Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships

PubMed Central

Booher, Nicholas J.; Carpenter, Sara C. D.; Sebra, Robert P.; Wang, Li; Salzberg, Steven L.; Leach, Jan E.

2015-01-01

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33–35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution. PMID:27148456

Engineered TAL Effector modulators for the large-scale gain-of-function screening

PubMed Central

Zhang, Hanshuo; Li, Juan; Hou, Sha; Wang, Gancheng; Jiang, Mingjun; Sun, Changhong; Hu, Xiongbing; Zhuang, Fengfeng; Dai, Zhifei; Dai, Junbiao; Xi, Jianzhong Jeff

2014-01-01

Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE–DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE–TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation. PMID:24939900

TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton

USDA-ARS?s Scientific Manuscript database

Bacterial blight of cotton (BBC), caused by Xanthomonas citri subsp. malvacearum (Xcm), is among the most destructive diseases in cotton (Gossypium spp.). Transcription activator-like (TAL) effectors from Xcm are essential for BBC disease progression. Here, we carried out whole-genome PacBio-seque...

Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors

PubMed Central

Wan, Hua; Hu, Jian-ping; Li, Kang-shun; Tian, Xu-hong; Chang, Shan

2013-01-01

TAL (transcriptional activator-like) effectors (TALEs) are DNA-binding proteins, containing a modular central domain that recognizes specific DNA sequences. Recently, the crystallographic studies of TALEs revealed the structure of DNA-recognition domain. In this article, molecular dynamics (MD) simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues) with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA), the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL). The conformational analysis of DNA indicates that the 5′ end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism. PMID:24130757

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

PubMed

Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

2016-01-01

Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

PubMed

Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agnès; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L; Certo, Michael T; Baker, David; Scharenberg, Andrew M

2014-02-01

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

A transcription activator-like effector from Xanthomonas oryzae pv. oryzicola elicits dose-dependent resistance in rice.

PubMed

Hummel, Aaron W; Wilkins, Katherine E; Wang, Li; Cernadas, R Andres; Bogdanove, Adam J

2017-01-01

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to

Target selection biases from recent experience transfer across effectors.

PubMed

Moher, Jeff; Song, Joo-Hyun

2016-02-01

Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions.

TALEs from a spring--superelasticity of Tal effector protein structures.

PubMed

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA.

TALEs from a Spring – Superelasticity of Tal Effector Protein Structures

PubMed Central

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA. PMID:25313859

The T-Cell Oncogene Tal2 Is a Target of PU.1 and Upregulated during Osteoclastogenesis

PubMed Central

Courtial, Nadine; Mücke, Christian; Herkt, Stefanie; Kolodziej, Stephan; Hussong, Helge; Lausen, Jörn

2013-01-01

Transcription factors play a crucial role in regulating differentiation processes during human life and are important in disease. The basic helix-loop-helix transcription factors Tal1 and Lyl1 play a major role in the regulation of gene expression in the hematopoietic system and are involved in human leukemia. Tal2, which belongs to the same family of transcription factors as Tal1 and Lyl1, is also involved in human leukaemia. However, little is known regarding the expression and regulation of Tal2 in hematopoietic cells. Here we show that Tal2 is expressed in hematopoietic cells of the myeloid lineage. Interestingly, we found that usage of the Tal2 promoter is different in human and mouse cells. Two promoters, hP1 and hP2 drive Tal2 expression in human erythroleukemia K562 cells, however in mouse RAW cells only the mP1 promoter is used. Furthermore, we found that Tal2 expression is upregulated during oesteoclastogenesis. We show that Tal2 is a direct target gene of the myeloid transcription factor PU.1, which is a key transcription factor for osteoclast gene expression. Strikingly, PU.1 binding to the P1 promoter is conserved between mouse and human, but PU.1 binding to P2 was only detected in human K562 cells. Additionally, we provide evidence that Tal2 influences the expression of the osteoclastic differentiation gene TRACP. These findings provide novel insight into the expression control of Tal2 in hematopoietic cells and reveal a function of Tal2 as a regulator of gene expression during osteoclast differentiation. PMID:24086757

Hitting the sweet spot-glycans as targets of fungal defense effector proteins.

PubMed

Künzler, Markus

2015-05-06

Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparably low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous) fungi against microbial competitors and animal predators.

Plant targets for Pseudomonas syringae type III effectors: virulence targets or guarded decoys?

PubMed

Block, Anna; Alfano, James R

2011-02-01

The phytopathogenic bacterium Pseudomonas syringae can suppress both pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) by the injection of type III effector (T3E) proteins into host cells. T3Es achieve immune suppression using a variety of strategies including interference with immune receptor signaling, blocking RNA pathways and vesicle trafficking, and altering organelle function. T3Es can be recognized indirectly by resistance proteins monitoring specific T3E targets resulting in ETI. It is presently unclear whether the monitored targets represent bona fide virulence targets or guarded decoys. Extensive overlap between PTI and ETI signaling suggests that T3Es may suppress both pathways through common targets and by possessing multiple activities. Copyright © 2010 Elsevier Ltd. All rights reserved.

A Plethora of Virulence Strategies Hidden Behind Nuclear Targeting of Microbial Effectors

PubMed Central

Rivas, Susana; Genin, Stéphane

2011-01-01

Plant immune responses depend on the ability to couple rapid recognition of the invading microbe to an efficient response. During evolution, plant pathogens have acquired the ability to deliver effector molecules inside host cells in order to manipulate cellular and molecular processes and establish pathogenicity. Following translocation into plant cells, microbial effectors may be addressed to different subcellular compartments. Intriguingly, a significant number of effector proteins from different pathogenic microorganisms, including viruses, oomycetes, fungi, nematodes, and bacteria, is targeted to the nucleus of host cells. In agreement with this observation, increasing evidence highlights the crucial role played by nuclear dynamics, and nucleocytoplasmic protein trafficking during a great variety of analyzed plant–pathogen interactions. Once in the nucleus, effector proteins are able to manipulate host transcription or directly subvert essential host components to promote virulence. Along these lines, it has been suggested that some effectors may affect histone packing and, thereby, chromatin configuration. In addition, microbial effectors may either directly activate transcription or target host transcription factors to alter their regular molecular functions. Alternatively, nuclear translocation of effectors may affect subcellular localization of their cognate resistance proteins in a process that is essential for resistance protein-mediated plant immunity. Here, we review recent progress in our field on the identification of microbial effectors that are targeted to the nucleus of host plant cells. In addition, we discuss different virulence strategies deployed by microbes, which have been uncovered through examination of the mechanisms that guide nuclear localization of effector proteins. PMID:22639625

Ancient class of translocated oomycete effectors targets the host nucleus.

PubMed

Schornack, Sebastian; van Damme, Mireille; Bozkurt, Tolga O; Cano, Liliana M; Smoker, Matthew; Thines, Marco; Gaulin, Elodie; Kamoun, Sophien; Huitema, Edgar

2010-10-05

Pathogens use specialized secretion systems and targeting signals to translocate effector proteins inside host cells, a process that is essential for promoting disease and parasitism. However, the amino acid sequences that determine host delivery of eukaryotic pathogen effectors remain mostly unknown. The Crinkler (CRN) proteins of oomycete plant pathogens, such as the Irish potato famine organism Phytophthora infestans, are modular proteins with predicted secretion signals and conserved N-terminal sequence motifs. Here, we provide direct evidence that CRN N termini mediate protein transport into plant cells. CRN host translocation requires a conserved motif that is present in all examined plant pathogenic oomycetes, including the phylogenetically divergent species Aphanomyces euteiches that does not form haustoria, specialized infection structures that have been implicated previously in delivery of effectors. Several distinct CRN C termini localized to plant nuclei and, in the case of CRN8, required nuclear accumulation to induce plant cell death. These results reveal a large family of ubiquitous oomycete effector proteins that target the host nucleus. Oomycetes appear to have acquired the ability to translocate effector proteins inside plant cells relatively early in their evolution and before the emergence of haustoria. Finally, this work further implicates the host nucleus as an important cellular compartment where the fate of plant-microbe interactions is determined.

The targeting of plant cellular systems by injected type III effector proteins.

PubMed

Lewis, Jennifer D; Guttman, David S; Desveaux, Darrell

2009-12-01

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector-host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.

Direct regulation of E-cadherin by targeted histone methylation of TALE-SET fusion protein in cancer cells.

PubMed

Cho, Hyun-Soo; Kang, Jeong Gu; Lee, Jae-Hye; Lee, Jeong-Ju; Jeon, Seong Kook; Ko, Jeong-Heon; Kim, Dae-Soo; Park, Kun-Hyang; Kim, Yong-Sam; Kim, Nam-Soon

2015-09-15

TALE-nuclease chimeras (TALENs) can bind to and cleave specific genomic loci and, are used to engineer gene knockouts and additions. Recently, instead of using the FokI domain, epigenetically active domains, such as TET1 and LSD1, have been combined with TAL effector domains to regulate targeted gene expression via DNA and histone demethylation. However, studies of histone methylation in the TALE system have not been performed. Therefore, in this study, we established a novel targeted regulation system with a TAL effector domain and a histone methylation domain. To construct a TALE-methylation fusion protein, we combined a TAL effector domain containing an E-Box region to act as a Snail binding site and the SET domain of EHMT 2 to allow for histone methylation. The constructed TALE-SET module (TSET) repressed the expression of E-cadherin via by increasing H3K9 dimethylation. Moreover, the cells that overexpressed TSET showed increased cell migration and invasion. This is the first phenotype-based study of targeted histone methylation by the TALE module, and this new system can be applied in new cancer therapies to reduce side effects.

Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy

PubMed Central

Tochio, Naoya; Umehara, Kohei; Uewaki, Jun-ichi; Flechsig, Holger; Kondo, Masaharu; Dewa, Takehisa; Sakuma, Tetsushi; Yamamoto, Takashi; Saitoh, Takashi; Togashi, Yuichi; Tate, Shin-ichi

2016-01-01

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats. PMID:27883072

Bacterial effectors target the plant cell nucleus to subvert host transcription.

PubMed

Canonne, Joanne; Rivas, Susana

2012-02-01

In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells.

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection.

PubMed

Jaouannet, Maëlle; Rosso, Marie-Noëlle

2013-09-01

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.

Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life

PubMed Central

Weßling, Ralf; Epple, Petra; Altmann, Stefan; He, Yijian; Yang, Li; Henz, Stefan R.; McDonald, Nathan; Wiley, Kristin; Bader, Kai Christian; Gläßer, Christine; Mukhtar, M. Shahid; Haigis, Sabine; Ghamsari, Lila; Stephens, Amber E.; Ecker, Joseph R.; Vidal, Marc; Jones, Jonathan D. G.; Mayer, Klaus F. X.; van Themaat, Emiel Ver Loren; Weigel, Detlef; Schulze-Lefert, Paul; Dangl, Jeffery L.; Panstruga, Ralph; Braun, Pascal

2014-01-01

SUMMARY While conceptual principles governing plant immunity are becoming clear, its systems-level organization and the evolutionary dynamic of the host-pathogen interface are still obscure. We generated a systematic protein-protein interaction network of virulence effectors from the ascomycete pathogen Golovinomyces orontii and Arabidopsis thaliana host proteins. We combined this dataset with corresponding data for the eubacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. The resulting network identifies host proteins onto which intraspecies and interspecies pathogen effectors converge. Phenotyping of 124 Arabidopsis effector-interactor mutants revealed a correlation between intra- and interspecies convergence and several altered immune response phenotypes. The effectors and most heavily targeted host protein co-localized in sub-nuclear foci. Products of adaptively selected Arabidopsis genes are enriched for interactions with effector targets. Our data suggest the existence of a molecular host-pathogen interface that is conserved across Arabidopsis accessions, while evolutionary adaptation occurs in the immediate network neighborhood of effector targets. PMID:25211078

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

PubMed

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

PubMed

Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

2017-03-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

Distinct Pseudomonas type-III effectors use a cleavable transit peptide to target chloroplasts.

