y181 to C) in reverse transcriptase (RT) that accounted for the resistance. BI-RG-587 induced a different mutation (V106-->A) in AZT resistance backgrounds. A series of viable HIV-1 variants was constructed by site-directed mutagenesis of the RT, which harbored multiple drug resistance mutations, including Y181 to C. HIV-1 that was co-resistant to NNRT inhibitors and 2',3'-dideoxyinosine resulted when a 2',3'-dideoxyinosine resistance mutation (L74 to V) was also present in RT. By contrast, however, the Y181 to C mutation in an AZT resistance background significantly suppressed resistance to AZT, while it conferred resistance to NNRT inhibitors. However, the V106-->A substitution did not cause suppression of preexisting AZT resistance. Since certain combinations of nucleoside analogs and NNRT inhibitors might result in the development of co-resistance, careful analysis of clinical isolates obtained during combination therapy will be needed to determine the potential significance of these observations. PMID:1282792

  11. Non-injurious neonatal hypoxia confers resistance to brain senescence in aged male rats.

    PubMed

    Martin, Nicolas; Bossenmeyer-Pourié, Carine; Koziel, Violette; Jazi, Rozat; Audonnet, Sandra; Vert, Paul; Guéant, Jean-Louis; Daval, Jean-Luc; Pourié, Grégory

    2012-01-01

    Whereas brief acute or intermittent episodes of hypoxia have been shown to exert a protective role in the central nervous system and to stimulate neurogenesis, other studies suggest that early hypoxia may constitute a risk factor that influences the future development of mental disorders. We therefore investigated the effects of a neonatal "conditioning-like" hypoxia (100% N₂, 5 min) on the brain and the cognitive outcomes of rats until 720 days of age (physiologic senescence). We confirmed that such a short hypoxia led to brain neurogenesis within the ensuing weeks, along with reduced apoptosis in the hippocampus involving activation of Erk1/2 and repression of p38 and death-associated protein (DAP) kinase. At 21 days of age, increased thicknesses and cell densities were recorded in various subregions, with strong synapsin activation. During aging, previous exposure to neonatal hypoxia was associated with enhanced memory retrieval scores specifically in males, better preservation of their brain integrity than controls, reduced age-related apoptosis, larger hippocampal cell layers, and higher expression of glutamatergic and GABAergic markers. These changes were accompanied with a marked expression of synapsin proteins, mainly of their phosphorylated active forms which constitute major players of synapse function and plasticity, and with increases of their key regulators, i.e. Erk1/2, the transcription factor EGR-1/Zif-268 and Src kinase. Moreover, the significantly higher interactions between PSD-95 scaffolding protein and NMDA receptors measured in the hippocampus of 720-day-old male animals strengthen the conclusion of increased synaptic functional activity and plasticity associated with neonatal hypoxia. Thus, early non-injurious hypoxia may trigger beneficial long term effects conferring higher resistance to senescence in aged male rats, with a better preservation of cognitive functions.

  12. The Batten disease gene CLN3 confers resistance to endoplasmic reticulum stress induced by tunicamycin

    SciTech Connect

    Wu, Dan; Liu, Jing; Wu, Baiyan; Tu, Bo; Zhu, Weiguo; Luo, Jianyuan

    2014-04-25

    Highlights: • The work reveals a protective properties of CLN3 towards TM-induced apoptosis. • CLN3 regulates expression of the GRP78 and the CHOP in response to the ER stress. • CLN3 plays a specific role in the ERS response. - Abstract: Mutations in CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early-onset neurodegenerative disorder that is characterized by the accumulation of ceroid lipofuscin within lysosomes. The function of the CLN3 protein remains unclear and is presumed to be related to Endoplasmic reticulum (ER) stress. To investigate the function of CLN3 in the ER stress signaling pathway, we measured proliferation and apoptosis in cells transfected with normal and mutant CLN3 after treatment with the ER stress inducer tunicamycin (TM). We found that overexpression of CLN3 was sufficient in conferring increased resistance to ER stress. Wild-type CLN3 protected cells from TM-induced apoptosis and increased cell proliferation. Overexpression of wild-type CLN3 enhanced expression of the ER chaperone protein, glucose-regulated protein 78 (GRP78), and reduced expression of the proapoptotic protein CCAAT/-enhancer-binding protein homologous protein (CHOP). In contrast, overexpression of mutant CLN3 or siRNA knockdown of CLN3 produced the opposite effect. Together, our data suggest that the lack of CLN3 function in cells leads to a failure of management in the response to ER stress and this may be the key deficit in JNCL that causes neuronal degeneration.

  13. Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem

    PubMed Central

    Yano, Hisakazu; Kuga, Akio; Okamoto, Ryoichi; Kitasato, Hidero; Kobayashi, Toshimitsu; Inoue, Matsuhisa

    2001-01-01

    In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries blaIMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The kcat/Km value of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme. PMID:11302793

  14. Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of Barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate.

    PubMed

    Riedel, Christine; Habekuss, Antje; Schliephake, Edgar; Niks, Rients; Broer, Inge; Ordon, Frank

    2011-06-01

    Barley yellow dwarf virus (BYDV) is an economically important pathogen of barley, which may become even more important due to global warming. In barley, several loci conferring tolerance to BYDV-PAV-ASL-1 are known, e.g. Ryd2, Ryd3 and a quantitative trait locus (QTL) on chromosome 2H. The aim of the present study was to get information whether the level of tolerance against this isolate of BYDV in barley can be improved by combining these loci. Therefore, a winter and a spring barley population of doubled haploid (DH) lines were genotyped by molecular markers for the presence of the susceptibility or the resistance encoding allele at respective loci (Ryd2, Ryd3, QTL on chromosome 2H) and were tested for their level of BYDV-tolerance after inoculation with viruliferous (BYDV-PAV-ASL-1) aphids in field trials. In DH-lines carrying the combination Ryd2 and Ryd3, a significant reduction of the virus titre was detected as compared to lines carrying only one of these genes. Furthermore, spring barley DH-lines with this allele combination also showed a significantly higher relative grain yield as compared to lines carrying only Ryd2 or Ryd3. The QTL on chromosome 2H had only a small effect on the level of tolerance in those lines carrying only Ryd2, or Ryd3 or a combination of both, but the effect in comparison to lines carrying no tolerance allele was significant. Overall, these results show that the combination of Ryd2 and Ryd3 leads to quantitative resistance against BYDV-PAV instead of tolerance.

  15. Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of Barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate.

    PubMed

    Riedel, Christine; Habekuss, Antje; Schliephake, Edgar; Niks, Rients; Broer, Inge; Ordon, Frank

    2011-06-01

    Barley yellow dwarf virus (BYDV) is an economically important pathogen of barley, which may become even more important due to global warming. In barley, several loci conferring tolerance to BYDV-PAV-ASL-1 are known, e.g. Ryd2, Ryd3 and a quantitative trait locus (QTL) on chromosome 2H. The aim of the present study was to get information whether the level of tolerance against this isolate of BYDV in barley can be improved by combining these loci. Therefore, a winter and a spring barley population of doubled haploid (DH) lines were genotyped by molecular markers for the presence of the susceptibility or the resistance encoding allele at respective loci (Ryd2, Ryd3, QTL on chromosome 2H) and were tested for their level of BYDV-tolerance after inoculation with viruliferous (BYDV-PAV-ASL-1) aphids in field trials. In DH-lines carrying the combination Ryd2 and Ryd3, a significant reduction of the virus titre was detected as compared to lines carrying only one of these genes. Furthermore, spring barley DH-lines with this allele combination also showed a significantly higher relative grain yield as compared to lines carrying only Ryd2 or Ryd3. The QTL on chromosome 2H had only a small effect on the level of tolerance in those lines carrying only Ryd2, or Ryd3 or a combination of both, but the effect in comparison to lines carrying no tolerance allele was significant. Overall, these results show that the combination of Ryd2 and Ryd3 leads to quantitative resistance against BYDV-PAV instead of tolerance. PMID:21416402

  16. Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH-NADH adduct formation activities

    PubMed Central

    Cade, Christine E; Dlouhy, Adrienne C; Medzihradszky, Katalin F; Salas-Castillo, Saida Patricia; Ghiladi, Reza A

    2010-01-01

    Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD+/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr-Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H2O2), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct. PMID:20054829

  17. tcrB, a Gene Conferring Transferable Copper Resistance in Enterococcus faecium: Occurrence, Transferability, and Linkage to Macrolide and Glycopeptide Resistance

    PubMed Central

    Hasman, Henrik; Aarestrup, Frank M.

    2002-01-01

    A newly discovered gene, designated tcrB, which is located on a conjugative plasmid conferring acquired copper resistance in Enterococcus faecium, was identified in an isolate from a pig. The tcrB gene encodes a putative protein belonging to the CPx-type ATPase family with homology (46%) to the CopB protein from Enterococcus hirae. The tcrB gene was found in E. faecium isolated from pigs (75%), broilers (34%), calves (16%), and humans (10%) but not in isolates from sheep. Resistant isolates, containing the tcrB gene, grew on brain heart infusion agar plates containing up to 28 mM CuSO4 compared to only 4 mM for the susceptible isolates. Copper resistance, and therefore the presence of the tcrB gene, was strongly correlated to macrolide and glycopeptide resistance in isolates from pigs, and the tcrB gene was shown to be located on the same conjugative plasmid as the genes responsible for resistance to these two antimicrobial agents. The frequent occurrence of this new copper resistance gene in isolates from pigs, where copper sulfate is being used in large amounts as feed additive, suggests that the use of copper has selected for resistance. PMID:11959576

  18. The Fd-GOGAT1 mutant gene lc7 confers resistance to Xanthomonas oryzae pv. Oryzae in rice

    PubMed Central

    Chen, Honglin; Li, Chunrong; Liu, Liping; Zhao, Jiying; Cheng, Xuzhen; Jiang, Guanghuai; Zhai, Wenxue

    2016-01-01

    Disease resistance is an important goal of crop improvement. The molecular mechanism of resistance requires further study. Here, we report the identification of a rice leaf color mutant, lc7, which is defective in chlorophyll synthesis and photosynthesis but confers resistance to Xanthomonas oryzae pv. Oryzae (Xoo). Map-based cloning revealed that lc7 encodes a mutant ferredoxin-dependent glutamate synthase1 (Fd-GOGAT1). Fd-GOGAT1 has been proposed to have great potential for improving nitrogen-use efficiency, but its function in bacterial resistance has not been reported. The lc7 mutant accumulates excessive levels of ROS (reactive oxygen species) in the leaves, causing the leaf color to become yellow after the four-leaf stage. Compared to the wild type, lc7 mutants have a broad-spectrum high resistance to seven Xoo strains. Differentially expressed genes (DEGs) and qRT-PCR analysis indicate that many defense pathways that are involved in this broad-spectrum resistance are activated in the lc7 mutant. These results suggest that Fd-GOGAT1 plays an important role in broad-spectrum bacterial blight resistance, in addition to modulating nitrogen assimilation and chloroplast development. PMID:27211925

  19. Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

    PubMed

    Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W; Gill, Bikram S

    2014-11-01

    Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

  20. Identification of regulated genes conferring resistance to high concentrations of glyphosate in a new strain of Enterobacter.

    PubMed

    Fei, Yun-Yan; Gai, Jun-Yi; Zhao, Tuan-Jie

    2013-12-01

    Glyphosate is a widely used herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity. Most plants and microbes are sensitive to glyphosate. However, transgenic-resistant crops that contain a modified epsps obtained from the resistant microbes have been commercially successful and therefore, new resistance genes and their adaptive regulatory mechanisms are of great interest. In this study, a soil-borne, glyphosate-resistant bacterium was selected and identified as Enterobacter. The EPSPS in this strain was found to have been altered to a resistant one. A total of 42 differentially expressed genes (DEGs) in the glyphosate were screened using microarray techniques. Under treatment, argF, sdhA, ivbL, rrfA-H were downregulated, whereas the transcripts of speA, osmY, pflB, ahpC, fusA, deoA, uxaC, rpoD and a few ribosomal protein genes were upregulated. Data were verified by quantitative real-time PCR on selected genes. All transcriptional changes appeared to protect the bacteria from glyphosate and associated osmotic, acidic and oxidative stresses. Many DEGs may have the potential to confer resistance to glyphosate alone, and some may be closely related to the shikimate pathway, reflecting the complex gene interaction network for glyphosate resistance.

  1. Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

    PubMed

    Jugulam, Mithila; Niehues, Kindsey; Godar, Amar S; Koo, Dal-Hoe; Danilova, Tatiana; Friebe, Bernd; Sehgal, Sunish; Varanasi, Vijay K; Wiersma, Andrew; Westra, Philip; Stahlman, Phillip W; Gill, Bikram S

    2014-11-01

    Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations. PMID:25037215

  2. TetAB(46), a predicted heterodimeric ABC transporter conferring tetracycline resistance in Streptococcus australis isolated from the oral cavity

    PubMed Central

    Warburton, Philip J.; Ciric, Lena; Lerner, Avigdor; Seville, Lorna A.; Roberts, Adam P.; Mullany, Peter; Allan, Elaine

    2013-01-01

    Objectives To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient. Methods Agar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms. A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame deletions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agar dilution and disc diffusion assay. Results The tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic library in E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity to multidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both the naturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46). Conclusions This is the first report of a predicted ABC transporter conferring tetracycline resistance in a member of the oral microbiota. PMID:22941900

  3. Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance

    PubMed Central

    Miao, Jianqiang; Cai, Meng; Dong, Xue; Liu, Li; Lin, Dong; Zhang, Can; Pang, Zhili; Liu, Xili

    2016-01-01

    The potential for oxathiapiprolin resistance in Phytophthora capsici was evaluated. The baseline sensitivities of 175 isolates to oxathiapiprolin were initially determinated and found to conform to a unimodal curve with a mean EC50 value of 5.61 × 10-4 μg/ml. Twelve stable oxathiapiprolin-resistant mutants were generated by fungicide adaptation in two sensitive isolates, LP3 and HNJZ10. The fitness of the LP3-mutants was found to be similar to or better than that of the parental isolate LP3, while the HNJZ10-mutants were found to have lost the capacity to produce zoospores. Taken together these results suggest that the risk of P. capsici developing resistance to oxathiapiprolin is moderate. Comparison of the PcORP1 genes in the LP3-mutants and wild-type parental isolate, which encode the target protein of oxathiapiprolin, revealed that a heterozygous mutation caused the amino acid substitution G769W. Transformation and expression of the mutated PcORP1-769W allele in the sensitive wild-type isolate BYA5 confirmed that the mutation in PcORP1 was responsible for the observed oxathiapiprolin resistance. Finally diagnostic tests including As-PCR and CAPs were developed to detect the oxathiapiprolin resistance resulting from the G769W point mutation in field populations of P. capsici. PMID:27199944

  4. Distinct Detoxification Mechanisms Confer Resistance to Mesotrione and Atrazine in a Population of Waterhemp1[C][W][OPEN

    PubMed Central

    Ma, Rong; Kaundun, Shiv S.; Tranel, Patrick J.; Riggins, Chance W.; McGinness, Daniel L.; Hager, Aaron G.; Hawkes, Tim; McIndoe, Eddie; Riechers, Dean E.

    2013-01-01

    Previous research reported the first case of resistance to mesotrione and other 4-hydroxyphenylpyruvate dioxygenase (HPPD) herbicides in a waterhemp (Amaranthus tuberculatus) population designated MCR (for McLean County mesotrione- and atrazine-resistant). Herein, experiments were conducted to determine if target site or nontarget site mechanisms confer mesotrione resistance in MCR. Additionally, the basis for atrazine resistance was investigated in MCR and an atrazine-resistant but mesotrione-sensitive population (ACR for Adams County mesotrione-sensitive but atrazine-resistant). A standard sensitive population (WCS for Wayne County herbicide-sensitive) was also used for comparison. Mesotrione resistance was not due to an alteration in HPPD sequence, HPPD expression, or reduced herbicide absorption. Metabolism studies using whole plants and excised leaves revealed that the time for 50% of absorbed mesotrione to degrade in MCR was significantly shorter than in ACR and WCS, which correlated with previous phenotypic responses to mesotrione and the quantity of the metabolite 4-hydroxy-mesotrione in excised leaves. The cytochrome P450 monooxygenase inhibitors malathion and tetcyclacis significantly reduced mesotrione metabolism in MCR and corn (Zea mays) excised leaves but not in ACR. Furthermore, malathion increased mesotrione activity in MCR seedlings in greenhouse studies. These results indicate that enhanced oxidative metabolism contributes significantly to mesotrione resistance in MCR. Sequence analysis of atrazine-resistant (MCR and ACR) and atrazine-sensitive (WCS) waterhemp populations detected no differences in the psbA gene. The times for 50% of absorbed atrazine to degrade in corn, MCR, and ACR leaves were shorter than in WCS, and a polar metabolite of atrazine was detected in corn, MCR, and ACR that cochromatographed with a synthetic atrazine-glutathione conjugate. Thus, elevated rates of metabolism via distinct detoxification mechanisms contribute to

  5. Common HLA Alleles Associated with Health, but Not with Facial Attractiveness

    PubMed Central

    Coetzee, Vinet; Barrett, Louise; Greeff, Jaco M.; Henzi, S. Peter; Perrett, David I.; Wadee, Ahmed A.

    2007-01-01

    Three adaptive hypotheses have been proposed to explain the link between the human leucocyte antigen (HLA) genes, health measures and facial attractiveness: inbreeding avoidance, heterozygote advantage and frequency-dependent selection. This paper reports findings that support a new hypothesis relating HLA to health. We suggest a new method to quantify the level of heterozygosity. HLA heterozygosity did not significantly predict health measures in women, but allele frequency did. Women with more common HLA alleles reported fewer cold and flu bouts per year, fewer illnesses in the previous year and rated themselves healthier than women with rare alleles. To our knowledge, this is the first study to show a positive correlation between HLA allele frequency and general health measures. We propose that certain common HLA alleles confer resistance to prevalent pathogens. Nevertheless, neither HLA heterozygosity nor allele frequency significantly predicted how healthy or attractive men rated the female volunteers. Three non-mutually exclusive explanations are put forward to explain this finding. PMID:17653267

  6. Development of pyramidal lines with two major QTLs conferring resistance to sheath blight in rice (Oryza sativa L.)

    NASA Astrophysics Data System (ADS)

    Hossain, Md Kamal; Jena, Kshirod; Bhuiyan, Md Atiqur Rahman; Ratnam, Wickneswari

    2014-09-01

    Sheath blight is an emerging threat in rice cultivation. It is animportant disease caused by the soil-borne necrotrophic pathogenic fungus, Rhizoctonia solani Kühn. However, to date neither known major genes for quantitative resistance, nor any rice lines immune to this disease has been identified. The disease resistance is quantitative in nature. Numerous genes are involved in this resistance process. There are few quantitative trait loci (QTLs) detected conferring improved resistance against the disease. Teqing and Tetepshowimproved resistance having QTLs, qSB-9 and qSBR11-1, respectively. Since, these QTLs demonstrates additive effects, pyramiding of these QTLs might be an option to increase the sheath blight resistance in rice. Nine rice cultivars were screened at greenhouse conditions. Results showed that Tetep and Teqing had the lowest disease ratings. UKMRC2a new high yielding cultivar was as recipient parent. Crosses between UKMRC2 and Teqing, and UKMRC2 and Tetep were made and confirmed. Subsequently 4-way crosses between the two F1s were performed to develop pyramidal lines.