PubMed

Li, Guangyong; Froehlich, John E; Elowsky, Christian; Msanne, Joseph; Ostosh, Andrew C; Zhang, Chi; Awada, Tala; Alfano, James R

2014-01-01

The pathogen Pseudomonas syringae requires a type-III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type-III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type-III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector-triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP-triggered immune responses compared with wild-type plants. An N-terminal region of HopK1 shares similarity with the corresponding region in the well-studied type-III effector AvrRps4; however, their C-terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast localized, indicating that the shared N-terminal regions of these type-III effectors represent a chloroplast transit peptide. The HopK1 contribution to virulence and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4-induced HR did not. Our results suggest that a primary virulence target of these type-III effectors resides in chloroplasts, and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type-III effectors use a cleavable transit peptide to localize to chloroplasts, and that targets within this organelle are important for immunity. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors.

PubMed

Konermann, Silvana; Lotfy, Peter; Brideau, Nicholas J; Oki, Jennifer; Shokhirev, Maxim N; Hsu, Patrick D

2018-04-19

Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development. Copyright © 2018 Elsevier Inc. All rights reserved.

A Xanthomonas oryzae pv. oryzae effector, XopR, associates with receptor-like cytoplasmic kinases and suppresses PAMP-triggered stomatal closure.

PubMed

Wang, Shuangfeng; Sun, Jianhang; Fan, Fenggui; Tan, Zhaoyun; Zou, Yanmin; Lu, Dongping

2016-09-01

Receptor-like kinases (RLKs) play important roles in plant immunity signaling; thus, many are hijacked by pathogen effectors to promote successful pathogenesis. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice leaf blight disease. The strain PXO99A has 18 non-TAL (transcription activation-like) effectors; however, their mechanisms of action and host target proteins remain largely unknown. Although the effector XopR from the Xoo strain MAFF311018 was shown to suppress PAMP-triggered immune responses in Arabidopsis, its target has not yet been identified. Here, we show that PXO99A XopR interacts with BIK1 at the plasma membrane. BIK1 is a receptor-like cytoplasmic kinase (RLCK) belonging to the RLK family of proteins and mediates PAMP-triggered stomatal immunity. In turn, BIK1 phosphorylates XopR. Furthermore, XopR suppresses PAMP-triggered stomatal closure in transgenic Arabidopsis expressing XopR. In addition, XopR is able to associate with RLCKs other than BIK1. These results suggest that XopR likely suppresses plant immunity by targeting BIK1 and other RLCKs.

Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators.

PubMed

Bhattacharjee, Saikat; Halane, Morgan K; Kim, Sang Hee; Gassmann, Walter

2011-12-09

Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll-interleukin-1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR-mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

PubMed Central

2017-01-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.

PubMed

Hummel, Aaron W; Doyle, Erin L; Bogdanove, Adam J

2012-09-01

Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

A systems biology perspective on plant-microbe interactions: biochemical and structural targets of pathogen effectors.

PubMed

Pritchard, Leighton; Birch, Paul

2011-04-01

Plants have biochemical defences against stresses from predators, parasites and pathogens. In this review we discuss the interaction of plant defences with microbial pathogens such as bacteria, fungi and oomycetes, and viruses. We examine principles of complex dynamic networks that allow identification of network components that are differentially and predictably sensitive to perturbation, thus making them likely effector targets. We relate these principles to recent developments in our understanding of known effector targets in plant-pathogen systems, and propose a systems-level framework for the interpretation and modelling of host-microbe interactions mediated by effectors. We describe this framework briefly, and conclude by discussing useful experimental approaches for populating this framework. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Bacterial virulence effectors and their activities.

PubMed

Hann, Dagmar R; Gimenez-Ibanez, Selena; Rathjen, John P

2010-08-01

The major virulence strategy for plant pathogenic bacteria is deployment of effector molecules within the host cytoplasm. Each bacterial strain possesses a set of 20-30 effectors which have overlapping activities, are functionally interchangeable, and diverge in composition between strains. Effectors target host molecules to suppress immunity. Two main strategies are apparent. Effectors that target host proteins seem to attack conserved structural domains but otherwise lack specificity. On the other hand, those that influence host gene transcription directly do so with extreme specificity. In both cases, examples are known where the host has exploited effector-target affinities to establish immune recognition of effectors. The molecular activity of each effector links virulence and immune outcomes. Copyright 2010 Elsevier Ltd. All rights reserved.

Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganusov, Vitaly V

2009-01-01

In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targetsmore » with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the

Xanthomonas TAL effectors hijack host basal transcription factor IIA α and γ subunits for invasion.

PubMed

Ma, Ling; Wang, Qiang; Yuan, Meng; Zou, Tingting; Yin, Ping; Wang, Shiping

2018-02-05

The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria, which infect a broad range of crops and wild plant species, cause symptoms with leaf blights, streaks, spots, stripes, necrosis, wilt, cankers and gummosis on leaves, stems and fruits in a wide variety of plants via injecting their effector proteins into the host cell during infection. Among these virulent effectors, transcription activator-like effectors (TALEs) interact with the γ subunit of host transcription factor IIA (TFIIAγ) to activate the transcription of host disease susceptibility genes. Functional TFIIA is a ternary complex comprising α, β and γ subunits. However, whether TALEs recruit TFIIAα, TFIIAβ, or both remains unknown. The underlying molecular mechanisms by which TALEs mediate host susceptibility gene activation require full elucidation. Here, we show that TALEs interact with the α+γ binary subcomplex but not the α+β+γ ternary complex of rice TFIIA (holo-OsTFIIA). The transcription factor binding (TFB) regions of TALEs, which are highly conserved in Xanthomonas species, have a dominant role in these interactions. Furthermore, the interaction between TALEs and the α+γ complex exhibits robust DNA binding activity in vitro. These results collectively demonstrate that TALE-carrying pathogens hijack the host basal transcription factors TFIIAα and TFIIAγ, but not TFIIAβ, to enhance host susceptibility during pathogen infection. The uncovered mechanism widens new insights on host-microbe interaction and provide an applicable strategy to breed high-resistance crop varieties. Copyright © 2018 Elsevier Inc. All rights reserved.

Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3.

PubMed

Song, Jing; Win, Joe; Tian, Miaoying; Schornack, Sebastian; Kaschani, Farnusch; Ilyas, Muhammad; van der Hoorn, Renier A L; Kamoun, Sophien

2009-02-03

Current models of plant-pathogen interactions stipulate that pathogens secrete effector proteins that disable plant defense components known as virulence targets. Occasionally, the perturbations caused by these effectors trigger innate immunity via plant disease resistance proteins as described by the "guard hypothesis." This model is nicely illustrated by the interaction between the fungal plant pathogen Cladosporium fulvum and tomato. C. fulvum secretes a protease inhibitor Avr2 that targets the tomato cysteine protease Rcr3(pim). In plants that carry the resistance protein Cf2, Rcr3(pim) is required for resistance to C. fulvum strains expressing Avr2, thus fulfilling one of the predictions of the guard hypothesis. Another prediction of the guard hypothesis has not yet been tested. Considering that virulence targets are important components of defense, different effectors from unrelated pathogens are expected to evolve to disable the same host target. In this study we confirm this prediction using a different pathogen of tomato, the oomycete Phytophthora infestans that is distantly related to fungi such as C. fulvum. This pathogen secretes an array of protease inhibitors including EPIC1 and EPIC2B that inhibit tomato cysteine proteases. Here we show that, similar to Avr2, EPIC1 and EPIC2B bind and inhibit Rcr3(pim). However, unlike Avr2, EPIC1 and EPIC2B do not trigger hypersensitive cell death or defenses on Cf-2/Rcr3(pim) tomato. We also found that the rcr3-3 mutant of tomato that carries a premature stop codon in the Rcr3 gene exhibits enhanced susceptibility to P. infestans, suggesting a role for Rcr3(pim) in defense. In conclusion, our findings fulfill a key prediction of the guard hypothesis and suggest that the effectors Avr2, EPIC1, and EPIC2B secreted by two unrelated pathogens of tomato target the same defense protease Rcr3(pim). In contrast to C. fulvum, P. infestans appears to have evolved stealthy effectors that carry inhibitory activity without

Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells.

PubMed

Quentin, Michaëel; Abad, Pierre; Favery, Bruno

2013-01-01

Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells (FCs). Effectors synthesized in the esophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized FCs requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defense responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalized within FC nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesized roles in the unique feeding behavior of these pests.

The Pseudomonas syringae type III effector HopG1 targets mitochondria, alters plant development, and suppresses plant innate immunity

PubMed Central

Block, Anna; Guo, Ming; Li, Guangyong; Elowsky, Christian; Clemente, Thomas E.; Alfano, James R.

2009-01-01

Summary The bacterial plant pathogen Pseudomonas syringae uses a type III protein secretion system to inject type III effectors into plant cells. Primary targets of these effectors appear to be effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). The type III effector HopG1 is a suppressor of ETI that is broadly conserved in bacterial plant pathogens. Here we show that HopG1 from P. syringae pv. tomato DC3000 also suppresses PTI. Interestingly, HopG1 localizes to plant mitochondria, suggesting that its suppression of innate immunity may be linked to a perturbation of mitochondrial function. While HopG1 possesses no obvious mitochondrial signal peptide, its N-terminal two-thirds was sufficient for mitochondrial localization. A HopG1-GFP fusion lacking HopG1’s N-terminal 13 amino acids was not localized to the mitochondria reflecting the importance of the N-terminus for targeting. Constitutive expression of HopG1 in Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Lycopersicon esculentum (tomato) dramatically alters plant development resulting in dwarfism, increased branching and infertility. Constitutive expression of HopG1 in planta leads to reduced respiration rates and an increased basal level of reactive oxygen species. These findings suggest that HopG1’s target is mitochondrial and that effector/target interaction promotes disease by disrupting mitochondrial functions. PMID:19863557

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors.

PubMed

Wels, Winfried; Biburger, Markus; Müller, Tina; Dälken, Benjamin; Giesübel, Ulrike; Tonn, Torsten; Uherek, Christoph

2004-03-01

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells' potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.

An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters

PubMed Central

Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

2015-01-01

Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters. PMID:26714171

An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

PubMed

Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

2015-12-01

Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

PubMed Central

Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

50 CFR 648.231 - Spiny dogfish Annual Catch Target (ACT) and Total Allowable Level of Landings (TAL).

Code of Federal Regulations, 2014 CFR

2014-10-01

... ACT and TAL recommendation process. (1) The ACT shall be identified as less than or equal to the ACL... conduct a detailed review of fishery performance relative to TALs in conjunction with any ACL performance...

50 CFR 648.231 - Spiny dogfish Annual Catch Target (ACT) and Total Allowable Level of Landings (TAL).

Code of Federal Regulations, 2012 CFR

2012-10-01

... ACT and TAL recommendation process. (1) The ACT shall be identified as less than or equal to the ACL... conduct a detailed review of fishery performance relative to TALs in conjunction with any ACL performance...

50 CFR 648.231 - Spiny dogfish Annual Catch Target (ACT) and Total Allowable Level of Landings (TAL).

Code of Federal Regulations, 2013 CFR

2013-10-01

... ACT and TAL recommendation process. (1) The ACT shall be identified as less than or equal to the ACL... conduct a detailed review of fishery performance relative to TALs in conjunction with any ACL performance...

A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.

PubMed

Jiang, Feng; Waterfield, Nicholas R; Yang, Jian; Yang, Guowei; Jin, Qi

2014-05-14

Widely found in animal and plant-associated proteobacteria, type VI secretion systems (T6SSs) are potentially capable of facilitating diverse interactions with eukaryotes and/or other bacteria. Pseudomonas aeruginosa encodes three distinct T6SS haemolysin coregulated protein (Hcp) secretion islands (H1, H2, and H3-T6SS), each involved in different aspects of the bacterium's interaction with other organisms. Here we describe the characterization of a P. aeruginosa H3-T6SS-dependent phospholipase D effector, PldB, and its three tightly linked cognate immunity proteins. PldB targets the periplasm of prokaryotic cells and exerts an antibacterial activity. Surprisingly, PldB also facilitates intracellular invasion of host eukaryotic cells by activation of the PI3K/Akt pathway, revealing it to be a trans-kingdom effector. Our findings imply a potentially widespread T6SS-mediated mechanism, which deploys a single phospholipase effector to influence both prokaryotic cells and eukaryotic hosts. Copyright © 2014 Elsevier Inc. All rights reserved.

Bacterial effectors target the common signaling partner BAK1 to disrupt multiple MAMP receptor-signaling complexes and impede plant immunity.

PubMed

Shan, Libo; He, Ping; Li, Jianming; Heese, Antje; Peck, Scott C; Nürnberger, Thorsten; Martin, Gregory B; Sheen, Jen

2008-07-17

Successful pathogens have evolved strategies to interfere with host immune systems. For example, the ubiquitous plant pathogen Pseudomonas syringae injects two sequence-distinct effectors, AvrPto and AvrPtoB, to intercept convergent innate immune responses stimulated by multiple microbe-associated molecular patterns (MAMPs). However, the direct host targets and precise molecular mechanisms of bacterial effectors remain largely obscure. We show that AvrPto and AvrPtoB bind the Arabidopsis receptor-like kinase BAK1, a shared signaling partner of both the flagellin receptor FLS2 and the brassinosteroid receptor BRI1. This targeting interferes with ligand-dependent association of FLS2 with BAK1 during infection. It also impedes BAK1-dependent host immune responses to diverse other MAMPs and brassinosteroid signaling. Significantly, the structural basis of AvrPto-BAK1 interaction appears to be distinct from AvrPto-Pto association required for effector-triggered immunity. These findings uncover a unique strategy of bacterial pathogenesis where virulence effectors block signal transmission through a key common component of multiple MAMP-receptor complexes.

The subclonal complexity of STIL-TAL1+ T-cell acute lymphoblastic leukaemia.

PubMed

Furness, Caroline L; Mansur, Marcela B; Weston, Victoria J; Ermini, Luca; van Delft, Frederik W; Jenkinson, Sarah; Gale, Rosemary; Harrison, Christine J; Pombo-de-Oliveira, Maria S; Sanchez-Martin, Marta; Ferrando, Adolfo A; Kearns, Pamela; Titley, Ian; Ford, Anthony M; Potter, Nicola E; Greaves, Mel

2018-03-20

Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.

Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection.

PubMed

Qiao, Yongli; Shi, Jinxia; Zhai, Yi; Hou, Yingnan; Ma, Wenbo

2015-05-05

A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate-glutamate-alanine-histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein.

PubMed

Yan, Winston X; Chong, Shaorong; Zhang, Huaibin; Makarova, Kira S; Koonin, Eugene V; Cheng, David R; Scott, David A

2018-04-19

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection. Copyright © 2018 Elsevier Inc. All rights reserved.

Deciphering Interplay between Salmonella Invasion Effectors

PubMed Central

Koronakis, Vassilis

2008-01-01

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction. PMID:18389058

Plant-bacterial pathogen interactions mediated by type III effectors.

PubMed

Feng, Feng; Zhou, Jian-Min

2012-08-01

Effectors secreted by the bacterial type III system play a central role in the interaction between Gram-negative bacterial pathogens and their host plants. Recent advances in the effector studies have helped cementing several key concepts concerning bacterial pathogenesis, plant immunity, and plant-pathogen co-evolution. Type III effectors use a variety of biochemical mechanisms to target specific host proteins or DNA for pathogenesis. The identifications of their host targets led to the identification of novel components of plant innate immune system. Key modules of plant immune signaling pathways such as immune receptor complexes and MAPK cascades have emerged as a major battle ground for host-pathogen adaptation. These modules are attacked by multiple type III effectors, and some components of these modules have evolved to actively sense the effectors and trigger immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

PADI4 acts as a coactivator of Tal1 by counteracting repressive histone arginine methylation

NASA Astrophysics Data System (ADS)

Kolodziej, Stephan; Kuvardina, Olga N.; Oellerich, Thomas; Herglotz, Julia; Backert, Ingo; Kohrs, Nicole; Buscató, Estel. La; Wittmann, Sandra K.; Salinas-Riester, Gabriela; Bonig, Halvard; Karas, Michael; Serve, Hubert; Proschak, Ewgenij; Lausen, Jörn

2014-05-01

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4, which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast, at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia.

LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

PubMed Central

Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

2017-01-01

Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

An effector of apple proliferation phytoplasma targets TCP transcription factors-a generalized virulence strategy of phytoplasma?

PubMed

Janik, Katrin; Mithöfer, Axel; Raffeiner, Margot; Stellmach, Hagen; Hause, Bettina; Schlink, Katja

2017-04-01

The plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple-growing areas within Europe. Despite its economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11-like effector ATP_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11-like proteins seem to be key players in phytoplasmal infection. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.

PubMed

Robin, Guillaume P; Kleemann, Jochen; Neumann, Ulla; Cabre, Lisa; Dallery, Jean-Félix; Lapalu, Nicolas; O'Connell, Richard J

2018-01-01

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum , encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium -mediated transformation to transiently express them as N -terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana , and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C -terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

PubMed Central

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-01-01

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

PubMed

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

PubMed Central

Liu, Yunxiao; Lan, Xia; Song, Shiren; Yin, Ling; Dry, Ian B.; Qu, Junjie; Xiang, Jiang; Lu, Jiang

2018-01-01

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria. PMID:29706971

CdiA Effectors from Uropathogenic Escherichia coli Use Heterotrimeric Osmoporins as Receptors to Recognize Target Bacteria

PubMed Central

Beck, Christina M.; Willett, Julia L. E.; Kim, Jeff J.; Low, David A.; Hayes, Christopher S.

2016-01-01

Many Gram-negative bacterial pathogens express contact-dependent growth inhibition (CDI) systems that promote cell-cell interaction. CDI+ bacteria express surface CdiA effector proteins, which transfer their C-terminal toxin domains into susceptible target cells upon binding to specific receptors. CDI+ cells also produce immunity proteins that neutralize the toxin domains delivered from neighboring siblings. Here, we show that CdiAEC536 from uropathogenic Escherichia coli 536 (EC536) uses OmpC and OmpF as receptors to recognize target bacteria. E. coli mutants lacking either ompF or ompC are resistant to CDIEC536-mediated growth inhibition, and both porins are required for target-cell adhesion to inhibitors that express CdiAEC536. Experiments with single-chain OmpF fusions indicate that the CdiAEC536 receptor is heterotrimeric OmpC-OmpF. Because the OmpC and OmpF porins are under selective pressure from bacteriophages and host immune systems, their surface-exposed loops vary between E. coli isolates. OmpC polymorphism has a significant impact on CDIEC536 mediated competition, with many E. coli isolates expressing alleles that are not recognized by CdiAEC536. Analyses of recombinant OmpC chimeras suggest that extracellular loops L4 and L5 are important recognition epitopes for CdiAEC536. Loops L4 and L5 also account for much of the sequence variability between E. coli OmpC proteins, raising the possibility that CDI contributes to the selective pressure driving OmpC diversification. We find that the most efficient CdiAEC536 receptors are encoded by isolates that carry the same cdi gene cluster as E. coli 536. Thus, it appears that CdiA effectors often bind preferentially to "self" receptors, thereby promoting interactions between sibling cells. As a consequence, these effector proteins cannot recognize nor suppress the growth of many potential competitors. These findings suggest that self-recognition and kin selection are important functions of CDI. PMID:27723824

How effectors promote beneficial interactions.

PubMed

Miwa, Hiroki; Okazaki, Shin

2017-08-01

Beneficial microbes such as rhizobia possess effector proteins that are secreted into the host cytoplasm where they modulate host-signaling pathways. Among these effectors, type 3 secreted effectors (T3Es) of rhizobia play roles in promoting nitrogen-fixing nodule symbiosis, suppressing host defenses and directly activating symbiosis-related processes. Rhizobia use the same strategy as pathogenic bacteria to suppress host defenses such as targeting the MAPK cascade. In addition, rhizobial T3E can promote root nodule symbiosis by directly activating Nod factor signaling, which bypasses Nod factor perception. The various strategies employed by beneficial microbes to promote infection and maintain viability in the host are therefore crucial for plant endosymbiosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity.

PubMed

Ma, Ka-Wai; Ma, Wenbo

2016-12-01

Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted "effector" proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

DOE PAGES

Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; ...

2014-12-08

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

PubMed

Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

2015-07-01

Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity

PubMed Central

2016-01-01

SUMMARY Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. PMID:27784797

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

PubMed Central

Shames, Stephanie R.; Liu, Luying; Havey, James C.; Schofield, Whitman B.; Goodman, Andrew L.; Roy, Craig R.

2017-01-01

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis. PMID:29133401

Ectopic activation of the rice NLR heteropair RGA4/RGA5 confers resistance to bacterial blight and bacterial leaf streak diseases.

PubMed

Hutin, Mathilde; Césari, Stella; Chalvon, Véronique; Michel, Corinne; Tran, Tuan Tu; Boch, Jens; Koebnik, Ralf; Szurek, Boris; Kroj, Thomas

2016-10-01

Bacterial blight (BB) and bacterial leaf streak (BLS) are important diseases in Oryza sativa caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. In both bacteria, transcription activator-like (TAL) effectors are major virulence determinants that act by transactivating host genes downstream of effector-binding elements (EBEs) bound in a sequence-specific manner. Resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance. For Xoc, no resistance sources are known in rice. Here, we investigated whether the recognition of effectors by nucleotide binding and leucine-rich repeat domain immune receptors (NLRs), the most widespread resistance mechanism in plants, is also able to stop BB and BLS. In one instance, transgenic rice lines harboring the AVR1-CO39 effector gene from the rice blast fungus Magnaporthe oryzae, under the control of an inducible promoter, were challenged with transgenic Xoo and Xoc strains carrying a TAL effector designed to transactivate the inducible promoter. This induced AVR1-CO39 expression and triggered BB and BLS resistance when the corresponding Pi-CO39 resistance locus was present. In a second example, the transactivation of an auto-active NLR by Xoo-delivered designer TAL effectors resulted in BB resistance, demonstrating that NLR-triggered immune responses efficiently control Xoo. This forms the foundation for future BB and BLS disease control strategies, whereupon endogenous TAL effectors will target synthetic promoter regions of Avr or NLR executor genes. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants

PubMed Central

Jwa, Nam-Soo; Hwang, Byung Kook

2017-01-01

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants. PMID:29033963

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants.

PubMed

Jwa, Nam-Soo; Hwang, Byung Kook

2017-01-01

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

PubMed

Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

2018-05-04

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

PubMed

Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

2016-04-01

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au. © 2015 CSIRO New Phytologist © 2015 New Phytologist Trust.

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.

Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT,more » NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of action.« less

Yersinia type III effectors perturb host innate immune responses

PubMed Central

Pha, Khavong; Navarro, Lorena

2016-01-01

The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts.

PubMed

Petre, Benjamin; Lorrain, Cécile; Saunders, Diane G O; Win, Joe; Sklenar, Jan; Duplessis, Sébastien; Kamoun, Sophien

2016-04-01

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast-targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N-terminal signal-dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells. © 2015 John Wiley & Sons Ltd.

A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

PubMed

Han, Wenyuan; Li, Yingjun; Deng, Ling; Feng, Mingxia; Peng, Wenfang; Hallstrøm, Søren; Zhang, Jing; Peng, Nan; Liang, Yun Xiang; White, Malcolm F; She, Qunxin

2017-02-28

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

PubMed

Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

2015-10-13

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida.

PubMed

Thorpe, Peter; Mantelin, Sophie; Cock, Peter Ja; Blok, Vivian C; Coke, Mirela C; Eves-van den Akker, Sebastian; Guzeeva, Elena; Lilley, Catherine J; Smant, Geert; Reid, Adam J; Wright, Kathryn M; Urwin, Peter E; Jones, John T

2014-10-23

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Killing of intrafamilial leukocytes by earthworm effector cells.

PubMed

Suzuki, M M; Cooper, E L

1995-01-01

When Lumbricus and Eisenia coelomocytes are cultured together in intrafamilial xenogeneic combinations, significant cytotoxicity occurs at 24 h but not at 5 nor 72 h, as shown by trypan blue assay. In a 4.5-h assay, measuring 51Cr release, using an effector/target ratio of 25:1, unpooled cells from a single Lumbricus killed Eisenia cells at levels of 6% and 14%. However, Eisenia coelomocyte survival was high and identical in either cell-free xenogeneic (Lumbricus) coelomic fluid or in artificial medium. In this 1-way assay, earthworm (Lumbricus) coelomocytes act as effector cells that kill non-self target cells, even those of other earthworms. Comparisons with previous results reveal greater reliability and consistently repeatable results when the 51Cr release assay is used to measure cytotoxicity regardless of the targets.