  7. A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper).

    PubMed

    Liu, Zewen; Williamson, Martin S; Lansdell, Stuart J; Denholm, Ian; Han, Zhaojun; Millar, Neil S

    2005-06-14

    Neonicotinoids, such as imidacloprid, are nicotinic acetylcholine receptor (nAChR) agonists with potent insecticidal activity. Since its introduction in the early 1990s, imidacloprid has become one of the most extensively used insecticides for both crop protection and animal health applications. As with other classes of insecticides, resistance to neonicotinoids is a significant threat and has been identified in several pest species, including the brown planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. In this study, radioligand binding experiments have been conducted with whole-body membranes prepared from imidacloprid-susceptible and imidacloprid-resistant strains of N. lugens. The results reveal a much higher level of [3H]imidacloprid-specific binding to the susceptible strain than to the resistant strain (16.7 +/- 1.0 and 0.34 +/- 0.21 fmol/mg of protein, respectively). With the aim of understanding the molecular basis of imidacloprid resistance, five nAChR subunits (Nlalpha1-Nlalpha4 and Nlbeta1) have been cloned from N. lugens.A comparison of nAChR subunit genes from imidacloprid-sensitive and imidacloprid-resistant populations has identified a single point mutation at a conserved position (Y151S) in two nAChR subunits, Nlalpha1 and Nlalpha3. A strong correlation between the frequency of the Y151S point mutation and the level of resistance to imidacloprid has been demonstrated by allele-specific PCR. By expression of hybrid nAChRs containing N. lugens alpha and rat beta2 subunits, evidence was obtained that demonstrates that mutation Y151S is responsible for a substantial reduction in specific [3H]imidacloprid binding. This study provides direct evidence for the occurrence of target-site resistance to a neonicotinoid insecticide. PMID:15937112

  8. Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent.

    PubMed

    Winglee, Kathryn; Lun, Shichun; Pieroni, Marco; Kozikowski, Alan; Bishai, William

    2015-11-01

    Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation. PMID:26303802

  9. Dissection of two soybean QTL conferring partial resistance to Phytophthora sojae through sequence and gene expression analysis

    PubMed Central

    2012-01-01

    Background Phytophthora sojae is the primary pathogen of soybeans that are grown on poorly drained soils. Race-specific resistance to P. sojae in soybean is gene-for-gene, although in many areas of the US and worldwide there are populations that have adapted to the most commonly deployed resistance to P. sojae ( Rps) genes. Hence, this system has received increased attention towards identifying mechanisms and molecular markers associated with partial resistance to this pathogen. Several quantitative trait loci (QTL) have been identified in the soybean cultivar ‘Conrad’ that contributes to the expression of partial resistance to multiple P. sojae isolates. Results In this study, two of the Conrad QTL on chromosome 19 were dissected through sequence and expression analysis of genes in both resistant (Conrad) and susceptible (‘Sloan’) genotypes. There were 1025 single nucleotide polymorphisms (SNPs) in 87 of 153 genes sequenced from Conrad and Sloan. There were 304 SNPs in 54 genes sequenced from Conrad compared to those from both Sloan and Williams 82, of which 11 genes had SNPs unique to Conrad. Eleven of 19 genes in these regions analyzed with qRT-PCR had significant differences in fold change of transcript abundance in response to infection with P. sojae in lines with QTL haplotype from the resistant parent compared to those with the susceptible parent haplotype. From these, 8 of the 11 genes had SNPs in the upstream, untranslated region, exon, intron, and/or downstream region. These 11 candidate genes encode proteins potentially involved in signal transduction, hormone-mediated pathways, plant cell structural modification, ubiquitination, and basal resistance. Conclusions These findings may indicate a complex defense network with multiple mechanisms underlying these two soybean QTL conferring resistance to P. sojae. SNP markers derived from these candidate genes can contribute to fine mapping of QTL and marker assisted breeding for resistance to P. sojae

  10. Characterization and mapping of Rpi1, a gene that confers dominant resistance to stalk rot in maize.

    PubMed

    Yang, D E; Jin, D M; Wang, B; Zhang, D S; Nguyen, H-T; Zhang, C L; Chen, S J

    2005-10-01

    The maize inbred lines 1145 (resistant) and Y331 (susceptible), and the F(1), F(2) and BC(1)F(1) populations derived from them were inoculated with the pathogen Pythium inflatum Matthews, which causes stalk rot in Zea mays. Field data revealed that the ratio of resistant to susceptible plants was 3:1 in the F(2) population, and 1:1 in the BC(1)F(1)population, indicating that the resistance to P. inflatum Matthews was controlled by a single dominant gene in the 1145xY331 cross. The gene that confers resistance to P. inflatum Matthews was designated Rpi1 for resistance to P. inflatum) according to the standard nomenclature for plant disease resistance genes. Fifty SSR markers from 10 chromosomes were first screened in the F(2) population to find markers linked to the Rpi1 gene. The results indicated that umc1702 and mmc0371 were both linked to Rpi1, placing the resistance gene on chromosome 4. RAPD (randomly amplified polymorphic DNA) markers were then tested in the F(2)population using bulked segregant analysis (BSA). Four RAPD products were found to show linkage to the Rpi1 gene. Then 27 SSR markers and 8 RFLP markers in the region encompassing Rpi1 were used for fine-scale mapping of the resistance gene. Two SSR markers and four RFLP markers were linked to the Rpi1 gene. Finally, the Rpi1 gene was mapped between the SSR markers bnlg1937 and agrr286 on chromosome 4, 1.6 cM away from the former and 4.1 cM distant from the latter. This is the first time that a dominant gene for resistance to maize stalk rot caused by P. inflatum Matthews has been mapped with molecular marker techniques. PMID:16133168

  11. Characterization and mapping of Rpi1, a gene that confers dominant resistance to stalk rot in maize.

    PubMed

    Yang, D E; Jin, D M; Wang, B; Zhang, D S; Nguyen, H-T; Zhang, C L; Chen, S J

    2005-10-01

    The maize inbred lines 1145 (resistant) and Y331 (susceptible), and the F(1), F(2) and BC(1)F(1) populations derived from them were inoculated with the pathogen Pythium inflatum Matthews, which causes stalk rot in Zea mays. Field data revealed that the ratio of resistant to susceptible plants was 3:1 in the F(2) population, and 1:1 in the BC(1)F(1)population, indicating that the resistance to P. inflatum Matthews was controlled by a single dominant gene in the 1145xY331 cross. The gene that confers resistance to P. inflatum Matthews was designated Rpi1 for resistance to P. inflatum) according to the standard nomenclature for plant disease resistance genes. Fifty SSR markers from 10 chromosomes were first screened in the F(2) population to find markers linked to the Rpi1 gene. The results indicated that umc1702 and mmc0371 were both linked to Rpi1, placing the resistance gene on chromosome 4. RAPD (randomly amplified polymorphic DNA) markers were then tested in the F(2)population using bulked segregant analysis (BSA). Four RAPD products were found to show linkage to the Rpi1 gene. Then 27 SSR markers and 8 RFLP markers in the region encompassing Rpi1 were used for fine-scale mapping of the resistance gene. Two SSR markers and four RFLP markers were linked to the Rpi1 gene. Finally, the Rpi1 gene was mapped between the SSR markers bnlg1937 and agrr286 on chromosome 4, 1.6 cM away from the former and 4.1 cM distant from the latter. This is the first time that a dominant gene for resistance to maize stalk rot caused by P. inflatum Matthews has been mapped with molecular marker techniques.

  12. Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles.

    PubMed

    Ohnishi, Yusuke; Tokunaga, Katsushi; Kaneko, Kiyotoshi; Hohjoh, Hirohiko

    2006-02-28

    Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASPRNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer's disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid beta 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

  13. A Novel Glycolipid Biosurfactant Confers Grazing Resistance upon Pantoea ananatis BRT175 against the Social Amoeba Dictyostelium discoideum.

    PubMed

    Smith, Derek D N; Nickzad, Arvin; Déziel, Eric; Stavrinides, John

    2016-01-01

    Pantoea is a versatile genus of bacteria with both plant- and animal-pathogenic strains, some of which have been suggested to cause human infections. There is, however, limited knowledge on the potential determinants used for host association and pathogenesis in animal systems. In this study, we used the model host Dictyostelium discoideum to show that isolates of Pantoea ananatis exhibit differential grazing susceptibility, with some being resistant to grazing by the amoebae. We carried out a high-throughput genetic screen of one grazing-resistant isolate, P. ananatis BRT175, using the D. discoideum pathosystem to identify genes responsible for the resistance phenotype. Among the 26 candidate genes involved in grazing resistance, we identified rhlA and rhlB, which we show are involved in the biosynthesis of a biosurfactant that enables swarming motility in P. ananatis BRT175. Using liquid chromatography-mass spectrometry (LC-MS), the biosurfactant was shown to be a glycolipid with monohexose-C10-C10 as the primary congener. We show that this novel glycolipid biosurfactant is cytotoxic to the amoebae and is capable of compromising cellular integrity, leading to cell lysis. The production of this biosurfactant may be important for bacterial survival in the environment and could contribute to the establishment of opportunistic infections. IMPORTANCE The genetic factors used for host interaction by the opportunistic human pathogen Pantoea ananatis are largely unknown. We identified two genes that are important for the production of a biosurfactant that confers grazing resistance against the social amoeba Dictyostelium discoideum. We show that the biosurfactant, which exhibits cytotoxicity toward the amoebae, is a glycolipid that incorporates a hexose rather than rhamnose. The production of this biosurfactant may confer a competitive advantage in the environment and could potentially contribute to the establishment of opportunistic infections. PMID:27303689

  14. A Novel Glycolipid Biosurfactant Confers Grazing Resistance upon Pantoea ananatis BRT175 against the Social Amoeba Dictyostelium discoideum.

    PubMed

    Smith, Derek D N; Nickzad, Arvin; Déziel, Eric; Stavrinides, John

    2016-01-01

    Pantoea is a versatile genus of bacteria with both plant- and animal-pathogenic strains, some of which have been suggested to cause human infections. There is, however, limited knowledge on the potential determinants used for host association and pathogenesis in animal systems. In this study, we used the model host Dictyostelium discoideum to show that isolates of Pantoea ananatis exhibit differential grazing susceptibility, with some being resistant to grazing by the amoebae. We carried out a high-throughput genetic screen of one grazing-resistant isolate, P. ananatis BRT175, using the D. discoideum pathosystem to identify genes responsible for the resistance phenotype. Among the 26 candidate genes involved in grazing resistance, we identified rhlA and rhlB, which we show are involved in the biosynthesis of a biosurfactant that enables swarming motility in P. ananatis BRT175. Using liquid chromatography-mass spectrometry (LC-MS), the biosurfactant was shown to be a glycolipid with monohexose-C10-C10 as the primary congener. We show that this novel glycolipid biosurfactant is cytotoxic to the amoebae and is capable of compromising cellular integrity, leading to cell lysis. The production of this biosurfactant may be important for bacterial survival in the environment and could contribute to the establishment of opportunistic infections. IMPORTANCE The genetic factors used for host interaction by the opportunistic human pathogen Pantoea ananatis are largely unknown. We identified two genes that are important for the production of a biosurfactant that confers grazing resistance against the social amoeba Dictyostelium discoideum. We show that the biosurfactant, which exhibits cytotoxicity toward the amoebae, is a glycolipid that incorporates a hexose rather than rhamnose. The production of this biosurfactant may confer a competitive advantage in the environment and could potentially contribute to the establishment of opportunistic infections.

  15. A Novel Glycolipid Biosurfactant Confers Grazing Resistance upon Pantoea ananatis BRT175 against the Social Amoeba Dictyostelium discoideum

    PubMed Central

    Smith, Derek D. N.; Nickzad, Arvin

    2016-01-01

    ABSTRACT Pantoea is a versatile genus of bacteria with both plant- and animal-pathogenic strains, some of which have been suggested to cause human infections. There is, however, limited knowledge on the potential determinants used for host association and pathogenesis in animal systems. In this study, we used the model host Dictyostelium discoideum to show that isolates of Pantoea ananatis exhibit differential grazing susceptibility, with some being resistant to grazing by the amoebae. We carried out a high-throughput genetic screen of one grazing-resistant isolate, P. ananatis BRT175, using the D. discoideum pathosystem to identify genes responsible for the resistance phenotype. Among the 26 candidate genes involved in grazing resistance, we identified rhlA and rhlB, which we show are involved in the biosynthesis of a biosurfactant that enables swarming motility in P. ananatis BRT175. Using liquid chromatography-mass spectrometry (LC-MS), the biosurfactant was shown to be a glycolipid with monohexose-C10-C10 as the primary congener. We show that this novel glycolipid biosurfactant is cytotoxic to the amoebae and is capable of compromising cellular integrity, leading to cell lysis. The production of this biosurfactant may be important for bacterial survival in the environment and could contribute to the establishment of opportunistic infections. IMPORTANCE The genetic factors used for host interaction by the opportunistic human pathogen Pantoea ananatis are largely unknown. We identified two genes that are important for the production of a biosurfactant that confers grazing resistance against the social amoeba Dictyostelium discoideum. We show that the biosurfactant, which exhibits cytotoxicity toward the amoebae, is a glycolipid that incorporates a hexose rather than rhamnose. The production of this biosurfactant may confer a competitive advantage in the environment and could potentially contribute to the establishment of opportunistic infections. PMID

  16. Integrative conjugative element ICE-βox confers oxidative stress resistance to Legionella pneumophila in vitro and in macrophages.

    PubMed

    Flynn, Kaitlin J; Swanson, Michele S

    2014-01-01

    ABSTRACT Integrative conjugative elements (ICEs) are mobile blocks of DNA that can contribute to bacterial evolution by self-directed transmission of advantageous traits. Here, we analyze the activity of a putative 65-kb ICE harbored by Legionella pneumophila using molecular genetics, conjugation assays, a phenotype microarray screen, and macrophage infections. The element transferred to a naive L. pneumophila strain, integrated site-specifically, and conferred increased resistance to oxacillin, penicillin, hydrogen peroxide, and bleach. Furthermore, the element increased survival of L. pneumophila within restrictive mouse macrophages. In particular, this ICE protects L. pneumophila from phagocyte oxidase activity, since mutation of the macrophage NADPH oxidase eliminated the fitness difference between strains that carried and those that lacked the mobile element. Renamed ICE-βox (for β-lactam antibiotics and oxidative stress), this transposable element is predicted to contribute to the emergence of L. pneumophila strains that are more fit in natural and engineered water systems and in macrophages. IMPORTANCE Bacteria evolve rapidly by acquiring new traits via horizontal gene transfer. Integrative conjugative elements (ICEs) are mobile blocks of DNA that encode the machinery necessary to spread among bacterial populations. ICEs transfer antibiotic resistance and other bacterial survival factors as cargo genes carried within the element. Here, we show that Legionella pneumophila, the causative agent of Legionnaires' disease, carries ICE-βox, which enhances the resistance of this opportunistic pathogen to bleach and β-lactam antibiotics. Moreover, L. pneumophila strains encoding ICE-βox are more resistant to macrophages that carry phagocyte oxidase. Accordingly, ICE-βox is predicted to increase the fitness of L. pneumophila in natural and engineered waters and in humans. To our knowledge, this is the first description of an ICE that confers oxidative

  17. Fine mapping of qSB-11(LE), the QTL that confers partial resistance to rice sheath blight.

    PubMed

    Zuo, Shimin; Yin, Yuejun; Pan, Cunhong; Chen, Zongxiang; Zhang, Yafang; Gu, Shiliang; Zhu, Lihuang; Pan, Xuebiao

    2013-05-01

    Sheath blight (SB), caused by Rhizoctonia solani kühn, is one of the most serious global rice diseases. No major resistance genes to SB have been identified so far. All discovered loci are quantitative resistance to rice SB. The qSB-11(LE) resistance quantitative trait locus (QTL) has been previously reported on chromosome 11 of Lemont (LE). In this study, we report the precise location of qSB-11 (LE) . We developed a near isogenic line, NIL-qSB11(TQ), by marker-assisted selection that contains susceptible allele(s) from Teqing (TQ) at the qSB-11 locus in the LE genetic background. NIL-qSB11(TQ) shows higher susceptibility to SB than LE in both field and greenhouse tests, suggesting that this region of LE contains a QTL contributing to SB resistance. In order to eliminate the genetic background effects and increase the accuracy of phenotypic evaluation, a total of 112 chromosome segment substitution lines (CSSLs) with the substituted segment specific to the qSB-11 (LE) region were produced as the fine mapping population. The genetic backgrounds and morphological characteristics of these CSSLs are similar to those of the recurrent parent LE. The donor TQ chromosomal segments in these CSSL lines contiguously overlap to bridge the qSB-11 (LE) region. Through artificial inoculation, all CSSLs were evaluated for resistance to SB in the field in 2005. For the recombinant lines, their phenotypes were evaluated in the field for another 3 years and during the final year were also evaluated in a controlled greenhouse environment, showing a consistent phenotype in SB resistance across years and conditions. After comparing the genotypic profile of each CSSL with its phenotype, we are able to localize qSB-11 (LE) to the region defined by two cleaved-amplified polymorphic sequence markers, Z22-27C and Z23-33C covering 78.871 kb, based on the rice reference genome. Eleven putative genes were annotated within this region and three of them were considered the most likely

  18. Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa.

    PubMed

    Jiwan, Derick; Roalson, Eric H; Main, Dorrie; Dhingra, Amit

    2013-12-01

    Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.

  19. Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa.

    PubMed

    Jiwan, Derick; Roalson, Eric H; Main, Dorrie; Dhingra, Amit

    2013-12-01

    Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae. PMID:23728780

  20. A Hepatitis C Virus Envelope Polymorphism Confers Resistance to Neutralization by Polyclonal Sera and Broadly Neutralizing Monoclonal Antibodies

    PubMed Central

    Wasilewski, Lisa N.; El-Diwany, Ramy; Munshaw, Supriya; Snider, Anna E.; Brady, Jillian K.; Osburn, William O.; Ray, Stuart C.

    2016-01-01

    ABSTRACT Hepatitis C virus (HCV) infection is a global health problem, with millions of chronically infected individuals at risk for cirrhosis and hepatocellular carcinoma. HCV vaccine development is vital in the effort toward disease control and eradication, an undertaking aided by an increased understanding of the mechanisms of resistance to broadly neutralizing antibodies (bNAbs). In this study, we identified HCV codons that vary deep in a phylogenetic tree of HCV sequences and showed that a polymorphism at one of these positions renders Bole1a, a computationally derived, ancestral genotype 1a HCV strain, resistant to neutralization by both polyclonal-HCV-infected plasma and multiple broadly neutralizing monoclonal antibodies with unique binding epitopes. This bNAb resistance mutation reduces replicative fitness, which may explain the persistence of both neutralization-sensitive and neutralization-resistant variants in circulating viral strains. This work identifies an important determinant of bNAb resistance in an ancestral, representative HCV genome, which may inform HCV vaccine development. IMPORTANCE Worldwide, more than 170 million people are infected with hepatitis C virus (HCV), the leading cause of hepatocellular carcinoma and liver transplantation in the United States. Despite recent significant advances in HCV treatment, a vaccine is needed. Control of the HCV pandemic with drug treatment alone is likely to fail due to limited access to treatment, reinfections in high-risk individuals, and the potential for resistance to direct-acting antivirals (DAAs). Broadly neutralizing antibodies (bNAbs) block infection by diverse HCV variants and therefore serve as a useful guide for vaccine development, but our understanding of resistance to bNAbs is incomplete. In this report, we identify a viral polymorphism conferring resistance to neutralization by both polyclonal plasma and broadly neutralizing monoclonal antibodies, which may inform HCV vaccine development

  1. Retroviral transfer of a murine cDNA for multidrug resistance confers pleiotropic drug resistance to cells without prior drug selection

    SciTech Connect

    Guild, B.C.; Mulligan, R.C.; Gros, P.; Housman, D.E.

    1988-03-01

    The authors have constructed a retrovirus expression vector that carries the murine mdr cDNA transcribed under the control of the human H4 histone promoter to examine the feasibility of efficiently transferring a multidrug resistance phenotype to cells without requiring drug selection. This approach will facilitate the transfer of mdr cDNA to hematopoietic progenitor cells for the study of multidrug resistance in vivo. The retrovirus vector pHmdr has been used for transmission and expression of the mdr cDNA in initially drug-sensitive NIH 3T3 fibroblasts. Selection of pHmdr infectants in the cytotoxic agents colchicine or doxorubicin gave rise to highly multidrug-resistant colonies containing a single gene copy of the vector. Moreover, in the analysis of 12 cloned unselected NIH 3T3 cell infectants, a multidrug resistance phenotype was conferred by as few as two copies of the pHmdr vector. Overexpression of the mdr cDNA in drug-selected and unselected pHmdr infectants was directly related to cell survival in three cytotoxic agents tested. These results hold significant implications for the study of multidrug resistance in vivo.

  2. FHIT loss confers cisplatin resistance in lung cancer via the AKT/NF-κB/Slug-mediated PUMA reduction.

    PubMed

    Wu, D-W; Lee, M-C; Hsu, N-Y; Wu, T-C; Wu, J-Y; Wang, Y-C; Cheng, Y-W; Chen, C-Y; Lee, H

    2015-05-01

    Fragile histidine triad (FHIT) loss by the two-hit mechanism of loss of heterozygosity and promoter hypermethylation commonly occurrs in non-small cell lung cancer (NSCLC) and may confer cisplatin resistance in NSCLC cells. However, the underlying mechanisms of FHIT loss in cisplatin resistance and the response to cisplatin-based chemotherapy in NSCLC patients have not yet been reported. In the present study, inhibition concentration of 50% cell viability induced by cisplatin (IC50) and soft agar growth and invasion capability were increased and decreased in FHIT-knockdown and -overexpressing cells, respectively. Mechanistically, Slug transcription is upregulated by AKT/NF-κB activation due to FHIT loss and, in turn, Slug suppresses PUMA expression; this decrease of PUMA by FHIT loss is responsible for cisplatin resistance. In addition, cisplatin resistance due to FHIT loss can be conquered by AKT inhibitor-perifosine in xenograft tumors. Among NSCLC patients, low FHIT, high p-AKT, high Slug and low PUMA were correlated with shorter overall survival, relapse-free survival and poorer response to cisplatin-based chemotherapy. Therefore, the AKT inhibitor perifosine might potentially overcome the resistance to cisplatin-based chemotherapy in NSCLC patients with low-FHIT tumors, and consequently improve the outcome. PMID:24998847

  3. FHIT loss confers cisplatin resistance in lung cancer via the AKT/NF-κB/Slug-mediated PUMA reduction.

    PubMed

    Wu, D-W; Lee, M-C; Hsu, N-Y; Wu, T-C; Wu, J-Y; Wang, Y-C; Cheng, Y-W; Chen, C-Y; Lee, H

    2015-05-01

    Fragile histidine triad (FHIT) loss by the two-hit mechanism of loss of heterozygosity and promoter hypermethylation commonly occurrs in non-small cell lung cancer (NSCLC) and may confer cisplatin resistance in NSCLC cells. However, the underlying mechanisms of FHIT loss in cisplatin resistance and the response to cisplatin-based chemotherapy in NSCLC patients have not yet been reported. In the present study, inhibition concentration of 50% cell viability induced by cisplatin (IC50) and soft agar growth and invasion capability were increased and decreased in FHIT-knockdown and -overexpressing cells, respectively. Mechanistically, Slug transcription is upregulated by AKT/NF-κB activation due to FHIT loss and, in turn, Slug suppresses PUMA expression; this decrease of PUMA by FHIT loss is responsible for cisplatin resistance. In addition, cisplatin resistance due to FHIT loss can be conquered by AKT inhibitor-perifosine in xenograft tumors. Among NSCLC patients, low FHIT, high p-AKT, high Slug and low PUMA were correlated with shorter overall survival, relapse-free survival and poorer response to cisplatin-based chemotherapy. Therefore, the AKT inhibitor perifosine might potentially overcome the resistance to cisplatin-based chemotherapy in NSCLC patients with low-FHIT tumors, and consequently improve the outcome.

  4. The A395T mutation in ERG11 gene confers fluconazole resistance in Candida tropicalis causing candidemia.

    PubMed

    Tan, Jingwen; Zhang, Jinqing; Chen, Wei; Sun, Yi; Wan, Zhe; Li, Ruoyu; Liu, Wei

    2015-04-01

    The mechanism of fluconazole resistance in Candida tropicalis is still unclear. Recently, we isolated a fluconazole-resistant strain of C. tropicalis from the blood specimen of a patient with candidemia in China. In vitro antifungal susceptibility of the isolate was determined by using CLSI M27-A3 and E-test methods. The sequence of ERG11 gene was then analyzed, and the three-dimensional model of Erg11p encoded by ERG11 gene was also investigated. The sequencing of ERG11 gene revealed the mutation of A395T in this fluconazole-resistant isolate of C. tropicalis, resulting in the Y132F substitution in Erg11p. Sequence alignment and three-dimensional model comparison of Erg11ps showed high similarity between fluconazole-susceptible isolates of C. tropicalis and Candida albicans. The comparison of the three-dimensional models of Erg11ps demonstrated that the position of the Y132F substitution in this isolate of C. tropicalis is identical to the isolate of C. albicans with fluconazole resistance resulting from Y132F substitution in Erg11p. Hence, we ascertain that the Y132F substitution of Erg11p caused by A395T mutation in ERG11 gene confers the fluconazole resistance in C. tropicalis.

  5. Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

    PubMed Central

    Balasubramanian, V; Pavelka, M S; Bardarov, S S; Martin, J; Weisbrod, T R; McAdam, R A; Bloom, B R; Jacobs, W R

    1996-01-01

    Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines. PMID:8550428

  6. Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity

    SciTech Connect

    Okawa, Hiromi; Motohashi, Hozumi; Kobayashi, Akira; Aburatani, Hiroyuki; Kensler, Thomas W.; Yamamoto, Masayuki . E-mail: masi@tara.tsukuba.ac.jp

    2006-01-06

    Nrf2 is a key regulator of many detoxifying enzyme genes, and cytoplasmic protein Keap1 represses the Nrf2 activity under quiescent conditions. Germ line deletion of the keap1 gene results in constitutive activation of Nrf2, but the pups unexpectedly died before weaning. To investigate how constitutive activation of Nrf2 influences the detoxification system in adult mice, we generated mice bearing a hepatocyte-specific disruption of the keap1 gene. Homozygous mice were viable and their livers displayed no apparent abnormalities, but nuclear accumulation of Nrf2 is elevated. Microarray analysis revealed that, while many detoxifying enzyme genes are highly expressed, some of the typical Nrf2-dependent genes are only marginally increased in the Keap1-deficient liver. The mutant mice were significantly more resistant to toxic doses of acetaminophen than control animals. These results demonstrate that chronic activation of Nrf2 confers animals with resistance to xenobiotics without affecting the morphological and physiological integrity of hepatocytes.

  7. Phloem-specific expression of the lectin gene from Allium sativum confers resistance to the sap-sucker Nilaparvata lugens.

    PubMed

    Chandrasekhar, Kottakota; Vijayalakshmi, Muvva; Vani, Kalasamudramu; Kaul, Tanushri; Reddy, Malireddy K

    2014-05-01

    Rice production is severely hampered by insect pests. Garlic lectin gene (ASAL) holds great promise in conferring protection against chewing (lepidopteran) and sap-sucking (homopteran) insect pests. We have developed transgenic rice lines resistant to sap-sucking brown hopper (Nilaparvata lugens) by ectopic expression of ASAL in their phloem tissues. Molecular analyses of T0 lines confirmed stable integration of transgene. T1 lines (NP 1-2, 4-3, 11-6 & 17-7) showed active transcription and translation of ASAL transgene. ELISA revealed ASAL expression was as high as 0.95% of total soluble protein. Insect bioassays on T2 homozygous lines (NP 18 & 32) revealed significant reduction (~74-83%) in survival rate, development and fecundity of brown hoppers in comparison to wild type. Transgenics exhibited enhanced resistance (1-2 score) against brown hoppers, minimal plant damage and no growth penalty or phenotypic abnormalities. PMID:24563293

  8. Structure and function of starch and resistant starch from corn with different doses of mutant amylose-extender and floury-1 alleles.

    PubMed

    Yao, Ni; Paez, Alix V; White, Pamela J

    2009-03-11

    Four corn types with different doses of mutant amylose-extender (ae) and floury-1 (fl1) alleles, in the endosperm, including no. 1, aeaeae; no. 2, fl1fl1fl1; no. 3, aeaefl1; and no. 4, fl1fl1ae, were developed for use in making Hispanic food products with high resistant starch (RS) content. The RS percentages in the native starch (NS) of 1-4 were 55.2, 1.1, 5.7, and 1.1%, respectively. All NS were evaluated for pasting properties with a rapid viscoanalyzer (RVA) and for thermal properties with a differential scanning calorimeter (DSC). NS 1 had a low peak viscosity (PV) caused by incomplete gelatinization, whereas NS 3 had the greatest PV and breakdown of all four starch types. On the DSC, NS 2 had the lowest onset temperature and greatest enthalpy. NS 1 and 3 had similar onset and peak temperatures, both higher than those of NS 2 and 4. The gel strength of NS heated with a RVA was evaluated by using a texture analyzer immediately after RVA heating (fresh, RVA-F) and after the gel had been stored at 4 degrees C for 10 days (retrograded, RVA-R). NS 1 gel was watery and had the lowest strength (30 g) among starch gel types. NS 3 gel, although exhibiting syneresis, had greater gel strength than NS 2 and 4. The structures of the NS, the RS isolated from the NS (RS-NS), the RS isolated from RVA-F (RS-RVA-F), and the RS isolated from RVA-R (RS-RVA-R) were evaluated by using size exclusion chromatography. NS 1 had a greater percentage of amylose (AM) (58.3%) than the other NS (20.4-26.8%). The RS from all NS types (RS-NS) had a lower percentage of amylopectin (AP) and a greater percentage of low molecular weight (MW) AM than was present in the original NS materials. The RS-RVA-R from all starches had no AP or high MW AM. The percentages of longer chain lengths (DP 35-60) of NS were greater in 1 and 3 than in 2 and 4, and the percentages of smaller chain lengths (DP 10-20) were greater in 2 and 4 than in 1 and 3. In general, NS 3 seemed to have inherited some pasting

  9. Keeping the Teacher at Arm's Length: Student Resistance in Writing Conferences in Two High School Classrooms

    ERIC Educational Resources Information Center

    Consalvo, Annamary; Maloch, Beth

    2015-01-01

    The purpose of this paper is to explore a subset of findings taken from yearlong qualitative study of writing conferences in two diversely populated, urban high school classrooms. Drawing on multiple data sources, we used case study and discourse analytic methods to follow two focal students across the year in order to examine instructional and…

  10. The Human Immunodeficiency Virus Type 1 Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation I132M Confers Hypersensitivity to Nucleoside Analogs▿

    PubMed Central

    Ambrose, Zandrea; Herman, Brian D.; Sheen, Chih-Wei; Zelina, Shannon; Moore, Katie L.; Tachedjian, Gilda; Nissley, Dwight V.; Sluis-Cremer, Nicolas

    2009-01-01

    We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity. PMID:19193782

  11. Molecular basis for increased susceptibility of isolates with atazanavir resistance-conferring substitution I50L to other protease inhibitors.

    PubMed

    Yanchunas, Joseph; Langley, David R; Tao, Li; Rose, Ronald E; Friborg, Jacques; Colonno, Richard J; Doyle, Michael L

    2005-09-01

    Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2- to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2' (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.

  12. 21-Hydroxylase gene mutant allele CYP21A2*15 strongly linked to the resistant HLA haplotype B*14:02-DRB1*01:02 in chronic Chagas disease.

    PubMed

    del Puerto, Florencia; Kikuchi, Mihoko; Nishizawa, Juan Eiki; Roca, Yelin; Avila, Cinthia; Gianella, Alberto; Lora, Javier; Gutierrez Velarde, Freddy Udalrico; Hirayama, Kenji

    2013-06-01

    We previously reported protective haplotype HLA-B*14:02-DRB1*01:02 against chronic Chagas disease in Bolivia. The V281L mutant allele of the 21-Hydroxylase gene, CYP21A2*15, is reported to be located in the Class III region of the Human leukocyte antigen region and linked to the haplotype HLA-B*14:02-DRB1*01:02. The mutant allele might play a primary role in the pathogenesis of chronic Chagas disease in the associated HLA region. We analyzed the frequency of this allele in the same subjects for the previous one. The statistical analysis showed a significant association of the CYP21A2*15 with resistance to severe chronic Chagas disease (OR=0.207273; Pv=0.0041). However, there is no significant tendency of the mutant gene contribution to the resistance after the elimination of the HLA-B*14:02-DRB1*01:02 linked mutants (OR=0.38; Pv=0.1533). Although the frequency of the CYP21A2*15 was small, we found no primary contribution of this mutation to the protection against chronic Chagas disease.

  13. Bordetella pertussis lipid A glucosamine modification confers resistance to cationic antimicrobial peptides and increases resistance to outer membrane perturbation.

    PubMed

    Shah, Nita R; Hancock, Robert E W; Fernandez, Rachel C

    2014-08-01

    Bordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

  14. Prevalence of mutations conferring resistance among multi- and extensively drug-resistant Mycobacterium tuberculosis isolates in China.

    PubMed

    Chen, Yan; Zhao, Bing; Liu, Hai-can; Sun, Qing; Zhao, Xiu-qin; Liu, Zhi-guang; Wan, Kang-lin; Zhao, Li-li

    2016-03-01

    To identify the mutations in multi- and extensively drug-resistant tuberculosis isolates and to evaluate the use of molecular markers of resistance, we analyzed 257 multi- and extensively drug-resistant isolates and 64 pan-sensitive isolates from 23 provinces in China. Seven loci associated with drug resistance, including rpoB for rifampin (RIF), katG, inhA and oxyR-ahpC for isoniazid (INH), gyrA and gyrB for ofloxacin (OFX), and rrs for kanmycin (KAN), were examined by DNA sequencing. Compared with the phenotypic data, the sensitivity and specificity for DNA sequencing were 91.1% and 98.4% for RIF, 80.2% and 98.4% for INH, 72.2% and 98.3% for OFX and 40% and 98.2% for KAN, respectively. The most common mutations found in RIF, INH, OFX and KAN resistance were Ser531Leu (48.2%) in rpoB, Ser315Thr (49.8%) in katG, C(-15)T (10.5%) in inhA, Asp94Gly (20.3%), Asp94Ala (12.7%) and Ala90Val (21.5%) in gyrA, and A1401G (40%) in rrs. This molecular information will be helpful to establish new molecular biology-based methods for diagnosing multi- and extensively drug-resistant tuberculosis in China.

  15. New susceptibility and resistance HLA-DP alleles to HBV-related diseases identified by a trans-ethnic association study in Asia.