Interaction of a Blumeria graminis f. sp. hordei effector candidate with a barley ARF-GAP suggests that host vesicle trafficking is a fungal pathogenicity target.

PubMed

Schmidt, Sarah M; Kuhn, Hannah; Micali, Cristina; Liller, Corinna; Kwaaitaal, Mark; Panstruga, Ralph

2014-08-01

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1-BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana-infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non-adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin-conjugating enzyme, and an ADP ribosylation factor-GTPase-activating protein (ARF-GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF-GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF-GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence-associated host vesicle trafficking. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

PubMed

Yang, Lina; McLellan, Hazel; Naqvi, Shaista; He, Qin; Boevink, Petra C; Armstrong, Miles; Giuliani, Licida M; Zhang, Wei; Tian, Zhendong; Zhan, Jiasui; Gilroy, Eleanor M; Birch, Paul R J

2016-05-01

Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease. © 2016 American Society of Plant Biologists. All Rights Reserved.

Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides.

PubMed

Salomon, Dor; Guo, Yirui; Kinch, Lisa N; Grishin, Nick V; Gardner, Kevin H; Orth, Kim

2013-01-01

Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

PubMed

Genga, Ryan M; Kearns, Nicola A; Maehr, René

2016-05-15

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

The small phytoplasma virulence effector SAP11 contains distinct domains required for nuclear targeting and CIN-TCP binding and destabilization.

PubMed

Sugio, Akiko; MacLean, Allyson M; Hogenhout, Saskia A

2014-05-01

Phytoplasmas are insect-transmitted bacterial phytopathogens that secrete virulence effectors and induce changes in the architecture and defense response of their plant hosts. We previously demonstrated that the small (± 10 kDa) virulence effector SAP11 of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) binds and destabilizes Arabidopsis CIN (CINCINNATA) TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 AND 2) transcription factors, resulting in dramatic changes in leaf morphogenesis and increased susceptibility to phytoplasma insect vectors. SAP11 contains a bipartite nuclear localization signal (NLS) that targets this effector to plant cell nuclei. To further understand how SAP11 functions, we assessed the involvement of SAP11 regions in TCP binding and destabilization using a series of mutants. SAP11 mutants lacking the entire N-terminal domain, including the NLS, interacted with TCPs but did not destabilize them. SAP11 mutants lacking the C-terminal domain were impaired in both binding and destabilization of TCPs. These SAP11 mutants did not alter leaf morphogenesis. A SAP11 mutant that did not accumulate in plant nuclei (SAP11ΔNLS-NES) was able to bind and destabilize TCP transcription factors, but instigated weaker changes in leaf morphogenesis than wild-type SAP11. Overall the results suggest that phytoplasma effector SAP11 has a modular organization in which at least three domains are required for efficient CIN-TCP destabilization in plants. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.

PubMed

Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang

2017-01-01

Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

Intestinal Effector T Cells in Health and Disease

PubMed Central

Maynard, Craig L.; Weaver, Casey T.

2011-01-01

Summary Crohn’s disease and ulcerative colitis are the two major forms of chronic relapsing inflammatory disorders of the human intestines collectively referred to as inflammatory bowel disease (IBD). Though a complex set of autoinflammatory disorders that can be precipitated by diverse genetic and environmental factors, a feature that appears common to IBD pathogenesis is a dysregulated effector T cell response to the commensal microbiota. Due to the heightened effector T cell activity in IBD, developmental and functional pathways that give rise to these cells are potential targets for therapeutic intervention. In this review, we highlight recent advances in our understanding of effector T cell biology in the context of intestinal immune regulation and speculate on their potential clinical significance. PMID:19766082

Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections

PubMed Central

Schiffer, Joshua T.; Aubert, Martine; Weber, Nicholas D.; Mintzer, Esther; Stone, Daniel

2012-01-01

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches. PMID:22718830

LMO2 is required for TAL1 DNA binding activity and initiation of definitive haematopoiesis at the haemangioblast stage.

PubMed

Stanulovic, Vesna S; Cauchy, Pierre; Assi, Salam A; Hoogenkamp, Maarten

2017-09-29

LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2-/- mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2-/- with Tal1-/- Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins.

PubMed

Hewezi, Tarek

2015-10-01

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. © 2015 American Society of Plant Biologists. All Rights Reserved.

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

PubMed Central

Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

Antibody distance from the cell membrane regulates antibody effector mechanisms

PubMed Central

Cleary, Kirstie L.S.; Chan, H.T. Claude; James, Sonja; Glennie, Martin J.; Cragg, Mark S.

2017-01-01

Immunotherapy using monoclonal antibodies (mAb) such as rituximab is an established means of treating haematological malignancies. Antibodies can elicit a number of mechanisms to delete target cells, including complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). The inherent properties of the target molecule help define which of these mechanisms are more important for efficacy. However, why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity, is often unclear. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules were generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains, to produce a panel of chimeric molecules which held the target epitope at different distances along the protein. It was shown that CDC and ADCC favoured a membrane proximal epitope, whilst ADCP favoured an epitope positioned further away. These findings were then confirmed using reagents targeting the membrane proximal or distal domains of CD137 itself before investigating these properties in vivo where a clear difference in the splenic clearance of transfected tumour cells was observed. Together, this work demonstrates how altering the position of the antibody epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents. PMID:28404636

Chromatin looping defines expression of TAL1, its flanking genes, and regulation in T-ALL.

PubMed

Zhou, Yan; Kurukuti, Sreenivasulu; Saffrey, Peter; Vukovic, Milica; Michie, Alison M; Strogantsev, Ruslan; West, Adam G; Vetrie, David

2013-12-19

TAL1 is an important regulator of hematopoiesis and its expression is tightly controlled despite complexities in its genomic organization. It is frequently misregulated in T-cell acute lymphoblastic leukemia (T-ALL), often due to deletions between TAL1 and the neighboring STIL gene. To better understand the events that lead to TAL1 expression in hematopoiesis and in T-ALL, we studied looping interactions at the TAL1 locus. In TAL1-expressing erythroid cells, the locus adopts a looping "hub" which brings into close physical proximity all known TAL1 cis-regulatory elements including CTCF-bound insulators. Loss of GATA1 results in disassembly of the hub and loss of CTCF/RAD21 from one of its insulators. Genes flanking TAL1 are partly dependent on hub integrity for their transcriptional regulation. We identified looping patterns unique to TAL1-expressing T-ALL cells, and, intriguingly, loops occurring between the TAL1 and STIL genes at the common TAL1/STIL breakpoints found in T-ALL. These findings redefine how TAL1 and neighboring genes communicate within the nucleus, and indicate that looping facilitates both normal and aberrant TAL1 expression and may predispose to structural rearrangements in T-ALL. We also propose that GATA1-dependent looping mechanisms may facilitate the conservation of TAL1 regulation despite cis-regulatory remodeling during vertebrate evolution.

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

PubMed Central

Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

2016-01-01

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

PubMed Central

Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

2016-01-01

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

Allogeneic killing by earthworm effector cells.

PubMed

Suzuki, M M; Cooper, E L

1995-01-01

We observed spontaneous allogeneic cytotoxicity by coelomocytes (Lumbricus terrestris) using three assays: trypan blue, lactate dehydrogenase release and chromium-51 release. Cell-cell contact may not be essential to effect cytotoxicity, since killing of allogeneic cells occurred in pooled allogeneic coelomic fluid derived from worms raised in two different geographic locales. We observed no significant spontaneous cytotoxicity against autogeneic target coelomocytes haptenated with 2,4,6-trinitrobenzene sulfonic acid; however, coelomocytes effected significant spontaneous cytotoxicity against haptenated allogeneic targets. These results support the view that earthworm coelomocytes can act as effector cells that can specifically kill nonself target cells.

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes.

PubMed

Win, Joe; Kamoun, Sophien

2008-04-01

Plant pathogenic microbes deliver effector proteins inside host cells to modulate plant defense circuitry and enable parasitic colonization. As genome sequences from plant pathogens become available, genome-wide evolutionary analyses will shed light on how pathogen effector genes evolved and adapted to the cellular environment of their host plants. In the August 2007 issue of Plant Cell, we described adaptive evolution (positive selection) in the cytoplasmic RXLR effectors of three recently sequenced oomycete plant pathogens. Here, we summarize our findings and describe additional data that further validate our approach.

At the Frontier; RXLR Effectors Crossing the Phytophthora-Host Interface.

PubMed

Bouwmeester, Klaas; Meijer, Harold J G; Govers, Francine

2011-01-01

Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens - like the potato late blight pathogen Phytophthora infestans - it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.

Targeting of RNA Polymerase II by a nuclear Legionella pneumophila Dot/Icm effector SnpL.

PubMed

Schuelein, Ralf; Spencer, Hugh; Dagley, Laura F; Li, Peng Fei; Luo, Lin; Stow, Jennifer L; Abraham, Gilu; Naderer, Thomas; Gomez-Valero, Laura; Buchrieser, Carmen; Sugimoto, Chihiro; Yamagishi, Junya; Webb, Andrew I; Pasricha, Shivani; Hartland, Elizabeth L

2018-04-24

The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localize to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localizes to the nucleus where they subvert host cell transcriptional responses to infection. Here we identified Lpg2519 (Lpp2587/Lpw27461), as a new nuclear-localized effector that we have termed SnpL. Upon ectopic expression or during L. pneumophila infection, SnpL showed strong nuclear localization by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, SUPT5H/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex (DSIF complex) that regulates RNA polymerase II (Pol II) dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central KOW motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L. pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression. This article is protected by copyright. All rights reserved.

Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions

PubMed Central

Skinner, Cassandra C.; McMichael, Elizabeth L.; Jaime-Ramirez, Alena C.; Abrams, Zachary B.; Lee, Robert J.; Carson, William E.

2016-01-01

The folate receptor (FR) is over-expressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is over-expressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis, using KB (human oral epithelial) and F01 (human melanoma) as a positive and negative control, respectively. FR-positive and negative cell lines were treated with F-IgG or control immunoglobulin G (C-IgG) in the presence or absence of cytokines in order to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells enhanced following treatment with F-IgG, as compared to C-IgG at all effector:target (E:T) ratios (p<0.01). This trend was further enhanced by NK cell stimulation with the activating cytokine interleukin-12 (IL-12). NK cell production of cytokines such as interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α), and regulated on activation normal T-cell expressed and secreted (RANTES) were also significantly increased in response to co-stimulation with IL-12 stimulation and F-IgG-coated Mel 39 target cells, as compared to controls (p<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells which can be further enhanced by the addition of cytokines. PMID:27035691

Minimal Mimicry: Mere Effector Matching Induces Preference

ERIC Educational Resources Information Center

Sparenberg, Peggy; Topolinski, Sascha; Springer, Anne; Prinz, Wolfgang

2012-01-01

Both mimicking and being mimicked induces preference for a target. The present experiments investigate the minimal sufficient conditions for this mimicry-preference link to occur. We argue that mere effector matching between one's own and the other person's movement is sufficient to induce preference, independent of which movement is actually…

Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system.

PubMed

Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

2013-10-11

Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

Identifying p53 Transactivation Domain 1-Specific Inhibitors to Alleviate the Side Effects of Prostate Cancer Therapy

DTIC Science & Technology

2013-09-01

further expanded with the exciting   7   development of Tal-effector and CRISPR guided nucleases. Transcription activator-like effector nucleases...also be achieved by the recently developed CRISPR -Cas9 system. CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) is widely believed...to be the most efficient method to engineer mammalian genomes. CRISPR RNAs (crRNA) that hybridize to a specific target DNA can be utilized to guide a

Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners

PubMed Central

Toruño, Tania Y.; Stergiopoulos, Ioannis; Coaker, Gitta

2017-01-01

Plants possess large arsenals of immune receptors capable of recognizing all pathogen classes. To cause disease, pathogenic organisms must be able to overcome physical barriers, suppress or evade immune perception, and derive nutrients from host tissues. Consequently, to facilitate some of these processes, pathogens secrete effector proteins that promote colonization. This review covers recent advances in the field of effector biology, focusing on conserved cellular processes targeted by effectors from diverse pathogens. The ability of effectors to facilitate pathogen entry into the host interior, suppress plant immune perception, and alter host physiology for pathogen benefit is discussed. Pathogens also deploy effectors in a spatial and temporal manner, depending on infection stage. Recent advances have also enhanced our understanding of effectors acting in specific plant organs and tissues. Effectors are excellent cellular probes that facilitate insight into biological processes as well as key points of vulnerability in plant immune signaling networks. PMID:27359369

An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus.