    PubMed

    Nishida, Nao; Sawai, Hiromi; Kashiwase, Koichi; Minami, Mutsuhiko; Sugiyama, Masaya; Seto, Wai-Kay; Yuen, Man-Fung; Posuwan, Nawarat; Poovorawan, Yong; Ahn, Sang Hoon; Han, Kwang-Hyub; Matsuura, Kentaro; Tanaka, Yasuhito; Kurosaki, Masayuki; Asahina, Yasuhiro; Izumi, Namiki; Kang, Jong-Hon; Hige, Shuhei; Ide, Tatsuya; Yamamoto, Kazuhide; Sakaida, Isao; Murawaki, Yoshikazu; Itoh, Yoshito; Tamori, Akihiro; Orito, Etsuro; Hiasa, Yoichi; Honda, Masao; Kaneko, Shuichi; Mita, Eiji; Suzuki, Kazuyuki; Hino, Keisuke; Tanaka, Eiji; Mochida, Satoshi; Watanabe, Masaaki; Eguchi, Yuichiro; Masaki, Naohiko; Murata, Kazumoto; Korenaga, Masaaki; Mawatari, Yoriko; Ohashi, Jun; Kawashima, Minae; Tokunaga, Katsushi; Mizokami, Masashi

    2014-01-01

    Previous studies have revealed the association between SNPs located on human leukocyte antigen (HLA) class II genes, including HLA-DP and HLA-DQ, and chronic hepatitis B virus (HBV) infection, mainly in Asian populations. HLA-DP alleles or haplotypes associated with chronic HBV infection or disease progression have not been fully identified in Asian populations. We performed trans-ethnic association analyses of HLA-DPA1, HLA-DPB1 alleles and haplotypes with hepatitis B virus infection and disease progression among Asian populations comprising Japanese, Korean, Hong Kong, and Thai subjects. To assess the association between HLA-DP and chronic HBV infection and disease progression, we conducted high-resolution (4-digit) HLA-DPA1 and HLA-DPB1 genotyping in a total of 3,167 samples, including HBV patients, HBV-resolved individuals and healthy controls. Trans-ethnic association analyses among Asian populations identified a new risk allele HLA-DPB1*09 ∶ 01 (P = 1.36 × 10(-6); OR= 1.97; 95% CI, 1.50-2.59) and a new protective allele DPB1*02 ∶ 01 (P = 5.22 × 10(-6); OR = 0.68; 95% CI, 0.58-0.81) to chronic HBV infection, in addition to the previously reported alleles. Moreover, DPB1*02 ∶ 01 was also associated with a decreased risk of disease progression in chronic HBV patients among Asian populations (P = 1.55 × 10(-7); OR = 0.50; 95% CI, 0.39-0.65). Trans-ethnic association analyses identified Asian-specific associations of HLA-DP alleles and haplotypes with HBV infection or disease progression. The present findings will serve as a base for future functional studies of HLA-DP molecules in order to understand the pathogenesis of HBV infection and the development of hepatocellular carcinoma. PMID:24520320

  16. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter

    PubMed Central

    Petersen, Ines; Gabryszewski, Stanislaw J.; Johnston, Geoffrey L.; Dhingra, Satish K.; Ecker, Andrea; Lewis, Rebecca E.; de Almeida, Mariana Justino; Straimer, Judith; Henrich, Philipp H.; Palatulan, Eugene; Johnson, David J.; Coburn-Flynn, Olivia; Sanchez, Cecilia; Lehane, Adele M.; Lanzer, Michael; Fidock, David A.

    2015-01-01

    Summary The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within-host competition with wild-type drug-sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC50 values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild-type pfcrt in co-culture competition assays. These three alleles mediated cross-resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first-line artemisinin-based combination therapy. These data reveal ongoing region-specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine-resistant malaria. PMID:25898991

  17. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter.

    PubMed

    Petersen, Ines; Gabryszewski, Stanislaw J; Johnston, Geoffrey L; Dhingra, Satish K; Ecker, Andrea; Lewis, Rebecca E; de Almeida, Mariana Justino; Straimer, Judith; Henrich, Philipp P; Palatulan, Eugene; Johnson, David J; Coburn-Flynn, Olivia; Sanchez, Cecilia; Lehane, Adele M; Lanzer, Michael; Fidock, David A

    2015-07-01

    The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within-host competition with wild-type drug-sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC50 values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild-type pfcrt in co-culture competition assays. These three alleles mediated cross-resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first-line artemisinin-based combination therapy. These data reveal ongoing region-specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine-resistant malaria.

  18. Expression of human glutathione S-transferases in Saccharomyces cerevisiae confers resistance to the anticancer drugs adriamycin and chlorambucil.

    PubMed Central

    Black, S M; Beggs, J D; Hayes, J D; Bartoszek, A; Muramatsu, M; Sakai, M; Wolf, C R

    1990-01-01

    Adaptation and resistance to chemicals in the environment is a critical part of the evolutionary process. As a result, a wide variety of defence systems that protect cells against chemical insult have evolved. Such chemical resistance mechanisms appear to play a central role in determining the sensitivity of human tumours to treatment with chemotherapeutic drugs. The glutathione S-transferases (GST) are important detoxification enzymes whose over-expression has been associated with drug-resistance. In order to evaluate this possibility we have expressed the human Alpha-class and Pi-class GST cDNAs that encode GST B1B1 and GST pi in the yeast Saccharomyces cerevisiae. The expression of GST B1B1 or GST pi resulted in a marked reduction in the cytotoxic effects of chlorambucil, a bifunctional alkylating agent, and an anthracycline, adriamycin. These data provide direct evidence that the over-expression of GST in cells can confer resistance to anticancer drugs. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:2194447

  19. Resistance to Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous Onchocerca species.

    PubMed

    Carlow, C K; Bianco, A E

    1987-06-01

    Embryonic stages of various Onchocerca species have been used to stimulate resistance in CBA mice to challenge injections with the microfilariae of Onchocerca lienalis. Comparable levels of resistance to challenge (29-37% reductions) were conferred by living, freeze-killed, or sonicated organisms administered with Freunds' Complete Adjuvant (FCA). Antigens extracted in saline, or with the detergent sodium deoxycholate, were also protective. Adjuvants enhanced the protective effect, particularly FCA (78% reduction), Freunds' Incomplete Adjuvant (74% reduction), aluminum hydroxide (70% reduction) and Bordetella pertussis (70% reduction). Detergent extracts prepared from intact embryos with n-octyl glucoside also stimulated significant levels of protection against microfilarial challenge when given with FCA (37-45% reductions). Levels of resistance induced by immunizations with intact organisms were greatest following subcutaneous (s.c.) injection over the neck or by intramuscular inoculation. Soluble extracts were also particially effective given by s.c. inguinal or intraperitoneal injection. A time-interval of greater than 3 weeks between the completion of immunization and challenge was required for the expression of immunity. Cross-protection against challenge with O. lienalis microfilariae was also afforded to mice by immunization with intact embryos or detergent extracts of Onchocerca gutturosa (45 and 34% reductions), Onchocerca gibsoni (66 and 47% reductions) or Onchocerca volvulus (58 and 41% reductions). It is concluded that the embryonic stages of both human and animal parasites provide a source of cross-protective antigens of value in studies on resistance to Onchocerca microfilariae in experimental hosts.

  20. Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation.

    PubMed

    Kim, Min Hwan; Kim, Jongshin; Hong, Hyowon; Lee, Si-Hyung; Lee, June-Koo; Jung, Eunji; Kim, Joon

    2016-03-01

    The activation of transcriptional coactivators YAP and its paralog TAZ has been shown to promote resistance to anti-cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton architecture. However, the influence of actin remodeling on cancer drug resistance remains largely unexplored. Here, we report a pivotal role of actin remodeling in YAP/TAZ-dependent BRAF inhibitor resistance in BRAF V600E mutant melanoma cells. Melanoma cells resistant to the BRAF inhibitor PLX4032 exhibit an increase in actin stress fiber formation, which appears to promote the nuclear accumulation of YAP/TAZ. Knockdown of YAP/TAZ reduces the viability of resistant melanoma cells, whereas overexpression of constitutively active YAP induces resistance. Moreover, inhibition of actin polymerization and actomyosin tension in melanoma cells suppresses both YAP/TAZ activation and PLX4032 resistance. Our siRNA library screening identifies actin dynamics regulator TESK1 as a novel vulnerable point of the YAP/TAZ-dependent resistance pathway. These results suggest that inhibition of actin remodeling is a potential strategy to suppress resistance in BRAF inhibitor therapies.

  1. HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction

    PubMed Central

    Kang, Hyo Jin; Yi, Yong Weon; Hong, Young Bin; Kim, Hee Jeong; Jang, Young-Joo; Seong, Yeon-Sun; Bae, Insoo

    2014-01-01

    Overexpression and/or activation of HER2 confers resistance of cancer cells to chemotherapeutic drugs. NRF2 also gives drug resistance of cancer cells through induction of detoxification and/or drug efflux proteins. Although several upstream effectors of NRF2 overlapped with the downstream molecules of HER2 pathway, no direct link between HER2 and NRF2 has ever been established. Here, we identified that co-expression of a constitutively active HER2 (HER2CA) and NRF2 increased the levels of NRF2 target proteins, HO-1 and MRP5. We also identified HER2CA activated the DNA-binding of NRF2 and the antioxidant response element (ARE)-mediated transcription in an NRF2-dependent manner. In addition, NRF2 and HER2CA cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes including GSTA2, GSTP1, CYP3A4, HO-1, MRP1, and MRP5. We also demonstrated that NRF2 binds to HER2 not only in transiently transfected HEK293T cells but also in HER2-amplified breast cancer cells. Functionally, overexpression of HER2CA gave resistance of MCF7 breast cancer cells to either paraquat or doxorubicin. Overexpression of dominant negative NRF2 (DN-NRF2) reduced the HER2CA-induced resistance of MCF7 cells to these agents. Taken together, these results suggest that active HER2 binds and regulates the NRF2-dependent transcriptional activation and induces drug resistance of cancer cells. PMID:25467193

  2. Towards Positional Isolation of Three Quantitative Trait Loci Conferring Resistance to Powdery Mildew in Two Spanish Barley Landraces

    PubMed Central

    Silvar, Cristina; Perovic, Dragan; Nussbaumer, Thomas; Spannagl, Manuel; Usadel, Björn; Casas, Ana; Igartua, Ernesto; Ordon, Frank

    2013-01-01

    Three quantitative trait loci (QTL) conferring broad spectrum resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei, were previously identified on chromosomes 7HS, 7HL and 6HL in the Spanish barley landrace-derived lines SBCC097 and SBCC145. In the present work, a genome-wide putative linear gene index of barley (Genome Zipper) and the first draft of the physical, genetic and functional sequence of the barley genome were used to go one step further in the shortening and explicit demarcation on the barley genome of these regions conferring resistance to powdery mildew as well as in the identification of candidate genes. First, a comparative analysis of the target regions to the barley Genome Zippers of chromosomes 7H and 6H allowed the development of 25 new gene-based molecular markers, which slightly better delimit the QTL intervals. These new markers provided the framework for anchoring of genetic and physical maps, figuring out the outline of the barley genome at the target regions in SBCC097 and SBCC145. The outermost flanking markers of QTLs on 7HS, 7HL and 6HL defined a physical area of 4 Mb, 3.7 Mb and 3.2 Mb, respectively. In total, 21, 10 and 16 genes on 7HS, 7HL and 6HL, respectively, could be interpreted as potential candidates to explain the resistance to powdery mildew, as they encode proteins of related functions with respect to the known pathogen defense-related processes. The majority of these were annotated as belonging to the NBS-LRR class or protein kinase family. PMID:23826271

  3. The miR9863 family regulates distinct Mla alleles in barley to attenuate NLR receptor-triggered disease resistance and cell-death signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley Mla alleles encode coiled-coil (CC), nucleotide binding and leucine-rich repeat (NB-LRR) intracellular receptors that trigger isolate-specific immune responses against the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). How Mla or NB-LRR genes in grass species are regulated at p...

  4. Evolution and diversity of the mechanisms endowing resistance to herbicides inhibiting acetolactate-synthase (ALS) in corn poppy (Papaver rhoeas L.).

    PubMed

    Délye, Christophe; Pernin, Fanny; Scarabel, Laura

    2011-02-01

    We investigated the diversity of mechanisms conferring resistance to herbicides inhibiting acetolactate synthase (ALS) in corn poppy (Papaver rhoeas L.) and the processes underlying the selection for resistance. Six mutant ALS alleles, Arg₁₉₇, His₁₉₇, Leu₁₉₇, Ser₁₉₇, Thr₁₉₇ and Leu₅₇₄ were identified in five Italian populations. Different alleles were found in a same population or a same plant. Comparison of individual plant phenotype (herbicide sensitivity) and genotype (amino-acid substitution(s) at codon 197) showed that all mutant ALS alleles conferred dominant resistance to the field rate of the sulfonylurea tribenuron and moderate or no resistance to the field rate of the triazolopyrimidine florasulam. Depending on the allele, dominant or partially dominant resistance to the field rate of the imidazolinone imazamox was observed. Putative non-target-site resistance mechanisms were also likely present in the populations investigated. The derived Cleaved Amplified Polymorphic Sequence assays targeting ALS codons crucial for herbicide sensitivity developed in this work will facilitate the detection of resistance due to mutant ALS alleles. Nucleotide variation around codon 197 indicated that mutant ALS alleles evolved by multiple, independent appearances. Resistance to ALS inhibitors in P. rhoeas clearly evolved by redundant evolution of a set of mutant ALS alleles and likely of non-target-site mechanisms.

  5. Evolution and diversity of the mechanisms endowing resistance to herbicides inhibiting acetolactate-synthase (ALS) in corn poppy (Papaver rhoeas L.).

    PubMed

    Délye, Christophe; Pernin, Fanny; Scarabel, Laura

    2011-02-01

    We investigated the diversity of mechanisms conferring resistance to herbicides inhibiting acetolactate synthase (ALS) in corn poppy (Papaver rhoeas L.) and the processes underlying the selection for resistance. Six mutant ALS alleles, Arg₁₉₇, His₁₉₇, Leu₁₉₇, Ser₁₉₇, Thr₁₉₇ and Leu₅₇₄ were identified in five Italian populations. Different alleles were found in a same population or a same plant. Comparison of individual plant phenotype (herbicide sensitivity) and genotype (amino-acid substitution(s) at codon 197) showed that all mutant ALS alleles conferred dominant resistance to the field rate of the sulfonylurea tribenuron and moderate or no resistance to the field rate of the triazolopyrimidine florasulam. Depending on the allele, dominant or partially dominant resistance to the field rate of the imidazolinone imazamox was observed. Putative non-target-site resistance mechanisms were also likely present in the populations investigated. The derived Cleaved Amplified Polymorphic Sequence assays targeting ALS codons crucial for herbicide sensitivity developed in this work will facilitate the detection of resistance due to mutant ALS alleles. Nucleotide variation around codon 197 indicated that mutant ALS alleles evolved by multiple, independent appearances. Resistance to ALS inhibitors in P. rhoeas clearly evolved by redundant evolution of a set of mutant ALS alleles and likely of non-target-site mechanisms. PMID:21421378

  6. A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis.

    PubMed

    Ando, Hiroki; Miyoshi-Akiyama, Tohru; Watanabe, Shinya; Kirikae, Teruo

    2014-02-01

    Drug resistance in Mycobacterium tuberculosis (Mtb) is caused by mutations in restricted regions of the genome. Mutations in katG, the promoter region of the mabA-inhA operon, and inhA are those most frequently responsible for isoniazid (INH) resistance. Several INH-resistant (INH(r) ) Mtb clinical isolates without mutations in these regions have been described, however, indicating that there are as yet undetermined mechanisms of INH resistance. We identified the mabA(g609a) silent mutation in a significant number of INH(r)  Mtb clinical isolates without known INH resistance mutations. A laboratory strain, H37Rv, constructed with mabA(g609a) , was resistant to INH. We show here that the mabA(g609a) mutation resulted in the upregulation of inhA, a gene encoding a target for INH, converting the region adjacent to the mutation into an alternative promoter for inhA. The mabA(g609a) silent mutation results in a novel mechanism of INH resistance, filling in a missing piece of INH resistance in Mtb.

  7. Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

    SciTech Connect

    Somerville, Chris R.; Scieble, Wolf

    2000-10-11

    Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  8. Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

    DOEpatents

    Somerville, Chris R.; Scheible, Wolf

    2007-07-10

    Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  9. Cyp12a4 confers lufenuron resistance in a natural population of Drosophila melanogaster.

    PubMed

    Bogwitz, Michael R; Chung, Henry; Magoc, Lorin; Rigby, Sheena; Wong, Wayn; O'Keefe, Melanie; McKenzie, John A; Batterham, Philip; Daborn, Phillip J

    2005-09-01

    Lufenuron is an insect growth regulator insecticide mainly used for the control of the cat flea. To understand mechanisms of resistance to lufenuron, we have characterized lufenuron resistance in a natural population of Drosophila melanogaster. In this study we have used precise genetic mapping to identify a mechanism of lufenuron resistance: the overexpression of the cytochrome P450 gene Cyp12a4. Cyp12a4 is predicted to encode a mitochondrial cytochrome P450 enzyme. Expression of Cyp12a4 in D. melanogaster third-instar larvae was detected in the midgut and Malpighian tubules of both lufenuron-resistant and wild-type strains. The level of Cyp12a4 expression in the midgut is higher in the lufenuron-resistant strain than in wild-type strains. Driving the expression of Cyp12a4 in the midgut and Malpighian tubules by using the GAL4/UAS gene expression system results in lufenuron resistance, but it does not result in resistance to three other insecticide classes. Transgenic expression of Cyp12a4 in a ubiquitous expression pattern results in late embryonic lethality, suggesting that high-level ectopic expression of Cyp12a4 is detrimental to development. PMID:16120680

  10. Cyp12a4 confers lufenuron resistance in a natural population of Drosophila melanogaster

    PubMed Central

    Bogwitz, Michael R.; Chung, Henry; Magoc, Lorin; Rigby, Sheena; Wong, Wayn; O'Keefe, Melanie; McKenzie, John A.; Batterham, Philip; Daborn, Phillip J.

    2005-01-01

    Lufenuron is an insect growth regulator insecticide mainly used for the control of the cat flea. To understand mechanisms of resistance to lufenuron, we have characterized lufenuron resistance in a natural population of Drosophila melanogaster. In this study we have used precise genetic mapping to identify a mechanism of lufenuron resistance: the overexpression of the cytochrome P450 gene Cyp12a4. Cyp12a4 is predicted to encode a mitochondrial cytochrome P450 enzyme. Expression of Cyp12a4 in D. melanogaster third-instar larvae was detected in the midgut and Malpighian tubules of both lufenuron-resistant and wild-type strains. The level of Cyp12a4 expression in the midgut is higher in the lufenuron-resistant strain than in wild-type strains. Driving the expression of Cyp12a4 in the midgut and Malpighian tubules by using the GAL4/UAS gene expression system results in lufenuron resistance, but it does not result in resistance to three other insecticide classes. Transgenic expression of Cyp12a4 in a ubiquitous expression pattern results in late embryonic lethality, suggesting that high-level ectopic expression of Cyp12a4 is detrimental to development. PMID:16120680

  11. Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens.

    PubMed

    Rivero, Mercedes; Furman, Nicolás; Mencacci, Nicolás; Picca, Pablo; Toum, Laila; Lentz, Ezequiel; Bravo-Almonacid, Fernando; Mentaberry, Alejandro

    2012-01-20

    Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.

  12. Rapid, high-throughput, multiplex, real-time PCR for identification of mutations in the cyp51A gene of Aspergillus fumigatus that confer resistance to itraconazole.

    PubMed

    Balashov, Sergey V; Gardiner, Rebecca; Park, Steven; Perlin, David S

    2005-01-01

    Aspergillus fumigatus is an important cause of life-threatening invasive fungal disease in patients with compromised immune systems. Resistance to itraconazole in A. fumigatus is closely linked to amino acid substitutions in Cyp51A that replace Gly54. In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established. This assay uses molecular beacons corresponding to the wild-type cyp51A gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54. Molecular beacon structure design and real-time PCR conditions were optimized to increase the assay specificity. The multiplex assay was applied to the analysis of chromosomal DNA samples from a collection of 48 A. fumigatus clinical and laboratory-derived isolates, most with reduced susceptibility to itraconazole. The cyp51A allelic identities for codon 54 were established for all of the strains tested, and mutations altering Gly54 in 23 strains were revealed. These mutations included G(54)W (n = 1), G(54)E (n = 12), G(54)K (n = 3), G(54)R (n = 3), and G(54)V (n = 4). Molecular beacon assay results were confirmed by DNA sequencing. Multiplex real-time PCR with molecular beacons is a powerful technique for allele differentiation and analysis of resistance mutations that is dynamic and suitable for rapid high-throughput assessment of drug resistance.

  13. New Susceptibility and Resistance HLA-DP Alleles to HBV-Related Diseases Identified by a Trans-Ethnic Association Study in Asia

    PubMed Central

    Kashiwase, Koichi; Minami, Mutsuhiko; Sugiyama, Masaya; Seto, Wai-Kay; Yuen, Man-Fung; Posuwan, Nawarat; Poovorawan, Yong; Ahn, Sang Hoon; Han, Kwang-Hyub; Matsuura, Kentaro; Tanaka, Yasuhito; Kurosaki, Masayuki; Asahina, Yasuhiro; Izumi, Namiki; Kang, Jong-Hon; Hige, Shuhei; Ide, Tatsuya; Yamamoto, Kazuhide; Sakaida, Isao; Murawaki, Yoshikazu; Itoh, Yoshito; Tamori, Akihiro; Orito, Etsuro; Hiasa, Yoichi; Honda, Masao; Kaneko, Shuichi; Mita, Eiji; Suzuki, Kazuyuki; Hino, Keisuke; Tanaka, Eiji; Mochida, Satoshi; Watanabe, Masaaki; Eguchi, Yuichiro; Masaki, Naohiko; Murata, Kazumoto; Korenaga, Masaaki; Mawatari, Yoriko; Ohashi, Jun; Kawashima, Minae; Tokunaga, Katsushi; Mizokami, Masashi

    2014-01-01

    Previous studies have revealed the association between SNPs located on human leukocyte antigen (HLA) class II genes, including HLA-DP and HLA-DQ, and chronic hepatitis B virus (HBV) infection, mainly in Asian populations. HLA-DP alleles or haplotypes associated with chronic HBV infection or disease progression have not been fully identified in Asian populations. We performed trans-ethnic association analyses of HLA-DPA1, HLA-DPB1 alleles and haplotypes with hepatitis B virus infection and disease progression among Asian populations comprising Japanese, Korean, Hong Kong, and Thai subjects. To assess the association between HLA-DP and chronic HBV infection and disease progression, we conducted high-resolution (4-digit) HLA-DPA1 and HLA-DPB1 genotyping in a total of 3,167 samples, including HBV patients, HBV-resolved individuals and healthy controls. Trans-ethnic association analyses among Asian populations identified a new risk allele HLA-DPB1*09∶01 (P = 1.36×10−6; OR = 1.97; 95% CI, 1.50–2.59) and a new protective allele DPB1*02∶01 (P = 5.22×10−6; OR = 0.68; 95% CI, 0.58–0.81) to chronic HBV infection, in addition to the previously reported alleles. Moreover, DPB1*02∶01 was also associated with a decreased risk of disease progression in chronic HBV patients among Asian populations (P = 1.55×10−7; OR = 0.50; 95% CI, 0.39–0.65). Trans-ethnic association analyses identified Asian-specific associations of HLA-DP alleles and haplotypes with HBV infection or disease progression. The present findings will serve as a base for future functional studies of HLA-DP molecules in order to understand the pathogenesis of HBV infection and the development of hepatocellular carcinoma. PMID:24520320

  14. Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system.

    PubMed

    Simkovsky, Ryan; Daniels, Emy F; Tang, Karen; Huynh, Stacey C; Golden, Susan S; Brahamsha, Bianca

    2012-10-01

    The grazing activity of predators on photosynthetic organisms is a major mechanism of mortality and population restructuring in natural environments. Grazing is also one of the primary difficulties in growing cyanobacteria and other microalgae in large, open ponds for the production of biofuels, as contaminants destroy valuable biomass and prevent stable, continuous production of biofuel crops. To address this problem, we have isolated a heterolobosean amoeba, HGG1, that grazes upon unicellular and filamentous freshwater cyanobacterial species. We have established a model predator-prey system using this amoeba and Synechococcus elongatus PCC 7942. Application of amoebae to a library of mutants of S. elongatus led to the identification of a grazer-resistant knockout mutant of the wzm ABC O-antigen transporter gene, SynPCC7942_1126. Mutations in three other genes involved in O-antigen synthesis and transport also prevented the expression of O-antigen and conferred resistance to HGG1. Complementation of these rough mutants returned O-antigen expression and susceptibility to amoebae. Rough mutants are easily identifiable by appearance, are capable of autoflocculation, and do not display growth defects under standard laboratory growth conditions, all of which are desired traits for a biofuel production strain. Thus, preventing the production of O-antigen is a pathway for producing resistance to grazing by certain amoebae.

  15. Diminished representation of HIV-1 variants containing select drug resistance-conferring mutations in primary HIV-1 infection.

    PubMed

    Turner, Dan; Brenner, Bluma; Routy, Jean-Pierre; Moisi, Daniela; Rosberger, Zeev; Roger, Michel; Wainberg, Mark A

    2004-12-15

    This study compared the incidence of HIV-1 variants harboring mutations conferring resistance to thymidine analogues, ie, thymidine analogue mutations (TAMs), nonnucleoside reverse transcriptase (RT) inhibitors (NNMs), lamivudine (3TC) (ie, M184V), and protease inhibitors (PIs) acquired in primary HIV infection (PHI) (n = 59) to their observed prevalence in a corresponding potential transmitter (PT) population of persons harboring resistant infections (n = 380). Both of these populations in the context of this cohort analysis possessed similar demographics. Whereas the frequencies of observed TAMs, NNMs, M184V, and protease-associated mutations (PRAMs) were similar in the PT groups, the prevalence of M184V and major PI mutations were significantly lower in the PHI group (PHI/PT ratios of 0.14 and 0.39, respectively). There was a decreased prevalence in the PHI population of resistant viruses co-expressing NNMs or TAMs with M184V compared with viruses that harbored NNMs or TAMs in the absence of M184V (P < 0.0001). It was also observed that individuals in the PT subgroups who harbored RT mutations or PRAMs with M184V had lower levels of plasma viremia than individuals who lacked M184V (P < 0.05). These findings suggest that both decreased viremia and viral fitness in the case of M184V-containing HIV-1 variants may impact on viral transmissibility.

  16. The RAC1 P29S Hotspot Mutation in Melanoma Confers Resistance to Pharmacological Inhibition of RAF

    PubMed Central

    Cabeceiras, Peter K.; Mahdavi, Mozhdeh; Gutschner, Tony; Genovese, Giannicola; Wang, Guocan; Fang, Zhuangna; Tepper, James M.; Stemke-Hale, Katherine; Tsai, Kenneth Y.; Davies, Michael A.; Mills, Gordon B.

    2014-01-01

    Following mutations in BRAF and NRAS, the RAC1 c.85C>T single nucleotide variant (SNV) encoding P29S amino acid change represents the next most frequently observed protein-coding hotspot mutation in melanoma. However, the biological and clinical significance of the RAC1 P29S somatic mutation in approximately 4–9% of patients remains unclear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (vemurafenib and dabrafenib). Enforced expression of RAC1 P29S in sensitive BRAF mutant melanoma cell lines confers resistance manifested by increased viability, decreased apoptosis and enhanced tumor growth in vivo upon treatment with RAF inhibitors. Conversely, RNAi mediated silencing of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to vemurafenib and dabrafenib. Our results suggest RAC1 P29S status may offer a predictive biomarker for RAF inhibitor resistance in melanoma patients, where it should be evaluated clinically. PMID:25056119

  17. Viral-inducible Argonaute18 confers broad-spectrum virus resistance in rice by sequestering a host microRNA

    PubMed Central

    Wu, Jianguo; Yang, Zhirui; Wang, Yu; Zheng, Lijia; Ye, Ruiqiang; Ji, Yinghua; Zhao, Shanshan; Ji, Shaoyi; Liu, Ruofei; Xu, Le; Zheng, Hong; Zhou, Yijun; Zhang, Xin; Cao, Xiaofeng; Xie, Lianhui; Wu, Zujian; Qi, Yijun; Li, Yi

    2015-01-01

    Viral pathogens are a major threat to rice production worldwide. Although RNA interference (RNAi) is known to mediate antiviral immunity in plant and animal models, the mechanism of antiviral RNAi in rice and other economically important crops is poorly understood. Here, we report that rice resistance to evolutionarily diverse viruses requires Argonaute18 (AGO18). Genetic studies reveal that the antiviral function of AGO18 depends on its activity to sequester microRNA168 (miR168) to alleviate repression of rice AGO1 essential for antiviral RNAi. Expression of miR168-resistant AGO1a in ago18 background rescues or increases rice antiviral activity. Notably, stable transgenic expression of AGO18 confers broad-spectrum virus resistance in rice. Our findings uncover a novel cooperative antiviral activity of two distinct AGO proteins and suggest a new strategy for the control of viral diseases in rice. DOI: http://dx.doi.org/10.7554/eLife.05733.001 PMID:25688565

  18. Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system.

    PubMed

    Simkovsky, Ryan; Daniels, Emy F; Tang, Karen; Huynh, Stacey C; Golden, Susan S; Brahamsha, Bianca

    2012-10-01

    The grazing activity of predators on photosynthetic organisms is a major mechanism of mortality and population restructuring in natural environments. Grazing is also one of the primary difficulties in growing cyanobacteria and other microalgae in large, open ponds for the production of biofuels, as contaminants destroy valuable biomass and prevent stable, continuous production of biofuel crops. To address this problem, we have isolated a heterolobosean amoeba, HGG1, that grazes upon unicellular and filamentous freshwater cyanobacterial species. We have established a model predator-prey system using this amoeba and Synechococcus elongatus PCC 7942. Application of amoebae to a library of mutants of S. elongatus led to the identification of a grazer-resistant knockout mutant of the wzm ABC O-antigen transporter gene, SynPCC7942_1126. Mutations in three other genes involved in O-antigen synthesis and transport also prevented the expression of O-antigen and conferred resistance to HGG1. Complementation of these rough mutants returned O-antigen expression and susceptibility to amoebae. Rough mutants are easily identifiable by appearance, are capable of autoflocculation, and do not display growth defects under standard laboratory growth conditions, all of which are desired traits for a biofuel production strain. Thus, preventing the production of O-antigen is a pathway for producing resistance to grazing by certain amoebae. PMID:23012457

  19. Impairment of O-antigen production confers resistance to grazing in a model amoeba–cyanobacterium predator–prey system

    PubMed Central

    Simkovsky, Ryan; Daniels, Emy F.; Tang, Karen; Huynh, Stacey C.; Golden, Susan S.; Brahamsha, Bianca

    2012-01-01

    The grazing activity of predators on photosynthetic organisms is a major mechanism of mortality and population restructuring in natural environments. Grazing is also one of the primary difficulties in growing cyanobacteria and other microalgae in large, open ponds for the production of biofuels, as contaminants destroy valuable biomass and prevent stable, continuous production of biofuel crops. To address this problem, we have isolated a heterolobosean amoeba, HGG1, that grazes upon unicellular and filamentous freshwater cyanobacterial species. We have established a model predator–prey system using this amoeba and Synechococcus elongatus PCC 7942. Application of amoebae to a library of mutants of S. elongatus led to the identification of a grazer-resistant knockout mutant of the wzm ABC O-antigen transporter gene, SynPCC7942_1126. Mutations in three other genes involved in O-antigen synthesis and transport also prevented the expression of O-antigen and conferred resistance to HGG1. Complementation of these rough mutants returned O-antigen expression and susceptibility to amoebae. Rough mutants are easily identifiable by appearance, are capable of autoflocculation, and do not display growth defects under standard laboratory growth conditions, all of which are desired traits for a biofuel production strain. Thus, preventing the production of O-antigen is a pathway for producing resistance to grazing by certain amoebae. PMID:23012457

  20. SU-C-303-01: Activation-Induced Cytidine Deaminase Confers Cancer Resistance to Radiation Therapy

    SciTech Connect

    Yi, S; La Count, S; Liu, J; Bai, X; Lu, L

    2015-06-15

    Purpose: To study the role of activation-induced cytidine deaminase (AID) in malignant cell resistance to radiation therapy. Methods: We first developed several small devices that could be used to adopt radiation beams from clinical high dose rate brachy therapy (HDR) or linac-based megavoltage machines to perform pre-clinical cell and mouse experiments. Then we used these devices to deliver radiation to AID-positive and AID-silenced cancer cells or tumors formed by these cells in mice. Cells and mice bearing tumors received the same dose under the same experimental conditions. For cells, we observed the apoptosis and the cell survival rate over time. For mice bearing tumors, we measured and recorded the tumor sizes every other day for 4 weeks. Results: For cell experiments, we found that the AID-positive cells underwent much less apoptosis compared with AID-silenced cells upon radiation. And for mouse experiments, we found that AID-positive tumors grew significantly faster than the AID-silenced tumors despite of receiving the same doses of radiation. Conclusion: Our study suggests that AID may confer cancer resistance to radiation therapy, and AID may be a significant biomarker predicting cancer resistance to radiation therapy for certain cancer types.

  1. Vat, an Amazing Gene Conferring Resistance to Aphids and Viruses They Carry: From Molecular Structure to Field Effects

    PubMed Central

    Boissot, Nathalie; Schoeny, Alexandra; Vanlerberghe-Masutti, Flavie

    2016-01-01

    We review half a century of research on Cucumis melo resistance to Aphis gossypii from molecular to field levels. The Vat gene is unique in conferring resistance to both A. gossypii and the viruses it transmits. This double phenotype is aphid clone-dependent and has been observed in 25 melon accessions, mostly from Asia. It is controlled by a cluster of genes including CC-NLR, which has been characterized in detail. Copy-number polymorphisms (for the whole gene and for a domain that stands out in the LLR region) and single-nucleotide polymorphisms have been identified in the Vat cluster. The role of these polymorphisms in plant/aphid interactions remains unclear. The Vat gene structure suggests a functioning with separate recognition and response phases. During the recognition phase, the VAT protein is thought to interact (likely indirectly) with an aphid effector introduced during cell puncture by the aphid. A few hours later, several miRNAs are upregulated in Vat plants. Peroxidase activity increases, and callose and lignin are deposited in the walls of the cells adjacent to the stylet path, disturbing aphid behavior. In aphids feeding on Vat plants, Piwi-interacting RNA-like sequences are abundant and the levels of other miRNAs are modified. At the plant level, resistance to aphids is quantitative (aphids escape the plant and display low rates of reproduction). Resistance to viruses is qualitative and local. Durability of NLR genes is highly variable. A. gossypii clones are adapted to Vat resistance, either by introducing a new effector that interferes with the deployment of plant defenses, or by adapting to the defenses it triggered. Viruses transmitted in a non-persistent manner cannot adapt to Vat resistance. At population level, Vat reduces aphid density and genetic diversity. The durability of Vat resistance to A. gossypii populations depends strongly on the agro-ecosystem, including, in particular, the presence of other cucurbit crops serving as

  2. Resistance to intestinal Entamoeba histolytica infection is conferred by innate immunity and Gr-1+ cells.

    PubMed

    Asgharpour, Amon; Gilchrist, Carol; Baba, Duza; Hamano, Shinjiro; Houpt, Eric

    2005-08-01

    Establishment of intestinal infection with Entamoeba histolytica depends on the mouse strain; C57BL/6 mice are highly resistant, and C3H/HeJ mice are relatively susceptible. We found that resistance to intestinal infection was independent of lymphocyte activity or H-2 haplotype and occurred in the first hours to days postchallenge according to in vivo imaging. At 18 h postchallenge, the ceca of resistant C57BL/6 mice were histologically unremarkable, in contrast to the severe inflammation observed in susceptible C3H/HeJ mice. Comparison of cecal gene expression in C3H/HeJ and C57BL/6 mice demonstrated that there was parasite-induced upregulation of proinflammatory and neutrophil chemotaxis transcripts and there was downregulation of transforming growth factor beta signaling molecules. Pretreatment with dexamethasone abrogated the partial resistance of C3H/HeJ or CBA mice through an innate, lymphocyte-independent mechanism, but it had no effect on the high-level resistance of C57BL/6 mice. Similarly, administration of a neutrophil-depleting anti-Gr-1 monoclonal antibody (RB6-8C5) decreased the partial resistance of CBA mice and led to severe pathology compared to control antibody-treated mice, but it had no effect on C57BL/6 resistance. These data indicate that there are discrete mechanisms of innate resistance to E. histolytica depending on the host background and, in contrast to other reports, imply that neutrophils are protective and not damaging in intestinal amebiasis.