PubMed

Shantharaj, Deepak; Römer, Patrick; Figueiredo, Jose F L; Minsavage, Gerald V; Krönauer, Christina; Stall, Robert E; Moore, Gloria A; Fisher, Latanya C; Hu, Yang; Horvath, Diana M; Lahaye, Thomas; Jones, Jeffrey B

2017-09-01

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs3 1EBE ), and fused this engineered promoter with multiple EBEs (ProBs3 14EBE ) to either the β-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs3 1EBE and ProBs3 14EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs3 14EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs3 14EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tilden, A.B.; Cauda, R.; Grossi, C.E.

1986-06-01

Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar tomore » those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.« less

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors.

PubMed

Wei, Hai-Lei; Collmer, Alan

2017-12-25

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Two-axis angular effector

DOEpatents

Vaughn, Mark R.; Robinett, III, Rush D.; Phelan, John R.; Van Zuiden, Don M.

1997-01-21

A new class of coplanar two-axis angular effectors. These effectors combine a two-axis rotational joint analogous to a Cardan joint with linear actuators in a manner to produce a wider range of rotational motion about both axes defined by the joint. This new class of effectors also allows design of robotic manipulators having very high strength and efficiency. These effectors are particularly suited for remote operation in unknown surroundings, because of their extraordinary versatility. An immediate application is to the problems which arise in nuclear waste remediation.

[Effector proteins of Clamidia].

PubMed

Kariagina, A S; Alekseevskiĭ, A V; Spirin, S A; Zigangirova, N A; Gintsburg, A L

2009-01-01

The review summarizes the recent published data on molecular mechanisms of Chlamidiae - host cell interaction, first of all on chlamydial effector proteins. Such proteins as well as III transport system proteins that transfer many effector proteins into host cytoplasm are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of the infection. Patogen proteins participating in the next stage, that is the intracellular inclusion traffic to the centrosome, are CT229 of C. trachomatis and Cpn0585 of C. pneumoniae, which interact with cellular Rab GTPases. In C. trachomatis, IncA protein plays a key role in chlamydial inclusions fusion, CT847 modulates life cycle of the host cell, LDA3 is essential in acquisition of nutrients. CPAF protease and inclusion membrane proteins IncG and CADD participate in suppression of apoptosis of infected cells. The proteases CPAF and CT441, as well as deubiquitinating ChlaDub1 protein, contribute to avoiding the immune response.

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana.

PubMed

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium -mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa , Psa -NZ V13 and Psa -NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana . Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium -mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1 , NDR1 , or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana . In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta .

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana

PubMed Central

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta. PMID:29326748

Intraspecies Competition in Serratia marcescens Is Mediated by Type VI-Secreted Rhs Effectors and a Conserved Effector-Associated Accessory Protein

PubMed Central

Alcoforado Diniz, Juliana

2015-01-01

ABSTRACT The type VI secretion system (T6SS) is widespread in Gram-negative bacteria and can deliver toxic effector proteins into eukaryotic cells or competitor bacteria. Antibacterial T6SSs are increasingly recognized as key mediators of interbacterial competition and may contribute to the outcome of many polymicrobial infections. Multiple antibacterial effectors can be delivered by these systems, with diverse activities against target cells and distinct modes of secretion. Polymorphic toxins containing Rhs repeat domains represent a recently identified and as-yet poorly characterized class of T6SS-dependent effectors. Previous work had revealed that the potent antibacterial T6SS of the opportunistic pathogen Serratia marcescens promotes intraspecies as well as interspecies competition (S. L. Murdoch, K. Trunk, G. English, M. J. Fritsch, E. Pourkarimi, and S. J. Coulthurst, J Bacteriol 193:6057–6069, 2011, http://dx.doi.org/10.1128/JB.05671-11). In this study, two new Rhs family antibacterial effectors delivered by this T6SS have been identified. One of these was shown to act as a DNase toxin, while the other contains a novel, cytoplasmic-acting toxin domain. Importantly, using S. marcescens, it has been demonstrated for the first time that Rhs proteins, rather than other T6SS-secreted effectors, can be the primary determinant of intraspecies competition. Furthermore, a new family of accessory proteins associated with T6SS effectors has been identified, exemplified by S. marcescens EagR1, which is specifically required for deployment of its associated Rhs effector. Together, these findings provide new insight into how bacteria can use the T6SS to deploy Rhs-family effectors and mediate different types of interbacterial interactions. IMPORTANCE Infectious diseases caused by bacterial pathogens represent a continuing threat to health and economic prosperity. To counter this threat, we must understand how such organisms survive and prosper. The type VI secretion

Evaluation of Secretion Prediction Highlights Differing Approaches Needed for Oomycete and Fungal Effectors.

PubMed

Sperschneider, Jana; Williams, Angela H; Hane, James K; Singh, Karam B; Taylor, Jennifer M

2015-01-01

The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen's advantage. Proteinaceous effectors are synthesized intracellularly and must be externalized to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score) and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localization predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes.

Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease

PubMed Central

Hu, Yang; Zhang, Junli; Jia, Hongge; Sosso, Davide; Li, Ting; Frommer, Wolf B.; Yang, Bing; White, Frank F.; Wang, Nian; Jones, Jeffrey B.

2014-01-01

Citrus bacterial canker (CBC) disease occurs worldwide and incurs considerable costs both from control measures and yield losses. Bacteria that cause CBC require one of six known type III transcription activator-like (TAL) effector genes for the characteristic pustule formation at the site of infection. Here, we show that Xanthomonas citri subspecies citri strain Xcc306, with the type III TAL effector gene pthA4 or with the distinct yet biologically equivalent gene pthAw from strain XccAw, induces two host genes, CsLOB1 and CsSWEET1, in a TAL effector-dependent manner. CsLOB1 is a member of the Lateral Organ Boundaries (LOB) gene family of transcription factors, and CsSWEET1 is a homolog of the SWEET sugar transporter and rice disease susceptibility gene. Both TAL effectors drive expression of CsLOB1 and CsSWEET1 promoter reporter gene fusions when coexpressed in citrus or Nicotiana benthamiana. Artificially designed TAL effectors directed to sequences in the CsLOB1 promoter region, but not the CsSWEET1 promoter, promoted pustule formation and higher bacterial leaf populations. Three additional distinct TAL effector genes, pthA*, pthB, and pthC, also direct pustule formation and expression of CsLOB1. Unlike pthA4 and pthAw, pthB and pthC do not promote the expression of CsSWEET1. CsLOB1 expression was associated with the expression of genes associated with cell expansion. The results indicate that CBC-inciting species of Xanthomonas exploit a single host disease susceptibility gene by altering the expression of an otherwise developmentally regulated gene using any one of a diverse set of TAL effector genes in the pathogen populations. PMID:24474801

Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease.

PubMed

Hu, Yang; Zhang, Junli; Jia, Hongge; Sosso, Davide; Li, Ting; Frommer, Wolf B; Yang, Bing; White, Frank F; Wang, Nian; Jones, Jeffrey B

2014-01-28

Citrus bacterial canker (CBC) disease occurs worldwide and incurs considerable costs both from control measures and yield losses. Bacteria that cause CBC require one of six known type III transcription activator-like (TAL) effector genes for the characteristic pustule formation at the site of infection. Here, we show that Xanthomonas citri subspecies citri strain Xcc306, with the type III TAL effector gene pthA4 or with the distinct yet biologically equivalent gene pthAw from strain XccA(w), induces two host genes, CsLOB1 and CsSWEET1, in a TAL effector-dependent manner. CsLOB1 is a member of the Lateral Organ Boundaries (LOB) gene family of transcription factors, and CsSWEET1 is a homolog of the SWEET sugar transporter and rice disease susceptibility gene. Both TAL effectors drive expression of CsLOB1 and CsSWEET1 promoter reporter gene fusions when coexpressed in citrus or Nicotiana benthamiana. Artificially designed TAL effectors directed to sequences in the CsLOB1 promoter region, but not the CsSWEET1 promoter, promoted pustule formation and higher bacterial leaf populations. Three additional distinct TAL effector genes, pthA*, pthB, and pthC, also direct pustule formation and expression of CsLOB1. Unlike pthA4 and pthAw, pthB and pthC do not promote the expression of CsSWEET1. CsLOB1 expression was associated with the expression of genes associated with cell expansion. The results indicate that CBC-inciting species of Xanthomonas exploit a single host disease susceptibility gene by altering the expression of an otherwise developmentally regulated gene using any one of a diverse set of TAL effector genes in the pathogen populations.

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

PubMed

Lee, Chang-Han; Romain, Gabrielle; Yan, Wupeng; Watanabe, Makiko; Charab, Wissam; Todorova, Biliana; Lee, Jiwon; Triplett, Kendra; Donkor, Moses; Lungu, Oana I; Lux, Anja; Marshall, Nicholas; Lindorfer, Margaret A; Goff, Odile Richard-Le; Balbino, Bianca; Kang, Tae Hyun; Tanno, Hidetaka; Delidakis, George; Alford, Corrine; Taylor, Ronald P; Nimmerjahn, Falk; Varadarajan, Navin; Bruhns, Pierre; Zhang, Yan Jessie; Georgiou, George

2017-08-01

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

PubMed

Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

2016-04-01

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

A type III effector protease NleC from enteropathogenic Escherichia coli targets NF-κB for degradation

PubMed Central

Pearson, Jaclyn S; Riedmaier, Patrice; Marchès, Olivier; Frankel, Gad; Hartland, Elizabeth L

2011-01-01

Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that enteropathogenic Escherichia coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IκB degradation and nuclear translocation of the p65 subunit of NF-κB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP–NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-κB components, c-Rel and p50. Recombinant His6-NleC induced p65 and p50 cleavage in HeLa cell lysates and mutation of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-κB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation. PMID:21306441

Proteomics of effector-triggered immunity (ETI) in plants.

PubMed

Hurley, Brenden; Subramaniam, Rajagopal; Guttman, David S; Desveaux, Darrell

2014-01-01

Effector-triggered immunity (ETI) was originally termed gene-for-gene resistance and dates back to fundamental observations of flax resistance to rust fungi by Harold Henry Flor in the 1940s. Since then, genetic and biochemical approaches have defined our current understanding of how plant "resistance" proteins recognize microbial effectors. More recently, proteomic approaches have expanded our view of the protein landscape during ETI and contributed significant advances to our mechanistic understanding of ETI signaling. Here we provide an overview of proteomic techniques that have been used to study plant ETI including both global and targeted approaches. We discuss the challenges associated with ETI proteomics and highlight specific examples from the literature, which demonstrate how proteomics is advancing the ETI research field.

Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

PubMed

Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

2015-07-15

Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Type III secretion system effector proteins: double agents in bacterial disease and plant defense.

PubMed

Alfano, James R; Collmer, Alan

2004-01-01

Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.

Type IV secretion system of Brucella spp. and its effectors

PubMed Central

Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

2015-01-01

Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis. PMID:26528442

Type IV secretion system of Brucella spp. and its effectors.

PubMed

Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

2015-01-01

Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis.

Oomycetes, effectors, and all that jazz.

PubMed

Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

2012-08-01

Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella

PubMed Central

Liesch, Marius

2017-01-01

Host-targeting type IV secretion systems (T4SS) evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP) domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal–the BID (Bep intracellular delivery) domain—similar to the Bartonella effector proteins (Beps) that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping stone in the

A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella.

PubMed

Harms, Alexander; Liesch, Marius; Körner, Jonas; Québatte, Maxime; Engel, Philipp; Dehio, Christoph

2017-10-01

Host-targeting type IV secretion systems (T4SS) evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP) domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery) domain-similar to the Bartonella effector proteins (Beps) that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping stone in the

Autonomous dexterous end-effectors for space robotics

NASA Technical Reports Server (NTRS)

Bekey, George A.; Iberall, Thea; Liu, Huan

1989-01-01

The development of a knowledge-based controller is summarized for the Belgrade/USC robot hand, a five-fingered end effector, designed for maximum autonomy. The biological principles of the hand and its architecture are presented. The conceptual and software aspects of the grasp selection system are discussed, including both the effects of the geometry of the target object and the task to be performed. Some current research issues are presented.

Phytophthora parasitica Effector PpRxLR2 Suppresses Nicotiana benthamiana Immunity.

PubMed

Dalio, R J D; Maximo, H J; Oliveira, T S; Dias, R O; Breton, M C; Felizatti, H; Machado, M

2018-04-01

Phytophthora species secrete several classes of effector proteins during interaction with their hosts. These proteins can have multiple functions including modulation of host physiology and immunity. The RxLR effectors have the ability to enter plant cells using the plant machinery. Some of these effectors have been characterized as immunity suppressors; however, very little is known about their functions in the interaction between Phytophthora parasitica and its hosts. Using a bioinformatics pipeline, we have identified 172 candidate RxLR effectors (CREs) in the isolate IAC 01_95 of P. parasitica. Of these 172 CREs, 93 were found to be also present in eight other genomes of P. parasitica, isolated from different hosts and continents. After transcriptomics and gene expression analysis, we have found five CREs to be up-regulated in in-vitro and in-planta samples. Subsequently, we selected three CREs for functional characterization in the model plant Nicotiana benthamiana. We show that PpRxLR2 is able to completely suppress INF-1-induced cell death, whereas PpRxLR3 and PpRxLR5 moderately suppressed N. benthamiana immunity in a less-extensive manner. Moreover, we confirmed the effector-triggered susceptibility activity of these proteins after transient transformation and infection of N. benthamiana plants. All three CREs enhanced virulence of P. parasitica during the interaction with N. benthamiana. These effectors, in particular PpRxLR2, can be targeted for the development of biotechnology-based control strategies of P. parasitica diseases.

Functional versus effector-specific organization of the human posterior parietal cortex: revisited

PubMed Central

Leone, Frank T. M.; Medendorp, W. Pieter

2016-01-01

It has been proposed that the posterior parietal cortex (PPC) is characterized by an effector-specific organization. However, strikingly similar functional MRI (fMRI) activation patterns have been found in the PPC for hand and foot movements. Because the fMRI signal is related to average neuronal activity, similar activation levels may result either from effector-unspecific neurons or from intermingled subsets of effector-specific neurons within a voxel. We distinguished between these possibilities using fMRI repetition suppression (RS). Participants made delayed, goal-directed eye, hand, and foot movements to visual targets. In each trial, the instructed effector was identical or different to that of the previous trial. RS effects indicated an attenuation of the fMRI signal in repeat trials. The caudal PPC was active during the delay but did not show RS, suggesting that its planning activity was effector independent. Hand and foot-specific RS effects were evident in the anterior superior parietal lobule (SPL), extending to the premotor cortex, with limb overlap in the anterior SPL. Connectivity analysis suggested information flow between the caudal PPC to limb-specific anterior SPL regions and between the limb-unspecific anterior SPL toward limb-specific motor regions. These results underline that both function and effector specificity should be integrated into a concept of PPC action representation not only on a regional but also on a fine-grained, subvoxel level. PMID:27466132

The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice.

PubMed

Park, Chan-Ho; Chen, Songbiao; Shirsekar, Gautam; Zhou, Bo; Khang, Chang Hyun; Songkumarn, Pattavipha; Afzal, Ahmed J; Ning, Yuese; Wang, Ruyi; Bellizzi, Maria; Valent, Barbara; Wang, Guo-Liang

2012-11-01

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

PubMed Central

Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

2016-01-01

Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants.

PubMed

MacLean, Allyson M; Sugio, Akiko; Makarova, Olga V; Findlay, Kim C; Grieve, Victoria M; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A

2011-10-01

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

PubMed

Mashimo, Tomoji

2014-01-01

The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed. © 2013 The Author Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Differential Effector Engagement by Oncogenic KRAS.

PubMed

Yuan, Tina L; Amzallag, Arnaud; Bagni, Rachel; Yi, Ming; Afghani, Shervin; Burgan, William; Fer, Nicole; Strathern, Leslie A; Powell, Katie; Smith, Brian; Waters, Andrew M; Drubin, David; Thomson, Ty; Liao, Rosy; Greninger, Patricia; Stein, Giovanna T; Murchie, Ellen; Cortez, Eliane; Egan, Regina K; Procter, Lauren; Bess, Matthew; Cheng, Kwong Tai; Lee, Chih-Shia; Lee, Liam Changwoo; Fellmann, Christof; Stephens, Robert; Luo, Ji; Lowe, Scott W; Benes, Cyril H; McCormick, Frank

2018-02-13

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

PubMed

Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

2016-02-01

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

Identification of legionella effectors using bioinformatic approaches.

PubMed

Segal, Gil

2013-01-01

Legionella pneumophila the causative agent of Legionnaires' disease, actively manipulates host cell processes to establish a replication niche inside host cells. The establishment of its replication niche requires a functional Icm/Dot type IV secretion system which translocates about 300 effector proteins into host cells during infection. Many of these effectors were first identified as effector candidates by several bioinformatic approaches, and these predicted effectors were later examined experimentally for translocation and a large number of which were validated as effector proteins. Here, I summarized the bioinformatic approaches that were used to identify these effectors.

Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

PubMed Central

Teper, Doron; Sunitha, Sukumaran; Martin, Gregory B; Sessa, Guido

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. PMID:26237448

Trafficking arms: oomycete effectors enter host plant cells.

PubMed

Birch, Paul R J; Rehmany, Anne P; Pritchard, Leighton; Kamoun, Sophien; Beynon, Jim L

2006-01-01

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.

Identification and Characterisation of a Hyper-Variable Apoplastic Effector Gene Family of the Potato Cyst Nematodes

PubMed Central

Eves-van den Akker, Sebastian; Lilley, Catherine J.; Jones, John T.; Urwin, Peter E.

2014-01-01

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism. PMID:25255291

Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

PubMed

Eves-van den Akker, Sebastian; Lilley, Catherine J; Jones, John T; Urwin, Peter E

2014-09-01

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.

A Conserved Basal Transcription Factor Is Required for the Function of Diverse TAL Effectors in Multiple Plant Hosts.

PubMed

Huang, Renyan; Hui, Shugang; Zhang, Meng; Li, Pei; Xiao, Jinghua; Li, Xianghua; Yuan, Meng; Wang, Shiping

2017-01-01

Many Xanthomonas bacteria use transcription activator-like effector (TALE) proteins to activate plant disease susceptibility ( S ) genes, and this activation contributes to disease. We recently reported that rice basal transcription factor IIA gamma subunit, OsTFIIAγ5, is hijacked by TALE-carrying Xanthomonas oryzae infecting the plants. However, whether TFIIAγs are also involved in TALE-carrying Xanthomonas -caused diseases in other plants is unknown. Here, molecular and genetic approaches were used to investigate the role of TFIIAγs in other plants. We found that TFIIAγs are also used by TALE-carrying Xanthomonas to cause disease in other plants. The TALEs of Xanthomonas citri pv. citri ( Xcc ) causing canker in citrus and Xanthomonas campestris pv. vesicatoria ( Xcv ) causing bacterial spot in pepper and tomato interacted with corresponding host TFIIAγs as in rice. Transcriptionally suppressing TFIIAγ led to resistance to Xcc in citrus and Xcv in pepper and tomato. The 39th residue of OsTFIIAγ5 and citrus CsTFIIAγ is vital for TALE-dependent induction of plant S genes. As mutated OsTFIIAγ5 V 39E , CsTFIIAγ V 39E , pepper CaTFIIAγ V 39E , and tomato SlTFIIAγ V 39E also did not interact with TALEs to prevent disease. These results suggest that TALE-carrying bacteria share a common mechanism for infecting plants. Using TFIIAγ V 39E -type mutation could be a general strategy for improving resistance to TALE-carrying pathogens in crops.

Advances in the Engineering of the Gene Editing Enzymes and the Genomes: Understanding and Handling the Off-Target Effects of CRISPR/Cas9.

PubMed

Yin, Yufang; Wang, Qian; Xiao, Li; Wang, Fengjiao; Song, Zhuo; Zhou, Cuilan; Liu, Xuan; Xing, Chungen; He, Nongyue; Li, Kai; Feng, Yan; Zhang, Jia

2018-03-01

In the past decades, significant progresses have been achieved in genetic engineering of nucleases. Among the genetically engineered nucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and CRIPSPR/Cas9 system form a new field of gene editing. The gene editing efficiency or targeting effect and the off-target effect are the two major determinant factors in evaluating the usefulness of a new enzyme. Engineering strategies in improving these gene editing enzymes, particularly in minimizing their off-target effects, are the focus of this paper. Examples of using these genetically engineered enzymes in genome modification are discussed in order to better understand the requirement of engineering efforts in obtaining more powerful and useful gene editing enzymes. In addition, the identification of naturally existed anti-Cas proteins has been employed in minimizing off-target effects. Considering the future application in human gene therapy, optimization of these well recognized gene editing enzymes and exploration of more novel enzymes are both required. Before people find an ideal gene editing system having virtually no off-target effect, technologies used to screen and identify off-target effects are of importance in clinical trials employing gene therapy.

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

PubMed

Teper, Doron; Salomon, Dor; Sunitha, Sukumaran; Kim, Jung-Gun; Mudgett, Mary Beth; Sessa, Guido

2014-01-01

Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein-protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14-3-3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14-3-3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

PubMed Central

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8–13% and reliably measures NK cell- or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. PMID:17617419

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

PubMed

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

A novel mode of enhancer evolution: The Tal1 stem cell enhancer recruited a MIR element to specifically boost its activity

PubMed Central

Smith, Aileen M.; Sanchez, Maria-Jose; Follows, George A.; Kinston, Sarah; Donaldson, Ian J.; Green, Anthony R.; Göttgens, Berthold

2008-01-01

Altered cis-regulation is thought to underpin much of metazoan evolution, yet the underlying mechanisms remain largely obscure. The stem cell leukemia TAL1 (also known as SCL) transcription factor is essential for the normal development of blood stem cells and we have previously shown that the Tal1 +19 enhancer directs expression to hematopoietic stem cells, hematopoietic progenitors, and to endothelium. Here we demonstrate that an adjacent region 1 kb upstream (+18 element) is in an open chromatin configuration and carries active histone marks but does not function as an enhancer in transgenic mice. Instead, it boosts activity of the +19 enhancer both in stable transfection assays and during differentiation of embryonic stem (ES) cells carrying single-copy reporter constructs targeted to the Hprt locus. The +18 element contains a mammalian interspersed repeat (MIR) which is essential for the +18 function and which was transposed to the Tal1 locus ∼160 million years ago at the time of the mammalian/marsupial branchpoint. Our data demonstrate a previously unrecognized mechanism whereby enhancer activity is modulated by a transposon exerting a “booster” function which would go undetected by conventional transgenic approaches. PMID:18687876

A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

DOE PAGES

Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...

2017-01-27

Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H 95E XXH 99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cellmore » rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Furthermore, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.« less

Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

PubMed

Truan, Daphné; Bjelić, Saša; Li, Xiao-Dan; Winkler, Fritz K

2014-07-29

The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Excitations Élémentaires au Voisinage de la Surface de Séparation d'un Métal Normal et d'un Métal Supraconducteur

NASA Astrophysics Data System (ADS)

Saint-James, Par D.

On étudie le spectre d'excitation pour une couche de métal normal déposée sur un supraconducteur. On montre que si l'interaction attractive électron-électron est négligeable dans le métal normal, il n'y a pas de gap d'énergie dans le spectre d'excitation, même si l'épaisseur de la couche normale est petite. Une étude analogue, conduisant à une conclusion similaire, est menée pour deux supraconducteurs accolés et pour des sphères de métal normal baignant dans un supraconducteur. L'effet prévu pourrait expliquer quelques résultats particuliers observés dans des mesures d'effet tunnel dans des supraconducteurs durs. The excitation spectrum of a layer of normal metal (N) deposited on a superconducting substrate (S) is discussed. It is shown that if the electron-electron attractive interaction is negligibly small in (N) there is no energy gap in the excitation spectrum even if the thickness of the layer (N) is small. A similar study, with equivalent conclusions, has been carried out for two superconductors and for normal metal spheres embedded in a superconductor. The effect may possibly explain some peculiar results of tunnelling experiments on hard superconductors.