  3. Structure and function of efflux pumps that confer resistance to drugs.

    PubMed Central

    Borges-Walmsley, M Ines; McKeegan, Kenneth S; Walmsley, Adrian R

    2003-01-01

    Resistance to therapeutic drugs encompasses a diverse range of biological systems, which all have a human impact. From the relative simplicity of bacterial cells, fungi and protozoa to the complexity of human cancer cells, resistance has become problematic. Stated in its simplest terms, drug resistance decreases the chance of providing successful treatment against a plethora of diseases. Worryingly, it is a problem that is increasing, and consequently there is a pressing need to develop new and effective classes of drugs. This has provided a powerful stimulus in promoting research on drug resistance and, ultimately, it is hoped that this research will provide novel approaches that will allow the deliberate circumvention of well understood resistance mechanisms. A major mechanism of resistance in both microbes and cancer cells is the membrane protein-catalysed extrusion of drugs from the cell. Resistant cells exploit proton-driven antiporters and/or ATP-driven ABC (ATP-binding cassette) transporters to extrude cytotoxic drugs that usually enter the cell by passive diffusion. Although some of these drug efflux pumps transport specific substrates, many are transporters of multiple substrates. These multidrug pumps can often transport a variety of structurally unrelated hydrophobic compounds, ranging from dyes to lipids. If we are to nullify the effects of efflux-mediated drug resistance, we must first of all understand how these efflux pumps can accommodate a diverse range of compounds and, secondly, how conformational changes in these proteins are coupled to substrate translocation. These are key questions that must be addressed. In this review we report on the advances that have been made in understanding the structure and function of drug efflux pumps. PMID:13678421

  4. Association of tellurium resistance and bacteriophage inhibition conferred by R plasmids.

    PubMed Central

    Taylor, D E; Summers, A O

    1979-01-01

    Concomitant resistance to tellurium compounds (Ter) and inhibition of coli-phage development (Phi) are properties mediated by many H2 incompatibility group R plasmids which have been isolated from diverse bacterial and geographic sources. Ter plasmids from tellurium-resistant bacteria that were isolated from sewage and industrial wastes also mediated phage inhibition. Of these Ter plasmids, three from Citrobacter freundii belonged to the H incompatibility group, whereas three from Klebsiella pneumoniae did not. Images PMID:374351

  5. ATG9A loss confers resistance to trastuzumab via c-Cbl mediated Her2 degradation

    PubMed Central

    Angelopoulos, Nicos; Chhetri, Jyoti; Osipo, Clodia; Grothey, Arnhild; Stebbing, Justin; Giamas, Georgios

    2016-01-01

    Acquired or de novo resistance to trastuzumab remains a barrier to patient survival and mechanisms underlying this still remain unclear. Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare proteome profiles between trastuzumab sensitive/resistant cells, we identified autophagy related protein 9A (ATG9A) as a down-regulated protein in trastuzumab resistant cells (BT474-TR). Interestingly, ATG9A ectopic expression markedly decreased the proliferative ability of BT474-TR cells but not that of the parental line (BT474). This was accompanied by a reduction of Her2 protein levels and AKT phosphorylation (S473), as well as a decrease in Her2 stability, which was also observed in JIMT1 and MDA-453, naturally trastuzumab-resistant cells. In addition, ATG9A indirectly promoted c-Cbl recruitment to Her2 on T1112, a known c-Cbl docking site, leading to increased K63 Her2 polyubiquitination. Whereas silencing c-Cbl abrogated ATG9A repressive effects on Her2 and downstream PI3K/AKT signaling, its depletion restored BT474-TR proliferative rate. Taken together, our findings show for this first time that ATG9A loss in trastuzumab resistant cells allowed Her2 to escape from lysosomal targeted degradation through K63 poly-ubiquitination via c-Cbl. This study identifies ATG9A as a potentially druggable target to overcome resistance to anti-Her2 blockade. PMID:27050377

  6. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    PubMed Central

    Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

    2016-01-01

    Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species. PMID:27187354

  7. Splice form variant and amino acid changes in MDR49 confers DDT resistance in transgenic Drosophila

    PubMed Central

    Seong, Keon Mook; Sun, Weilin; Clark, John M.; Pittendrigh, Barry R.

    2016-01-01

    The ATP-binding cassette (ABC) transporters represent a superfamily of proteins that have important physiological roles in both prokaryotes and eukaryotes. In insects, ABC transporters have previously been implicated in insecticide resistance. The 91-R strain of Drosophila melanogaster has been intensely selected with DDT over six decades. A recent selective sweeps analysis of 91-R implicated the potential role of MDR49, an ABC transporter, in DDT resistance, however, to date the details of how MDR49 may play a role in resistance have not been elucidated. In this study, we investigated the impact of structural changes and an alternative splicing event in MDR49 on DDT-resistance in 91-R, as compared to the DDT susceptible strain 91-C. We observed three amino acid differences in MDR49 when 91-R was compared with 91-C, and only one isoform (MDR49B) was implicated in DDT resistance. A transgenic Drosophila strain containing the 91-R-MDR49B isoform had a significantly higher LD50 value as compared to the 91-C-MDR49B isoform at the early time points (6 h to 12 h) during DDT exposure. Our data support the hypothesis that the MDR49B isoform, with three amino acid mutations, plays a role in the early aspects of DDT resistance in 91-R. PMID:27003579

  8. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

    PubMed

    Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

    2016-01-01

    Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species. PMID:27187354

  9. The second annual conference of International ovarian cancer consortium and the symposium on tumor microenvironment and therapeutic resistance

    PubMed Central

    Isidoro, Ciro; Song, Yong Sang; Surh, Young-Joon; Dhanasekaran, Danny N.

    2016-01-01

    The second Annual Meeting of the International Ovarian Cancer Consortium (IOCC) was held in conjunction with the Symposium on Tumor Microenvironment and Therapeutic Resistance at the Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, and USA. A brief welcoming event along with the banquet on Aug 16th was followed by the eight thematic scientific sessions from August 16 to 18, 2015. Forty-three lectures, organized in eight sessions, were discussed in front of an audience of more than hundred attendees. Emphasis was put on oncogene signaling in cancer genesis and progression, new approaches in Precision Medicine and therapy of ovarian cancer, the role of tumor microenvironment in carcinogenesis, and preventive/curative potential of natural products. In this meeting-report, we highlight the findings and the perspectives in cancer biology and therapeutic strategies that emerged during the conference.

  10. Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis.

    PubMed

    Larsen, Michelle H; Vilchèze, Catherine; Kremer, Laurent; Besra, Gurdyal S; Parsons, Linda; Salfinger, Max; Heifets, Leonid; Hazbon, Manzour H; Alland, David; Sacchettini, James C; Jacobs, William R

    2002-10-01

    The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre--lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species. PMID:12406221

  11. A Genetic Pathway Conferring Life Extension and Resistance to Uv Stress in Caenorhabditis Elegans

    PubMed Central

    Murakami, S.; Johnson, T. E.

    1996-01-01

    A variety of mechanisms have been proposed to explain the extension of adult life span (Age) seen in several mutants in Caenorhabditis elegans (age-1: an altered aging rate; daf-2 and daf-23: activation of a dauer-specific longevity program; spe-26: reduced fertility; clk-1: an altered biological clock). Using an assay for ultraviolet (UV) resistance in young adult hermaphrodites (survival after UV irradiation), we observed that all these Age mutants show increased resistance to UV. Moreover, mutations in daf-16 suppressed the UV resistance as well as the increased longevity of all the Age mutants. In contrast to the multiple mechanisms initially proposed, these results suggest that a single, daf-16-dependent pathway, specifies both extended life span and increased UV resistance. The mutations in daf-16 did not alter the reduced fertility of spe-26 and interestingly a daf-16 mutant is more fertile than wild type. We propose that life span and some aspects of stress resistance are jointly negatively regulated by a set of gerontogenes (genes whose alteration causes life extension) in C. elegans. PMID:8807294

  12. Identification of QTLs Conferring Resistance to Deltamethrin in Culex pipiens pallens

    PubMed Central

    Zhong, Daibin; Shen, Bo; Zhang, Donghui; Guo, Qin; Wang, Weijie; Yu, Jing; Lv, Yuan; Lei, Zhentao; Ma, Kai; Ma, Lei; Zhu, Changliang; Yan, Guiyun

    2015-01-01

    Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and West Nile virus. Deltamethrin is an insecticide that is widely used for mosquito control, however resistance to this and other insecticides has become a major challenge in the control of vector-borne diseases that appear to be inherited quantitatively. Furthermore, the genetic basis of insecticide resistance remains poorly understood. In this study, quantitative trait loci (QTL) mapping of resistance to deltamethrin was conducted in F2 intercross segregation populations using bulked segregation analysis (BSA) and amplified fragment length polymorphism markers (AFLP) in Culex pipiens pallens. A genetic linkage map covering 381 cM was constructed and a total of seven QTL responsible for resistance to deltamethrin were detected by composite interval mapping (CIM), which explained 95% of the phenotypic variance. The major QTL in linkage group 2 accounted for 62% of the variance and is worthy of further study. 12 AFLP markers in the map were cloned and the genomic locations of these marker sequences were determined by applying the Basic Local Alignment Search Tool (BLAST) tool to the genome sequence of the closely related Culex quinquefasciatus. Our results suggest that resistance to deltamethrin is a quantitative trait under the control of a major QTL in Culex pipiens pallens. Cloning of related AFLP markers confirm the potential utility for anchoring the genetic map to the physical map. The results provide insight into the genetic architecture of the trait. PMID:26484540

  13. Exosomes derived from human mesenchymal stem cells confer drug resistance in gastric cancer.

    PubMed

    Ji, Runbi; Zhang, Bin; Zhang, Xu; Xue, Jianguo; Yuan, Xiao; Yan, Yongmin; Wang, Mei; Zhu, Wei; Qian, Hui; Xu, Wenrong

    2015-08-01

    Mesenchymal stem cells (MSCs) play an important role in chemoresistance. Exosomes have been reported to modify cellular phenotype and function by mediating cell-cell communication. In this study, we aimed to investigate whether exosomes derived from MSCs (MSC-exosomes) are involved in mediating the resistance to chemotherapy in gastric cancer and to explore the underlying molecular mechanism. We found that MSC-exosomes significantly induced the resistance of gastric cancer cells to 5-fluorouracil both in vivo and ex vivo. MSC-exosomes antagonized 5-fluorouracil-induced apoptosis and enhanced the expression of multi-drug resistance associated proteins, including MDR, MRP and LRP. Mechanistically, MSC-exosomes triggered the activation of calcium/calmodulin-dependent protein kinases (CaM-Ks) and Raf/MEK/ERK kinase cascade in gastric cancer cells. Blocking the CaM-Ks/Raf/MEK/ERK pathway inhibited the promoting role of MSC-exosomes in chemoresistance. Collectively, MSC-exosomes could induce drug resistance in gastric cancer cells by activating CaM-Ks/Raf/MEK/ERK pathway. Our findings suggest that MSC-exosomes have profound effects on modifying gastric cancer cells in the development of drug resistance. Targeting the interaction between MSC-exosomes and cancer cells may help improve the efficacy of chemotherapy in gastric cancer.

  14. MEK Inhibition Overcomes Cisplatin Resistance Conferred by SOS/MAPK Pathway Activation in Squamous Cell Carcinoma.

    PubMed

    Kong, Li Ren; Chua, Kian Ngiap; Sim, Wen Jing; Ng, Hsien Chun; Bi, Chonglei; Ho, Jingshan; Nga, Min En; Pang, Yin Huei; Ong, Weijie Richard; Soo, Ross Andrew; Huynh, Hung; Chng, Wee Joo; Thiery, Jean-Paul; Goh, Boon Cher

    2015-07-01

    Genomic analyses of squamous cell carcinoma (SCC) have yet to yield significant strategies against pathway activation to improve treatment. Platinum-based chemotherapy remains the mainstay of treatment for SCC of different histotypes either as a single-agent or alongside other chemotherapeutic drugs or radiotherapy; however, resistance inevitably emerges, which limits the duration of treatment response. To elucidate mechanisms that mediate resistance to cisplatin, we compared drug-induced perturbations to gene and protein expression between cisplatin-sensitive and -resistant SCC cells, and identified MAPK-ERK pathway upregulation and activation in drug-resistant cells. ERK-induced resistance appeared to be activated by Son of Sevenless (SOS) upstream, and mediated through Bim degradation downstream. Clinically, elevated p-ERK expression was associated with shorter disease-free survival in patients with locally advanced head and neck SCC treated with concurrent chemoradiation. Inhibition of MEK/ERK, but not that of EGFR or RAF, augmented cisplatin sensitivity in vitro and demonstrated efficacy and tolerability in vivo. Collectively, these findings suggest that inhibition of the activated SOS-MAPK-ERK pathway may augment patient responses to cisplatin treatment.

  15. Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease.

    PubMed

    Namukwaya, B; Tripathi, L; Tripathi, J N; Arinaitwe, G; Mukasa, S B; Tushemereirwe, W K

    2012-08-01

    Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic.

  16. A mutation in reverse transcriptase of bis(heteroaryl)piperazine-resistant human immunodeficiency virus type 1 that confers increased sensitivity to other nonnucleoside inhibitors.

    PubMed Central

    Dueweke, T J; Pushkarskaya, T; Poppe, S M; Swaney, S M; Zhao, J Q; Chen, I S; Stevenson, M; Tarpley, W G

    1993-01-01

    Several nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been described, including Nevirapine, thiobenzimidazolone (TIBO) derivatives, pyridinone derivatives such as L-697,661, and the bis(heteroaryl)piperazines (BHAPs). HIV-1 resistant to L-697,661 or Nevirapine emerges rapidly in infected patients treated with these drugs, and the resistance is caused primarily by substitutions at amino acids 181 and 103 of RT that also confer cross resistance to the other nonnucleoside inhibitors. We describe derivation and characterization of two BHAP-resistant HIV-1 variants that differ from this pattern of cross resistance. With both variants, HIV-1 resistance to BHAP RT inhibitors was caused by a RT mutation that results in a proline-to-leucine substitution at amino acid 236 (P236L). Rather than conferring cross resistance to other RT inhibitors, this substitution sensitized RT 7- to 10-fold to Nevirapine, TIBO R82913, and L-697,661 without influencing sensitivity to nucleoside analogue RT inhibitors. This sensitization caused by P236L was also observed in cell culture with BHAP-resistant HIV-1. The effects of the P236L RT substitution suggest that emergence of BHAP-resistant virus in vivo could produce a viral population sensitized to inhibition by these other nonnucleoside RT inhibitors. PMID:7685109

  17. Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

    PubMed

    Wendlandt, Sarah; Kadlec, Kristina; Feßler, Andrea T; Schwarz, Stefan

    2015-06-12

    The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance.

  18. Mycobacterium tuberculosis pncA Polymorphisms That Do Not Confer Pyrazinamide Resistance at a Breakpoint Concentration of 100 Micrograms per Milliliter in MGIT

    PubMed Central

    Whitfield, Michael G.; Streicher, Elizabeth M.; Sampson, Samantha L.; Sirgel, Frik A.; van Helden, Paul D.; Mercante, Alexandra; Willby, Melisa; Hughes, Kelsey; Birkness, Kris; Morlock, Glenn; van Rie, Annelies; Posey, James E.

    2015-01-01

    Sequencing of the Mycobacterium tuberculosis pncA gene allows for pyrazinamide susceptibility testing. We summarize data on pncA polymorphisms that do not confer resistance at a susceptibility breakpoint of 100 μg/ml pyrazinamide in MGIT within a cohort of isolates from South Africa and the U.S. Centers for Disease Control and Prevention. PMID:26292310

  19. The Gastrodia anti-fungal protein confers increased resistance to Phytophthora root rot and the root-knot nematode in a fruit tree species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Gastrodia Anti-Fungal Protein (GAFP) is a monocot mannose-binding lectin isolated from the Asiatic orchid Gastrodia elata. This protein, among others, enables the orchid to live parasitically off the basidiomycete pathogen Armillaria mellea. GAFP has been shown to confer resistance to transgenic...

  20. UCP-3 uncoupling protein confers hypoxia resistance to renal epithelial cells and is upregulated in renal cell carcinoma

    PubMed Central

    Braun, Norbert; Klumpp, Dominik; Hennenlotter, Jörg; Bedke, Jens; Duranton, Christophe; Bleif, Martin; Huber, Stephan M.

    2015-01-01

    Tumor cells can adapt to a hostile environment with reduced oxygen supply. The present study aimed to identify mechanisms that confer hypoxia resistance. Partially hypoxia/reoxygenation (H/R)-resistant proximal tubular (PT) cells were selected by exposing PT cultures to repetitive cycles of H/R. Thereafter, H/R-induced changes in mRNA and protein expression, inner mitochondrial membrane potential (ΔΨm), formation of superoxide, and cell death were compared between H/R-adapted and control PT cultures. As a result, H/R-adapted PT cells exhibited lower H/R-induced hyperpolarization of ΔΨm and produced less superoxide than the control cultures. Consequently, H/R triggered ΔΨm break-down and DNA degradation in a lower percentage of H/R-adapted than control PT cells. Moreover, H/R induced upregulation of mitochondrial uncoupling protein-3 (UCP-3) in H/R-adapted PT but not in control cultures. In addition, ionizing radiation killed a lower percentage of H/R-adapted as compared to control cells suggestive of an H/R-radiation cross-resistance developed by the selection procedure. Knockdown of UCP-3 decreased H/R- and radioresitance of the H/R-adapted cells. Finally, UCP-3 protein abundance of PT-derived clear cell renal cell carcinoma and normal renal tissue was compared in human specimens indicating upregulation of UCP-3 during tumor development. Combined, our data suggest functional significance of UCP-3 for H/R resistance. PMID:26304588

  1. Transcription factor StWRKY1 regulates phenylpropanoid metabolites conferring late blight resistance in potato

    PubMed Central

    Yogendra, Kalenahalli N.; Kumar, Arun; Sarkar, Kobir; Li, Yunliang; Pushpa, Doddaraju; Mosa, Kareem A.; Duggavathi, Raj; Kushalappa, Ajjamada C.