Manipulation of host membranes by bacterial effectors.

PubMed

Ham, Hyeilin; Sreelatha, Anju; Orth, Kim

2011-07-18

Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R-mediated immunity in Arabidopsis.

PubMed

Tasset, Céline; Bernoux, Maud; Jauneau, Alain; Pouzet, Cécile; Brière, Christian; Kieffer-Jacquinod, Sylvie; Rivas, Susana; Marco, Yves; Deslandes, Laurent

2010-11-18

Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as "guards". The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity.

Repeat-containing protein effectors of plant-associated organisms

PubMed Central

Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

Repeat-containing protein effectors of plant-associated organisms.

PubMed

Mesarich, Carl H; Bowen, Joanna K; Hamiaux, Cyril; Templeton, Matthew D

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Spatial resolution limits for the isotropic-3D PET detector X’tal cube

NASA Astrophysics Data System (ADS)

Yoshida, Eiji; Tashima, Hideaki; Hirano, Yoshiyuki; Inadama, Naoko; Nishikido, Fumihiko; Murayama, Hideo; Yamaya, Taiga

2013-11-01

Positron emission tomography (PET) has become a popular imaging method in metabolism, neuroscience, and molecular imaging. For dedicated human brain and small animal PET scanners, high spatial resolution is needed to visualize small objects. To improve the spatial resolution, we are developing the X’tal cube, which is our new PET detector to achieve isotropic 3D positioning detectability. We have shown that the X’tal cube can achieve 1 mm3 uniform crystal identification performance with the Anger-type calculation even at the block edges. We plan to develop the X’tal cube with even smaller 3D grids for sub-millimeter crystal identification. In this work, we investigate spatial resolution of a PET scanner based on the X’tal cube using Monte Carlo simulations for predicting resolution performance in smaller 3D grids. For spatial resolution evaluation, a point source emitting 511 keV photons was simulated by GATE for all physical processes involved in emission and interaction of positrons. We simulated two types of animal PET scanners. The first PET scanner had a detector ring 14.6 cm in diameter composed of 18 detectors. The second PET scanner had a detector ring 7.8 cm in diameter composed of 12 detectors. After the GATE simulations, we converted the interacting 3D position information to digitalized positions for realistic segmented crystals. We simulated several X’tal cubes with cubic crystals from (0.5 mm)3 to (2 mm)3 in size. Also, for evaluating the effect of DOI resolution, we simulated several X’tal cubes with crystal thickness from (0.5 mm)3 to (9 mm)3. We showed that sub-millimeter spatial resolution was possible using cubic crystals smaller than (1.0 mm)3 even with the assumed physical processes. Also, the weighted average spatial resolutions of both PET scanners with (0.5 mm)3 cubic crystals were 0.53 mm (14.6 cm ring diameter) and 0.48 mm (7.8 cm ring diameter). For the 7.8 cm ring diameter, spatial resolution with 0.5×0.5×1.0 mm3 crystals

Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor

PubMed Central

Aurass, Philipp; Oates, Clare V.; Tate, Edward W.; Hartland, Elizabeth L.; Flieger, Antje

2015-01-01

Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo. PMID:26216420

Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia1[W][OA

PubMed Central

Wroblewski, Tadeusz; Caldwell, Katherine S.; Piskurewicz, Urszula; Cavanaugh, Keri A.; Xu, Huaqin; Kozik, Alexander; Ochoa, Oswaldo; McHale, Leah K.; Lahre, Kirsten; Jelenska, Joanna; Castillo, Jose A.; Blumenthal, Daniel; Vinatzer, Boris A.; Greenberg, Jean T.; Michelmore, Richard W.

2009-01-01

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell. PMID:19571308

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

DOE PAGES

Weyman, Philip D.; Beeri, Karen; Lefebvre, Stephane C.; ...

2014-10-10

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow onmore » urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.« less

Functions and impact of tal-like genes in animals with regard to applied aspects.

PubMed

Zhu, Min; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2018-06-16

A large number of DNAs in eukaryote genomes can code for atypical transcripts, and their functions are controversial. It has been reported that the transcripts contain many small open reading frames (sORFs), which were originally considered as non-translatable RNAs. However, increasing evidence has suggested that some of these sORFs can encode for small peptides and some are conserved across large evolutionary distances. It has been reported that the small peptides have functions and may be involved in varieties of cellular processes, playing important roles in development, physiology, and metabolism. Among the sORFs, studies of the non-canonical gene polished rice/tarsal-less (pri/tal) in Drosophila and mille-pattes(mlpt) in Tribolium have been more thoroughly studied. The genes similar to pri/tal in other species have been defined as the tarsal-less-related gene family, tal-like gene. In this review, we described recent progress in the discovery and functional characterization of the small peptides encoded by the tal-like gene and their possible functional potentials.

Innate immunity in rice

PubMed Central

Chen, Xuewei; Ronald, Pamela C.

2011-01-01

Advances in studies of rice innate immunity have led to the identification and characterization of host sensors encoding receptor kinases that perceive conserved microbial signatures. The non-RD domain, a newly recognized hallmark of these receptor kinases is highly expanded in rice (Oryza sativa) compared with Arabidopsis (Arabidopsis thaliana). Researchers have also identified a diverse array of microbial effectors from bacterial and fungal pathogens that triggers immune responses upon perception. These include both, effectors that indirectly target host Nucleotide binding site/Leucine rice repeat (NBS-LRR) proteins and transcription activator-like (TAL) effectors that directly bind promoters of host genes. Here we review the recognition and signaling events that govern rice innate immunity. PMID:21602092

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments[OPEN

PubMed Central

2017-01-01

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses. PMID:28619883

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

PubMed Central

Charpentier, Xavier; Gabay, Joëlle E.; Reyes, Moraima; Zhu, Jing W.; Weiss, Arthur; Shuman, Howard A.

2009-01-01

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions. PMID:19578436

Autoreactive T effector memory differentiation mirrors β-cell function in type 1 diabetes.

PubMed

Yeo, Lorraine; Woodwyk, Alyssa; Sood, Sanjana; Lorenc, Anna; Eichmann, Martin; Pujol-Autonell, Irma; Melchiotti, Rossella; Skowera, Ania; Fidanis, Efthymios; Dolton, Garry M; Tungatt, Katie; Sewell, Andrew K; Heck, Susanne; Saxena, Alka; Beam, Craig A; Peakman, Mark

2018-05-31

In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Space Station end effector strategy study

NASA Technical Reports Server (NTRS)

Katzberg, Stephen J.; Jensen, Robert L.; Willshire, Kelli F.; Satterthwaite, Robert E.

1987-01-01

The results of a study are presented for terminology definition, identification of functional requirements, technolgy assessment, and proposed end effector development strategies for the Space Station Program. The study is composed of a survey of available or under-developed end effector technology, identification of requirements from baselined Space Station documents, a comparative assessment of the match between technology and requirements, and recommended strategies for end effector development for the Space Station Program.

Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

PubMed Central

Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

2013-01-01

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

PubMed

Schroeder, Gunnar N; Aurass, Philipp; Oates, Clare V; Tate, Edward W; Hartland, Elizabeth L; Flieger, Antje; Frankel, Gad

2015-10-01

Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Scleroderma pathogenesis: a pivotal role for fibroblasts as effector cells

PubMed Central

2013-01-01

Scleroderma (systemic sclerosis; SSc) is characterised by fibrosis of the skin and internal organs in the context of autoimmunity and vascular perturbation. Overproduction of extracellular matrix components and loss of specialised epithelial structures are analogous to the process of scar formation after tissue injury. Fibroblasts are the resident cells of connective tissue that become activated at sites of damage and are likely to be important effector cells in SSc. Differentiation into myofibroblasts is a hallmark process, although the mechanisms and cellular origins of this important fibroblastic cell are still unclear. This article reviews fibroblast biology in the context of SSc and highlights the potentially important place of fibroblast effector cells in fibrosis. Moreover, the heterogeneity of fibroblast properties, multiplicity of regulatory pathways and diversity of origin for myofibroblasts may underpin clinical diversity in SSc, and provide novel avenues for targeted therapy. PMID:23796020

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

PubMed

Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

2015-05-01

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Revisiting the TALE repeat.

PubMed

Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

2014-04-01

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.

Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

PubMed

Heidrich, Katharina; Wirthmueller, Lennart; Tasset, Céline; Pouzet, Cécile; Deslandes, Laurent; Parker, Jane E

2011-12-09

Pathogen effectors are intercepted by plant intracellular nucleotide binding-leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll-interleukin-1 receptor)-NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment-specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

Local sensory control of a dexterous end effector

NASA Technical Reports Server (NTRS)

Pinto, Victor H.; Everett, Louis J.; Driels, Morris

1990-01-01

A numerical scheme was developed to solve the inverse kinematics for a user-defined manipulator. The scheme was based on a nonlinear least-squares technique which determines the joint variables by minimizing the difference between the target end effector pose and the actual end effector pose. The scheme was adapted to a dexterous hand in which the joints are either prismatic or revolute and the fingers are considered open kinematic chains. Feasible solutions were obtained using a three-fingered dexterous hand. An algorithm to estimate the position and orientation of a pre-grasped object was also developed. The algorithm was based on triangulation using an ideal sensor and a spherical object model. By choosing the object to be a sphere, only the position of the object frame was important. Based on these simplifications, a minimum of three sensors are needed to find the position of a sphere. A two dimensional example to determine the position of a circle coordinate frame using a two-fingered dexterous hand was presented.

[Tale nucleases--new tool for genome editing].

PubMed

Glazkova, D V; Shipulin, G A

2014-01-01

The ability to introduce targeted changes in the genome of living cells or entire organisms enables researchers to meet the challenges of basic life sciences, biotechnology and medicine. Knockdown of target genes in the zygotes gives the opportunity to investigate the functions of these genes in different organisms. Replacement of single nucleotide in the DNA sequence allows to correct mutations in genes and thus to cure hereditary diseases. Adding transgene to specific genomic.loci can be used in biotechnology for generation of organisms with certain properties or cell lines for biopharmaceutical production. Such manipulations of gene sequences in their natural chromosomal context became possible after the emergence of the technology called "genome editing". This technology is based on the induction of a double-strand break in a specific genomic target DNA using endonucleases that recognize the unique sequences in the genome and on subsequent recovery of DNA integrity through the use of cellular repair mechanisms. A necessary tool for the genome editing is a custom-designed endonuclease which is able to recognize selected sequences. The emergence of a new type of programmable endonucleases, which were constructed on the basis of bacterial proteins--TAL-effectors (Transcription activators like effector), has become an important stage in the development of technology and promoted wide spread of the genome editing. This article reviews the history of the discovery of TAL effectors and creation of TALE nucleases, and describes their advantages over zinc finger endonucleases that appeared earlier. A large section is devoted to description of genetic modifications that can be performed using the genome editing.

An effector Peptide family required for Drosophila toll-mediated immunity.

PubMed

Clemmons, Alexa W; Lindsay, Scott A; Wasserman, Steven A

2015-04-01

In Drosophila melanogaster, recognition of an invading pathogen activates the Toll or Imd signaling pathway, triggering robust upregulation of innate immune effectors. Although the mechanisms of pathogen recognition and signaling are now well understood, the functions of the immune-induced transcriptome and proteome remain much less well characterized. Through bioinformatic analysis of effector gene sequences, we have defined a family of twelve genes - the Bomanins (Boms) - that are specifically induced by Toll and that encode small, secreted peptides of unknown biochemical activity. Using targeted genome engineering, we have deleted ten of the twelve Bom genes. Remarkably, inactivating these ten genes decreases survival upon microbial infection to the same extent, and with the same specificity, as does eliminating Toll pathway function. Toll signaling, however, appears unaffected. Assaying bacterial load post-infection in wild-type and mutant flies, we provide evidence that the Boms are required for resistance to, rather than tolerance of, infection. In addition, by generating and assaying a deletion of a smaller subset of the Bom genes, we find that there is overlap in Bom activity toward particular pathogens. Together, these studies deepen our understanding of Toll-mediated immunity and provide a new in vivo model for exploration of the innate immune effector repertoire.