    2015-01-01

    Quantitative resistance is polygenically controlled and durable, but the underlying molecular and biochemical mechanisms are poorly understood. Secondary cell wall thickening is a critical process in quantitative resistance, regulated by transcriptional networks. This paper provides compelling evidence on the functionality of StWRKY1 transcription factor, in a compatible interaction of potato–Phytophthora infestans, to extend our knowledge on the regulation of the metabolic pathway genes leading to strengthening the secondary cell wall. A metabolomics approach was used to identify resistance-related metabolites belonging to the phenylpropanoid pathway and their biosynthetic genes regulated by StWRKY1. The StWRKY1 gene in resistant potato was silenced to decipher its role in the regulation of phenylpropanoid pathway genes to strengthen the secondary cell wall. Sequencing of the promoter region of StWRKY1 in susceptible genotypes revealed the absence of heat shock elements (HSEs). Simultaneous induction of both the heat shock protein (sHSP17.8) and StWRKY1 following pathogen invasion enables functioning of the latter to interact with the HSE present in the resistant StWRKY1 promoter region. EMSA and luciferase transient expression assays further revealed direct binding of StWRKY1 to promoters of hydroxycinnamic acid amide (HCAA) biosynthetic genes encoding 4-coumarate:CoA ligase and tyramine hydroxycinnamoyl transferase. Silencing of the StWRKY1 gene was associated with signs of reduced late blight resistance by significantly increasing the pathogen biomass and decreasing the abundance of HCAAs. This study provides convincing evidence on the role of StWRKY1 in the regulation of downstream genes to biosynthesize HCAAs, which are deposited to reinforce secondary cell walls. PMID:26417019

  2. The efficiency of RNA interference for conferring stable resistance to Plum Pox Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plum transformed with an intron hairpin RNA CP (ihRNA-CP) were resistant to PPV infection through the specific process of RNA silencing involving both small interfering -RNA interfering (siRNA) and a methylated virus transgene. This recognition process specifically targeted the triggered PPV genome...

  3. Transgenic Resistance Confers Effective Field Level Control of Bacterial Spot Disease in Tomato

    PubMed Central

    Horvath, Diana M.; Stall, Robert E.; Jones, Jeffrey B.; Pauly, Michael H.; Vallad, Gary E.; Dahlbeck, Doug; Staskawicz, Brian J.; Scott, John W.

    2012-01-01

    We investigated whether lines of transgenic tomato (Solanum lycopersicum) expressing the Bs2 resistance gene from pepper, a close relative of tomato, demonstrate improved resistance to bacterial spot disease caused by Xanthomonas species in replicated multi-year field trials under commercial type growing conditions. We report that the presence of the Bs2 gene in the highly susceptible VF 36 background reduced disease to extremely low levels, and VF 36-Bs2 plants displayed the lowest disease severity amongst all tomato varieties tested, including commercial and breeding lines with host resistance. Yields of marketable fruit from transgenic lines were typically 2.5 times that of the non-transformed parent line, but varied between 1.5 and 11.5 fold depending on weather conditions and disease pressure. Trials were conducted without application of any copper-based bactericides, presently in wide use despite negative impacts on the environment. This is the first demonstration of effective field resistance in a transgenic genotype based on a plant R gene and provides an opportunity for control of a devastating pathogen while eliminating ineffective copper pesticides. PMID:22870280

  4. Campylobacter capsule and lipooligosaccharide confer resistance to serum and cationic antimicrobials

    PubMed Central

    Keo, Thormika; Collins, Jennifer; Kunwar, Pratima; Blaser, Martin J

    2011-01-01

    The innate immune system plays a critical role in host defense against mucosal bacteria. Campylobacter jejuni is a major cause of human gastroenteritis that usually resolves spontaneously within several days, suggesting that innate mechanisms are important to control the infection. However, the specific means by which this occurs is not well understood. While diarrheal isolates of C. jejuni usually are susceptible to human serum, we found that a systemic strain of C. jejuni, isolated from the cerebrospinal fluid of an infant with meningitis, is relatively more resistant to human serum, the Bactericidal/Permeability-Increasing Protein (BPI), an endogenous cationic antimicrobial protein, and the cationic peptide antibiotic polymyxin B. To test the hypothesis that the surface properties of this strain contributed to its ability to withstand these innate host defenses, we constructed isogenic mutants in capsule (kpsM) and lipooligosaccharide (waaF) and complemented these mutants by insertion of the complementation construct in trans into hipO, a chromosomal locus. We found that capsule expression was essential for serum resistance, whereas lipooligosaccharide played no substantial role. In contrast, the lipooligosaccharide mutant showed increased sensitivity to polymyxin B, α-defensins, cathelicidins and BPI. These findings suggest that the polysaccharides of C. jejuni strains contribute differently to resistance against host innate immunity, whereby capsule is more important for resisting human complement and lipooligosaccharide is more important for protection against killing mediated by cationic antimicrobial peptides and proteins. PMID:21266840

  5. Powdery mildew resistant cucurbit rootstocks confer tolerance to grafted susceptible watermelon scions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cucurbit powdery mildew (PM) caused by Podosphaera xanthii can impact seedling growth and cause serious losses in greenhouse and open fields. We have developed watermelon and bottle gourd germplasm lines with high levels of resistance to PM. A PM susceptible watermelon cultivar Mickey Lee (ML) was g...

  6. Tolerance to powdery mildew conferred in susceptible watermelon scion by grafting on resistant rootstocks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cucurbit powdery mildew (PM) caused by Podosphaera xanthii, can impact seedling growth and cause serious losses in greenhouse and open field production. We have developed several watermelon and bottle gourd germplasm lines with high levels of resistance to PM. A PM susceptible cultivar Mickey Lee ...

  7. Overexpression of a soybean salicylic acid methlyltransferase gene confers resistance to soybean cyst nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean cyst nematode (Heterodera glycines Ichinohe, SCN) is the most pervasive pest of soybean [Glycine max (L.) Merr.] in the USA and worldwide. SCN reduced soybean yields worldwide by an estimated billion dollars annually. These losses remained stable with the use of resistant cultivars but over ...

  8. Novel plasmid conferring kanamycin and tetracycline resistance in turkey-derived Campylobacter jejuni strain 11601MD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Campylobacter spp., resistance to the antibiotics kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095 bp.) harboring tet(O) was identified in...

  9. Potentially Deceptive Health Nutrition-Related Advertising Claims: The Role of Inoculation in Conferring Resistance

    ERIC Educational Resources Information Center

    Mason, Alicia M.; Miller, Claude H.

    2016-01-01

    Objective: This study sought to examine the efficacy of inoculation message treatments to facilitate resistance to health nutrition-related (HNR) commercial food advertising claims. Design: Data were collected across three phases extending across a 5-week period conducted over two semesters at a Midwest US university. A 2 × 3 between-subjects…

  10. Identification of genes conferring genetic resistance to Marek’s disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic resistance to Marek’s disease (MD) is complex and controlled by many genes with the majority having small effect making them difficult to detect. Thus, to identify specific genes, we have been employing and integrating a variety of genomic and functional genomic approaches that capitalize on...

  11. Identification of QTLs and possible candidate genes conferring sheath blight resistance in rice (Oryza sativa L.).

    PubMed

    Yadav, Shailesh; Anuradha, Ghanta; Kumar, Ravi Ranjan; Vemireddy, Lakshminaryana Reddy; Sudhakar, Ravuru; Donempudi, Krishnaveni; Venkata, Durgarani; Jabeen, Farzana; Narasimhan, Yamini Kalinati; Marathi, Balram; Siddiq, Ebrahimali Abubacker

    2015-01-01

    Sheath blight, caused by the pathogenic fungus Rhizoctonia solani Kühn, is one of the most devastating diseases in rice. Breeders have always faced challenges in acquiring reliable and absolute resistance to this disease in existing rice germplasm. In this context, 40 rice germplasm including eight wild, four landraces, twenty- six cultivated and two advanced breeding lines were screened utilizing the colonized bits of typha. Except Tetep and ARC10531 which expressed moderate level of resistance to the disease, none could be found to be authentically resistant. In order to map the quantitative trait loci (QTLs) governing the sheath blight resistance, two mapping populations (F2 and BC1F2) were developed from the cross BPT-5204/ARC10531. Utilizing composite interval mapping analysis, 9 QTLs mapped to five different chromosomes were identified with phenotypic variance ranging from 8.40 to 21.76%. Two SSR markers namely RM336 and RM205 were found to be closely associated with the major QTLs qshb7.3 and qshb9.2 respectively and were attested as well in BC1F2 population by bulk segregant analysis approach. A hypothetical β 1-3 glucanase with other 31 candidate genes were identified in silico utilizing rice database RAP-DB within the identified QTL region qshb9.2. A detailed insight into these candidate genes will facilitate at molecular level the intricate nature of sheath blight, a step forward towards functional genomics.

  12. Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt) is a devastating disease that can cause severe yield losses. A new Pgt race designated Ug99 has overcome most of the widely used resistance genes and is spreading through Africa and Asia threatening major wheat production areas. We re...

  13. Transfer of Clostridium difficile Genetic Elements Conferring Resistance to Macrolide-Lincosamide-Streptogramin B (MLSB) Antibiotics.

    PubMed

    Barbanti, Fabrizio; Wasels, François; Spigaglia, Patrizia

    2016-01-01

    Molecular analysis is an important tool to investigate Clostridium difficile resistance to macrolide-lincosamide-streptogramin B (MLSB). In particular, the protocols described in this chapter have been designed to investigate the genetic organization of erm(B)-containing elements and to evaluate the capability of these elements to transfer in C. difficile recipient strains using filter mating assay. PMID:27507342

  14. Identification of QTLs conferring resistance to downy mildew in legacy cultivars of lettuce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many cultivars of lettuce (Lactuca sativa L.), the most popular leafy vegetable, are susceptible to downy mildew disease caused by Bremia lactucae. Cultivars Iceberg and Grand Rapids that were released in 18th and 19th century, respectively, have high levels of quantitative resistance to downy milde...

  15. [Construction of a vector conferring herbicide and pest resistance in tobacco plant].

    PubMed

    Xie, Long-Xu; Xu, Pei-Lin; Nie, Yan-Fang; Tian, Ying-Chuan

    2003-09-01

    A binary plant expression vector, pCM12-slm, carrying the aroAM12 mutant gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Bts1m recombinant gene consisting of 331 N-terminal amino acids of CryIAc and 284 C-terminal amino acids of CryIAb has been constructed. The truncated Bts1 gene was fused with the PR1b signal peptide sequence and expressed in tobacco plants under the control of 2E-CaMV35S promoter and the omega (omega) translation enhancer sequence from tobacco mosaic virus. The mutant aroAM12 was fused with the transit sequence of tobacco EPSPS and expressed in tobacco plants under the control of the CaMV35S promoter. Tobacco leaves were transformed with Agrobacterium tumefaciens LBA4404 harboring the pCM12-slm plasmid, and the transgenic plants were selected directly on medium containing the herbicide. Forty glyphosate resistant plants were regenerated, with a transformation frequency of 27%. Transgenic plants were initially assessed for glyphosate resistance by placing leaf discs on shoot induction media containing the herbicide. Rooted plantlets, propagated from selected transgenic tobacco, were transferred to soil in a greenhouse and tested for glyphosate resistance by spraying them with Roundup at a commercial recommended dose. The glyphosate resistance assay indicated that all the transgenic plants showed highly resistant to the herbicide. The PCR assay showed that the aroAM12 gene was present in all of the 40 T0 transfer plants, and Bts1m genes present in 28 of 40 of the transgenic plants. Southern blot analysis further confirmed that the copy number of the transgenes varied from one to three copies in different transgenic plants. Northern blot and immunodot blot showed that the aroAM12 and Bts1m genes were expressed at the transcription and translation levels. Transgenic plants containing both the aroA M12 and Bts1m genes were further assessed for insect resistance. Tobacco leaves of T0 transgenic plants were infested

  16. M233I Mutation in the β-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide.

    PubMed

    Cai, Meng; Lin, Dong; Chen, Lei; Bi, Yang; Xiao, Lu; Liu, Xi-li

    2015-11-24

    Three phenotypes were detected in 161 Botrytis cinerea field isolates, including Zox(S)Car(S) (sensitive to zoxamide and carbendazim), Zox(S)Car(R) (sensitive to zoxamide and resistant to carbendazim), and Zox(R)Car(R) (resistant to zoxamide and carbendazim), but not Zox(R)Car(S) (resistant to zoxamide and sensitive to carbendazim). The baseline sensitivity to zoxamide was determined with a mean EC50 of 0.76 μg/ml. Two stable Zox(R)Car(S) isolates were obtained with a resistance factor of 13.28 and 20.43; there was a fitness penalty in mycelial growth rate, sporulation, virulence and sclerotium production. The results suggest that the resistance risk of B. cinerea to zoxamide is low where benzimidazoles have not been used. E198V, E198K and M233I, were detected in the β-tubulin of Zox(S)Car(R), Zox(R)Car(R), Zox(R)Car(S), respectively. Molecular docking indicated that position 198 in β-tubulin were targets for both zoxamide and carbendazim. The mutations at 198 prevented formation of hydrogen bonds between β-tubulin and carbendazim (E198V/K), and changed the conformation of the binding pocket of zoxamide (E198K). M233I had no effect on the binding of carbendazim but resulted in loss of a hydrogen bond between zoxamide and F200. M233 is suggested to be a unique target site for zoxamide and be very important in the function of β tubulin.

  17. M233I Mutation in the β-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide

    PubMed Central

    Cai, Meng; Lin, Dong; Chen, Lei; Bi, Yang; Xiao, Lu; Liu, Xi-li

    2015-01-01

    Three phenotypes were detected in 161 Botrytis cinerea field isolates, including ZoxSCarS (sensitive to zoxamide and carbendazim), ZoxSCarR (sensitive to zoxamide and resistant to carbendazim), and ZoxRCarR (resistant to zoxamide and carbendazim), but not ZoxRCarS (resistant to zoxamide and sensitive to carbendazim). The baseline sensitivity to zoxamide was determined with a mean EC50 of 0.76 μg/ml. Two stable ZoxRCarS isolates were obtained with a resistance factor of 13.28 and 20.43; there was a fitness penalty in mycelial growth rate, sporulation, virulence and sclerotium production. The results suggest that the resistance risk of B. cinerea to zoxamide is low where benzimidazoles have not been used. E198V, E198K and M233I, were detected in the β-tubulin of ZoxSCarR, ZoxRCarR, ZoxRCarS, respectively. Molecular docking indicated that position 198 in β-tubulin were targets for both zoxamide and carbendazim. The mutations at 198 prevented formation of hydrogen bonds between β-tubulin and carbendazim (E198V/K), and changed the conformation of the binding pocket of zoxamide (E198K). M233I had no effect on the binding of carbendazim but resulted in loss of a hydrogen bond between zoxamide and F200. M233 is suggested to be a unique target site for zoxamide and be very important in the function of β tubulin. PMID:26596626

  18. Salicylic acid confers enhanced resistance to Glomerella leaf spot in apple.

    PubMed

    Zhang, Ying; Shi, Xiangpeng; Li, Baohua; Zhang, Qingming; Liang, Wenxing; Wang, Caixia

    2016-09-01

    Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emergent disease that results in severe defoliation and fruit spots in apple. Currently, there are no effective means to control this disease except for the traditional fungicide sprays. Induced resistance by elicitors against pathogens infection is a widely accepted eco-friendly strategy. In the present study, we investigated whether exogenous application of salicylic acid (SA) could improve resistance to GLS in a highly susceptible apple cultivar (Malus domestica Borkh. cv. 'Gala') and the underlying mechanisms. The results showed that pretreatment with SA, at 0.1-1.0 mM, induced strong resistance against GLS in 'Gala' apple leaves, with SA treated leaves showing significant reduction in lesion numbers and disease index. Concurrent with the enhanced disease resistance, SA treatment markedly increased the total antioxidant capacity (T-AOC) and defence-related enzyme activities, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO). As expected, SA treatment also induced the expression levels of five pathogenesis-related (PR) genes including PR1, PR5, PR8, Chitinase and β-1,3-glucanase. Furthermore, the most pronounced and/or rapid increase was observed in leaves treated with SA and subsequently inoculated with G. cingulata compared to the treatment with SA or inoculation with the pathogen. Together, these results suggest that exogenous SA triggered increase in reactive oxygen species levels and the antioxidant system might be responsible for enhanced resistance against G. cingulata in 'Gala' apple leaves. PMID:27139585

  19. Resistance Mechanisms to Chlorpyrifos and F392W Mutation Frequencies in the Acetylcholine Esterase Ace1 Allele of Field Populations of the Tobacco Whitefly, Bemisia tabaci in China

    PubMed Central

    Zhang, Ning-ning; Liu, Cai-feng; Yang, Fang; Dong, Shuang-lin; Han, Zhao-jun

    2012-01-01

    The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides. PMID:22954331

  20. SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage ( Brassica rapa ssp. pekinensis).

    PubMed

    Piao, Z Y; Deng, Y Q; Choi, S R; Park, Y J; Lim, Y P

    2004-05-01

    Clubroot disease, caused by Plasmodiophora brassicae Wor., is highly damaging for Chinese cabbage. The CR (clubroot resistant) Shinki DH (doubled haploid) line of Chinese cabbage carries a single dominant gene, CRb, which confers resistance to the P. brassicae races 2, 4, and 8. An F(2) population derived from a cross between the CR Shinki DH line and a susceptible line, 94SK, was used to map the CRb gene. Inoculation of F(3) families with SSI (single-spore isolate) resulted in a 1:2:1 segregation ratio. Use of the AFLP technique combined with bulked segregant analysis allowed five co-dominant AFLP markers, and four and seven dominant AFLP markers linked in coupling and repulsion, respectively, to be identified. Six of the 16 AFLP markers showing low frequencies of recombination with the CRb locus among 138 F(2) lines were cloned. A reliable conversion procedure allowed five AFLP markers to be successfully converted into CAPS and SCAR markers. An F(2) population (143 plants) was analyzed with these markers and a previously identified SCAR marker, and a genetic map around CRb covering a total distance of 6.75 cM was constructed. One dominant marker, TCR09, was located 0.78 cM from CRb. The remaining markers (TCR05, TCR01, TCR10, TCR08, and TCR03) were located on the other side of CRb, and the nearest of these was TCR05, at a distance of 1.92 cM.

  1. A novel mechanism of protein thermostability: a unique N-terminal domain confers heat resistance to Fe/Mn-SODs

    PubMed Central

    Wang, Wei; Ma, Ting; Zhang, Baoliang; Yao, Nana; Li, Mingchang; Cui, Lianlei; Li, Guoqiang; Ma, Zhenping; Cheng, Jiansong

    2014-01-01

    Superoxide dismutases (SODs), especially thermostable SODs, are widely applied in medical treatments, cosmetics, food, agriculture, and other industries given their excellent antioxidant properties. A novel thermostable cambialistic SOD from Geobacillus thermodenitrificans NG80-2 exhibits maximum activity at 70°C and high thermostability over a broad range of temperatures (20–80°C). Unlike other reported SODs, this enzyme contains an extra repeat-containing N-terminal domain (NTD) of 244 residues adjacent to the conserved functional SODA domain. Deletion of the NTD dramatically decreased its optimum active temperature (OAT) to 30°C and also impaired its thermostability. Conversely, appending the NTD to a mesophilic counterpart from Bacillus subtilis led to a moderately thermophilic enzyme (OAT changed from 30 to 55°C) with improved heat resistance. Temperature-dependant circular dichroism analysis revealed the enhanced conformational stability of SODs fused with this NTD. Furthermore, the NTD also contributes to the stress resistance of host proteins without altering their metal ion specificity or oligomerisation form except for a slight effect on their pH profile. We therefore demonstrate that the NTD confers outstanding thermostability to the host protein. To our knowledge, this is the first discovery of a peptide capable of remarkably improving protein thermostability and provides a novel strategy for bioengineering thermostable SODs. PMID:25445927

  2. Environmentally co-occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context-dependent fitness effects.

    PubMed

    Hall, James P J; Harrison, Ellie; Lilley, Andrew K; Paterson, Steve; Spiers, Andrew J; Brockhurst, Michael A

    2015-12-01

    Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co-occurring pQBR family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid-borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus-encoding cluster and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity among co-occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity.

  3. Epigenetic inactivation of the putative DNA/RNA helicase SLFN11 in human cancer confers resistance to platinum drugs

    PubMed Central

    Nogales, Vanesa; Reinhold, William C.; Varma, Sudhir; Martinez-Cardus, Anna; Moutinho, Catia; Moran, Sebastian; Heyn, Holger; Sebio, Ana; Barnadas, Agusti; Pommier, Yves; Esteller, Manel

    2016-01-01

    Platinum-derived drugs such as cisplatin and carboplatin are among the most commonly used cancer chemotherapy drugs, but very few specific molecular and cellular markers predicting differential sensitivity to these agents in a given tumor type have been clearly identified. Epigenetic gene silencing is increasingly being recognized as a factor conferring distinct tumoral drug sensitivity, so we have used a comprehensive DNA methylation microarray platform to interrogate the widely characterized NCI60 panel of human cancer cell lines with respect to CpG methylation status and cisplatin/carboplatin sensitivity. Using this approach, we have found promoter CpG island hypermethylation-associated silencing of the putative DNA/RNA helicase Schlafen-11 (SLFN11) to be associated with increased resistance to platinum compounds. We have also experimentally validated these findings in vitro. In this setting, we also identified the BRCA1 interacting DHX9 RNA helicase (also known as RHA) as a protein partner for SLFN11, suggesting a mechanistic pathway for the observed chemoresistance effect. Most importantly, we have been able to extend these findings clinically, following the observation that those patients with ovarian and non-small cell lung cancer carrying SLFN11 hypermethylation had a poor response to both cisplatin and carboplatin treatments. Overall, these results identify SLFN11 epigenetic inactivation as a predictor of resistance to platinum drugs in human cancer. PMID:26625211

  4. Expression of the sigmaB-dependent general stress regulon confers multiple stress resistance in Bacillus subtilis.

    PubMed

    Völker, U; Maul, B; Hecker, M

    1999-07-01

    The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B. subtilis. sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of sigmaB-dependent genes provides nonsporulated B. subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.

  5. Identification and validation of QTLs conferring resistance to sorghum downy mildew (Peronosclerospora sorghi) and Rajasthan downy mildew (P. heteropogoni) in maize.

    PubMed

    Nair, S K; Prasanna, B M; Garg, A; Rathore, R S; Setty, T A S; Singh, N N

    2005-05-01

    We have mapped the quantitative trait loci (QTLs) conferring resistance to sorghum downy mildew (Peronosclerospora sorghi; SDM) and Rajasthan downy mildew (P. heteropogoni; RDM), two species of DM prevalent throughout India. QTL mapping was carried out on a backcross population of 151 individuals derived from a cross between CM139 (susceptible parent) and NAI116 (highly resistant to both SDM and RDM). Heritability estimates were 0.74 for SDM and 0.67 for RDM. Composite interval mapping combined with a linkage map constructed with 80 simple sequence repeat (SSR) markers resulted in the identification of three QTLs (one each on chromosomes 2, 3 and 6) for SDM resistance and two QTLs (one each on chromosomes 3 and 6) for RDM resistance, all of which were contributed by NAI116. The significance of the major QTL on chromosome 6 (bin 6.05) that confers resistance to diverse DMs in tropical Asia, including SDM and RDM in India, was also verified. The results confirmed that some common QTLs contribute to both SDM and RDM resistance, while additional loci might specifically govern resistance to SDM. The QTL information generated in this study provide information that will aid in undertaking an integrated breeding strategy for the transfer of resistance to SDM and RDM in maize lines using marker-assisted selection.

  6. Heterologous expression of a Tpo1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast.

    PubMed

    Cabrito, Tânia R; Teixeira, Miguel C; Duarte, Alexandra A; Duque, Paula; Sá-Correia, Isabel

    2009-10-01

    The understanding of the molecular mechanisms underlying acquired herbicide resistance is crucial in dealing with the emergence of resistant weeds. Saccharomyces cerevisiae has been used as a model system to gain insights into the mechanisms underlying resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The TPO1 gene, encoding a multidrug resistance (MDR) plasma membrane transporter of the major facilitator superfamily (MFS), was previously found to confer resistance to 2,4-D in yeast and to be transcriptionally activated in response to the herbicide. In this work, we demonstrate that Tpo1p is required to reduce the intracellular concentration of 2,4-D. ScTpo1p homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed for a possible role in 2,4-D resistance. At5g13750 was chosen for further analysis, as its transcript levels were found to increase in 2,4-D stressed plants. The functional heterologous expression of this plant open reading frame in yeast was found to confer increased resistance to the herbicide in Deltatpo1 and wild-type cells, through the reduction of the intracellular concentration of 2,4-D. Heterologous expression of At5g13750 in yeast also leads to increased resistance to indole-3-acetic acid (IAA), Al(3+) and Tl(3+). At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR.

  7. Overexpression of the pathogen-inducible wheat TaWRKY45 gene confers disease resistance to multiple fungi in transgenic wheat plants

    PubMed Central

    Bahrini, Insaf; Ogawa, Taiichi; Kobayashi, Fuminori; Kawahigashi, Hiroyuki; Handa, Hirokazu

    2011-01-01

    Recently we cloned and characterized the gene for the wheat transcription factor TaWRKY45 and showed that TaWRKY45 was upregulated in response to benzothiadiazole (BTH) and Fusarium head blight (FHB) and that its overexpression conferred enhanced resistance against F. graminearum. To characterize the functional role of TaWRKY45 in the disease resistance of wheat, in the present study we conducted expression analyses of TaWRKY45 with inoculations of powdery mildew and leaf rust and evaluated TaWRKY45-overexpressing wheat plants for resistance to these diseases. TaWRKY45 was upregulated in response to infections with Blumeria graminis, a causal fungus for powdery mildew, and Puccinia triticina, a causal fungus for leaf rust. Constitutive overexpression of the TaWRKY45 transgene conferred enhanced resistance against these two fungi on transgenic wheat plants grown under greenhouse conditions. However, the expression of two resistance-related genes, Pm3 and Lr34, was not induced by the inoculation with powdery mildew in TaWRKY45-overexpressing wheat plants. These results suggest that TaWRKY45 is involved in the defense responses for multiple fungal diseases in wheat but that resistance involving TaWRKY45 differs from at least Pm3 and/or Lr34-related resistance. Our present and previous studies indicate that TaWRKY45 may be potentially utilized to improve a wide range of disease resistance in wheat. PMID:23136468

  8. Development of a novel strategy for fungal transformation based on a mutant locus conferring carboxin-resistance in Magnaporthe oryzae.

    PubMed

    Guo, Min; Zhu, Xiaolei; Li, Hongxia; Tan, Leyong; Pan, Yuemin

    2016-12-01

    The accurate manipulation of genomic integration of mutant alleles or fluorescent fusion-protein constructs is necessary for understanding of pathogenic mechanism of Magnaporthe oryzae. Recently, this can be achieved by integrating of exogenous DNA randomly into genome of this pathogen, but ectopic integration may result in alteration of gene expression or gene disruption due to unpredictable position effects and/or disruption of protein-coding regions. In this study, we establish a novel strategy for locus-specific integration of exogenous DNA via carboxin-resistance reconstitution by a point-mutation (H245L) on succinate dehydrogenase subunit Mosdi1. Independent transformants derived from the same reconstitution construct showed consistent fluorescent signal and undiversified phenotypes, including hyphae growth, conidiation and pathogenicity, in M. oryzae. Meanwhile, 96 % of all transformants integrate correctly into the Mosdi1 locus as a single copy. Furthermore, we provide a vector carrying yeast recombination cassette and thus allow assembly of multiple overlapping DNA fragments by yeast in vivo recombination for gene complementation and protein localization assay. PMID:27558019

  9. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae.

    PubMed

    Bossé, Janine T; Li, Yanwen; Atherton, Tom G; Walker, Stephanie; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Holden, Matthew T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-08-01

    The complete nucleotide sequence of a 7.7kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally. PMID:26049592

  10. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae

    PubMed Central

    Bossé, Janine T; Li, Yanwen; Atherton, Tom G; Walker, Stephanie; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Holden, Matthew TG; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-01-01

    The complete nucleotide sequence of a 7.7 kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally. PMID:26049592

  11. Resistance Mutations in Human Immunodeficiency Virus Type 1 Integrase Selected with Elvitegravir Confer Reduced Susceptibility to a Wide Range of Integrase Inhibitors▿

    PubMed Central

    Goethals, Olivia; Clayton, Reginald; Van Ginderen, Marcia; Vereycken, Inge; Wagemans, Elisabeth; Geluykens, Peggy; Dockx, Koen; Strijbos, Rudy; Smits, Veerle; Vos, Ann; Meersseman, Geert; Jochmans, Dirk; Vermeire, Kurt; Schols, Dominique; Hallenberger, Sabine; Hertogs, Kurt

    2008-01-01

    Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds. PMID:18715920

  12. Modulation of auxin content in Arabidopsis confers improved drought stress resistance.

    PubMed

    Shi, Haitao; Chen, Li; Ye, Tiantian; Liu, Xiaodong; Ding, Kejian; Chan, Zhulong

    2014-09-01

    Auxin is a well-known plant phytohormone that is involved in multiple plant growth processes and stress responses. In this study, auxin response was significantly modulated under drought stress condition. The iaaM-OX transgenic lines with higher endogenous indole-3-acetic acid (IAA) level and IAA pre-treated wild type (WT) plants exhibited enhanced drought stress resistance, while the yuc1yuc2yuc6 triple mutants with lower endogenous IAA level showed decreased stress resistance in comparison to non-treated WT plants. Additionally, endogenous and exogenous auxin positively modulated the expression levels of multiple abiotic stress-related genes (RAB18, RD22, RD29A, RD29B, DREB2A, and DREB2B), and positively affected reactive oxygen species (ROS) metabolism and underlying antioxidant enzyme activities. Moreover, auxin significantly modulated some carbon metabolites including amino acids, organic acids, sugars, sugar alcohols and aromatic amines. Notably, endogenous and exogenous auxin positively modulated root architecture especially the lateral root number. Taken together, this study demonstrated that auxin might participate in the positive regulation of drought stress resistance, through regulation of root architecture, ABA-responsive genes expression, ROS metabolism, and metabolic homeostasis, at least partially. PMID:24992887

  13. Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions.

    PubMed

    Lee, Changhan; Kim, Jihong; Kwon, Minsuk; Lee, Kihyun; Min, Haeyoung; Kim, Seong Hun; Kim, Dongkyu; Lee, Nayoung; Kim, Jiyeun; Kim, Doyun; Ko, Changmin; Park, Chankyu

    2016-09-01

    Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of TnphoA-132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE, gidA and truA, (ii) DNA repair; recA and recC, (iii) toxin-antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase (cya) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD, gloA and sodB The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO.

  14. Modulation of auxin content in Arabidopsis confers improved drought stress resistance.

    PubMed

    Shi, Haitao; Chen, Li; Ye, Tiantian; Liu, Xiaodong; Ding, Kejian; Chan, Zhulong

    2014-09-01

    Auxin is a well-known plant phytohormone that is involved in multiple plant growth processes and stress responses. In this study, auxin response was significantly modulated under drought stress condition. The iaaM-OX transgenic lines with higher endogenous indole-3-acetic acid (IAA) level and IAA pre-treated wild type (WT) plants exhibited enhanced drought stress resistance, while the yuc1yuc2yuc6 triple mutants with lower endogenous IAA level showed decreased stress resistance in comparison to non-treated WT plants. Additionally, endogenous and exogenous auxin positively modulated the expression levels of multiple abiotic stress-related genes (RAB18, RD22, RD29A, RD29B, DREB2A, and DREB2B), and positively affected reactive oxygen species (ROS) metabolism and underlying antioxidant enzyme activities. Moreover, auxin significantly modulated some carbon metabolites including amino acids, organic acids, sugars, sugar alcohols and aromatic amines. Notably, endogenous and exogenous auxin positively modulated root architecture especially the lateral root number. Taken together, this study demonstrated that auxin might participate in the positive regulation of drought stress resistance, through regulation of root architecture, ABA-responsive genes expression, ROS metabolism, and metabolic homeostasis, at least partially.

  15. MIMIVIRE is a defence system in mimivirus that confers resistance to virophage.

    PubMed

    Levasseur, Anthony; Bekliz, Meriem; Chabrière, Eric; Pontarotti, Pierre; La Scola, Bernard; Raoult, Didier

    2016-03-10

    Since their discovery, giant viruses have revealed several unique features that challenge the conventional definition of a virus, such as their large and complex genomes, their infection by virophages and their presence of transferable short element transpovirons. Here we investigate the sensitivity of mimivirus to virophage infection in a collection of 59 viral strains and demonstrate lineage specificity in the resistance of mimivirus to Zamilon, a unique virophage that can infect lineages B and C of mimivirus but not lineage A. We hypothesized that mimiviruses harbour a defence mechanism resembling the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system that is widely present in bacteria and archaea. We performed de novo sequencing of 45 new mimivirus strains and searched for sequences specific to Zamilon in a total of 60 mimivirus genomes. We found that lineage A strains are resistant to Zamilon and contain the insertion of a repeated Zamilon sequence within an operon, here named the 'mimivirus virophage resistance element' (MIMIVIRE). Further analyses of the surrounding sequences showed that this locus is reminiscent of a defence mechanism related to the CRISPR-Cas system. Silencing the repeated sequence and the MIMIVIRE genes restores mimivirus susceptibility to Zamilon. The MIMIVIRE proteins possess the typical functions (nuclease and helicase) involved in the degradation of foreign nucleic acids. The viral defence system, MIMIVIRE, represents a nucleic-acid-based immunity against virophage infection. PMID:26934229

  16. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores.

    PubMed

    Possik, Elite; Ajisebutu, Andrew; Manteghi, Sanaz; Gingras, Marie-Claude; Vijayaraghavan, Tarika; Flamand, Mathieu; Coull, Barry; Schmeisser, Kathrin; Duchaine, Thomas; van Steensel, Maurice; Hall, David H; Pause, Arnim

    2015-10-01

    Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

  17. MIMIVIRE is a defence system in mimivirus that confers resistance to virophage.

    PubMed

    Levasseur, Anthony; Bekliz, Meriem; Chabrière, Eric; Pontarotti, Pierre; La Scola, Bernard; Raoult, Didier

    2016-03-10

    Since their discovery, giant viruses have revealed several unique features that challenge the conventional definition of a virus, such as their large and complex genomes, their infection by virophages and their presence of transferable short element transpovirons. Here we investigate the sensitivity of mimivirus to virophage infection in a collection of 59 viral strains and demonstrate lineage specificity in the resistance of mimivirus to Zamilon, a unique virophage that can infect lineages B and C of mimivirus but not lineage A. We hypothesized that mimiviruses harbour a defence mechanism resembling the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system that is widely present in bacteria and archaea. We performed de novo sequencing of 45 new mimivirus strains and searched for sequences specific to Zamilon in a total of 60 mimivirus genomes. We found that lineage A strains are resistant to Zamilon and contain the insertion of a repeated Zamilon sequence within an operon, here named the 'mimivirus virophage resistance element' (MIMIVIRE). Further analyses of the surrounding sequences showed that this locus is reminiscent of a defence mechanism related to the CRISPR-Cas system. Silencing the repeated sequence and the MIMIVIRE genes restores mimivirus susceptibility to Zamilon. The MIMIVIRE proteins possess the typical functions (nuclease and helicase) involved in the degradation of foreign nucleic acids. The viral defence system, MIMIVIRE, represents a nucleic-acid-based immunity against virophage infection.

  18. Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions.

    PubMed

    Lee, Changhan; Kim, Jihong; Kwon, Minsuk; Lee, Kihyun; Min, Haeyoung; Kim, Seong Hun; Kim, Dongkyu; Lee, Nayoung; Kim, Jiyeun; Kim, Doyun; Ko, Changmin; Park, Chankyu

    2016-09-01

    Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of TnphoA-132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE, gidA and truA, (ii) DNA repair; recA and recC, (iii) toxin-antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase (cya) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD, gloA and sodB The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO. PMID:27535647

  19. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress

    PubMed Central

    Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

    2016-01-01

    Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria. PMID:27077115

  20. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress.

    PubMed

    Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

    2016-03-01

    Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria. PMID:27077115

  1. Expression of a chitinase gene from Metarhizium anisopliae in tobacco plants confers resistance against Rhizoctonia solani.

    PubMed

    Kern, Marcelo Fernando; Maraschin, Simone de Faria; Vom Endt, Débora; Schrank, Augusto; Vainstein, Marilene Henning; Pasquali, Giancarlo

    2010-04-01

    The chit1 gene from the entomopathogenic fungus Metarhizium anisopliae, encoding the endochitinase CHIT42, was placed under the control of the CaMV 35S promoter, and the resulting construct was transferred to tobacco. Seventeen kanamycin-resistant transgenic lines were recovered, and the presence of the transgene was confirmed by polymerase c