Impact of end effector technology on telemanipulation performance

NASA Technical Reports Server (NTRS)

Bejczy, A. K.; Szakaly, Z.; Ohm, T.

1990-01-01

Generic requirements for end effector design are briefly summarized as derived from generic functional and operational requirements. Included is a brief summary of terms and definitions related to end effector technology. The second part contains a brief overview of end effector technology work as JPL during the past ten years, with emphasis on the evolution of new mechanical, sensing and control capabilities of end effectors. The third and major part is devoted to the description of current end effector technology. The ongoing work addresses mechanical, sensing and control details with emphasis on mechanical ruggedness, increased resolution in sensing, and close electronic and control integration with overall telemanipulator control system.

End effector with astronaut foot restraint

NASA Technical Reports Server (NTRS)

Monford, Leo G., Jr. (Inventor)

1991-01-01

The combination of a foot restraint platform designed primarily for use by an astronaut being rigidly and permanently attached to an end effector which is suitable for attachment to the manipulator arm of a remote manipulating system is described. The foot restraint platform is attached by a brace to the end effector at a location away from the grappling interface of the end effector. The platform comprises a support plate provided with a pair of stirrups for receiving the toe portion of an astronaut's boots when standing on the platform and a pair of heel retainers in the form of raised members which are fixed to the surface of the platform and located to provide abutment surfaces for abutting engagement with the heels of the astronaut's boots when his toes are in the stirrups. The heel retainers preclude a backward sliding movement of the feet on the platform and instead require a lifting of the heels in order to extract the feet. The brace for attaching the foot restraint platform to the end effector may include a pivot or swivel joint to permit various orientations of the platform with respect to the end effector.

Effector-Triggered Self-Replication in Coupled Subsystems.

PubMed

Komáromy, Dávid; Tezcan, Meniz; Schaeffer, Gaël; Marić, Ivana; Otto, Sijbren

2017-11-13

In living systems processes like genome duplication and cell division are carefully synchronized through subsystem coupling. If we are to create life de novo, similar control over essential processes such as self-replication need to be developed. Here we report that coupling two dynamic combinatorial subsystems, featuring two separate building blocks, enables effector-mediated control over self-replication. The subsystem based on the first building block shows only self-replication, whereas that based on the second one is solely responsive toward a specific external effector molecule. Mixing the subsystems arrests replication until the effector molecule is added, resulting in the formation of a host-effector complex and the liberation of the building block that subsequently engages in self-replication. The onset, rate and extent of self-replication is controlled by the amount of effector present. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

PubMed Central

Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

2013-01-01

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

Genome-Wide Analysis of Type VI System Clusters and Effectors in Burkholderia Species.

PubMed

Nguyen, Thao Thi; Lee, Hyun-Hee; Park, Inmyoung; Seo, Young-Su

2018-02-01

Type VI secretion system (T6SS) has been discovered in a variety of gram-negative bacteria as a versatile weapon to stimulate the killing of eukaryotic cells or prokaryotic competitors. Type VI secretion effectors (T6SEs) are well known as key virulence factors for important pathogenic bacteria. In many Burkholderia species, T6SS has evolved as the most complicated secretion pathway with distinguished types to translocate diverse T6SEs, suggesting their essential roles in this genus. Here we attempted to detect and characterize T6SSs and potential T6SEs in target genomes of plant-associated and environmental Burkholderia species based on computational analyses. In total, 66 potential functional T6SS clusters were found in 30 target Burkholderia bacterial genomes, of which 33% possess three or four clusters. The core proteins in each cluster were specified and phylogenetic trees of three components (i.e., TssC, TssD, TssL) were constructed to elucidate the relationship among the identified T6SS clusters. Next, we identified 322 potential T6SEs in the target genomes based on homology searches and explored the important domains conserved in effector candidates. In addition, using the screening approach based on the profile hidden Markov model (pHMM) of T6SEs that possess markers for type VI effectors (MIX motif) (MIX T6SEs), 57 revealed proteins that were not included in training datasets were recognized as novel MIX T6SE candidates from the Burkholderia species. This approach could be useful to identify potential T6SEs from other bacterial genomes.

Structure of the WipA protein reveals a novel tyrosine protein phosphatase effector from Legionella pneumophila.

PubMed

Pinotsis, Nikos; Waksman, Gabriel

2017-06-02

Legionnaires' disease is a severe form of pneumonia caused by the bacterium Legionella pneumophila. L. pneumophila pathogenicity relies on secretion of more than 300 effector proteins by a type IVb secretion system. Among these Legionella effectors, WipA has been primarily studied because of its dependence on a chaperone complex, IcmSW, for translocation through the secretion system, but its role in pathogenicity has remained unknown. In this study, we present the crystal structure of a large fragment of WipA, WipA435. Surprisingly, this structure revealed a serine/threonine phosphatase fold that unexpectedly targets tyrosine-phosphorylated peptides. The structure also revealed a sequence insertion that folds into an α-helical hairpin, the tip of which adopts a canonical coiled-coil structure. The purified protein was a dimer whose dimer interface involves interactions between the coiled coil of one WipA molecule and the phosphatase domain of another. Given the ubiquity of protein-protein interaction mediated by interactions between coiled-coils, we hypothesize that WipA can thereby transition from a homodimeric state to a heterodimeric state in which the coiled-coil region of WipA is engaged in a protein-protein interaction with a tyrosine-phosphorylated host target. In conclusion, these findings help advance our understanding of the molecular mechanisms of an effector involved in Legionella virulence and may inform approaches to elucidate the function of other effectors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

TAL Performance and Mission Analysis in a CDL Capacitor Powered Direct-Drive Configuration

NASA Technical Reports Server (NTRS)

Hrbud, Ivana; Rose, M. Frank; Oleson, Steve R.; Jenkins, Rhonald M.

1999-01-01

The goals of this research are (1) to prove the concept feasibility of a direct-drive electric propulsion system, and (2) to evaluate the performance and characteristics of a Russian TAL (Thruster with Anode Layer) operating in a long-pulse mode, powered by a capacitor-based power source developed at Space Power Institute. The TAL, designated D-55, is characterized by an external acceleration zone and is powered by a unique chemical double layer (CDL) capacitor bank with a capacitance of 4 F at a charge voltage of 400 V. Performance testing of this power supply on the TAL was conducted at NASA Lewis Research Center in Cleveland, OH. Direct thrust measurements of the TAL were obtained at CDL power levels ranging from 450 to 1750 W. The specific impulse encompassed a range from 1150 s to 2200 s, yielding thruster system efficiencies between 50 and 60%. Preliminary mission analysis of the CDL direct-drive concept and other electric propulsion options was performed for the ORACLE spacecraft in 6am/6pm and 12am/12pm, 300 km sun-synchronous orbits. The direct-drive option was competitive with the other systems by increasing available net mass between 5 and 42% and reducing two-year system wet mass between 18 and 63%. Overall, the electric propulsion power requirements for the satellite solar array were reduced between 57 and 91% depending oil the orbit evaluated The direct-drive, CDL capacitor-based concept in electric propulsion thus promises to be a highly-efficient, viable alternative for satellite operations in specific near-Earth missions.

Characterization of effectors from Fusarium graminearum

USDA-ARS?s Scientific Manuscript database

Fusarium graminearum is the causal agent of Fusarium head blight (FHB), which reduces crop yield and quality by producing various mycotoxins. Effectors play an important role in the pathogenesis of many bacterial and fungal pathogens. In this study, 26 effector candidates were selected for investiga...

Acute myeloid leukemia targets for bispecific antibodies

PubMed Central

Hoseini, S S; Cheung, N K

2017-01-01

Despite substantial gains in our understanding of the genomics of acute myelogenous leukemia (AML), patient survival remains unsatisfactory especially among the older age group. T cell-based therapy of lymphoblastic leukemia is rapidly advancing; however, its application in AML is still lagging behind. Bispecific antibodies can redirect polyclonal effector cells to engage chosen targets on leukemia blasts. When the effector cells are natural-killer cells, both antibody-dependent and antibody-independent mechanisms could be exploited. When the effectors are T cells, direct tumor cytotoxicity can be engaged followed by a potential vaccination effect. In this review, we summarize the AML-associated tumor targets and the bispecific antibodies that have been studied. The potentials and limitations of each of these systems will be discussed. PMID:28157217

RUNX1 promotes cell growth in human T-cell acute lymphoblastic leukemia by transcriptional regulation of key target genes.

PubMed

Jenkins, Catherine E; Gusscott, Samuel; Wong, Rachel J; Shevchuk, Olena O; Rana, Gurneet; Giambra, Vincenzo; Tyshchenko, Kateryna; Islam, Rashedul; Hirst, Martin; Weng, Andrew P

2018-05-04

RUNX1 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). The spectrum of RUNX1 mutations has led to the notion that it acts as a tumor suppressor in this context; however, other studies have placed RUNX1 along with transcription factors TAL1 and NOTCH1 as core drivers of an oncogenic transcriptional program. To reconcile these divergent roles, we knocked down RUNX1 in human T-ALL cell lines and deleted Runx1 or Cbfb in primary mouse T-cell leukemias. RUNX1 depletion consistently resulted in reduced cell proliferation and increased apoptosis. RUNX1 upregulated variable sets of target genes in each cell line, but consistently included a core set of oncogenic effectors including IGF1R and NRAS. Our results support the conclusion that RUNX1 has a net positive effect on cell growth in the context of established T-ALL. Copyright © 2018. Published by Elsevier Inc.

Uncovering the Legionella genus effector repertoire - strength in diversity and numbers

PubMed Central

Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

2016-01-01

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution. PMID:26752266

Accurate prediction of bacterial type IV secreted effectors using amino acid composition and PSSM profiles.

PubMed

Zou, Lingyun; Nan, Chonghan; Hu, Fuquan

2013-12-15

Various human pathogens secret effector proteins into hosts cells via the type IV secretion system (T4SS). These proteins play important roles in the interaction between bacteria and hosts. Computational methods for T4SS effector prediction have been developed for screening experimental targets in several isolated bacterial species; however, widely applicable prediction approaches are still unavailable In this work, four types of distinctive features, namely, amino acid composition, dipeptide composition, .position-specific scoring matrix composition and auto covariance transformation of position-specific scoring matrix, were calculated from primary sequences. A classifier, T4EffPred, was developed using the support vector machine with these features and their different combinations for effector prediction. Various theoretical tests were performed in a newly established dataset, and the results were measured with four indexes. We demonstrated that T4EffPred can discriminate IVA and IVB effectors in benchmark datasets with positive rates of 76.7% and 89.7%, respectively. The overall accuracy of 95.9% shows that the present method is accurate for distinguishing the T4SS effector in unidentified sequences. A classifier ensemble was designed to synthesize all single classifiers. Notable performance improvement was observed using this ensemble system in benchmark tests. To demonstrate the model's application, a genome-scale prediction of effectors was performed in Bartonella henselae, an important zoonotic pathogen. A number of putative candidates were distinguished. A web server implementing the prediction method and the source code are both available at http://bioinfo.tmmu.edu.cn/T4EffPred.

Double lead spiral platen parallel jaw end effector

NASA Technical Reports Server (NTRS)

Beals, David C.

1989-01-01

The double lead spiral platen parallel jaw end effector is an extremely powerful, compact, and highly controllable end effector that represents a significant improvement in gripping force and efficiency over the LaRC Puma (LP) end effector. The spiral end effector is very simple in its design and has relatively few parts. The jaw openings are highly predictable and linear, making it an ideal candidate for remote control. The finger speed is within acceptable working limits and can be modified to meet the user needs; for instance, greater finger speed could be obtained by increasing the pitch of the spiral. The force relaxation is comparable to the other tested units. Optimization of the end effector design would involve a compromise of force and speed for a given application.

Phytoplasma Effector SAP54 Induces Indeterminate Leaf-Like Flower Development in Arabidopsis Plants1[C][W][OA

PubMed Central

MacLean, Allyson M.; Sugio, Akiko; Makarova, Olga V.; Findlay, Kim C.; Grieve, Victoria M.; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A.

2011-01-01

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host. PMID:21849514

Effector proteins of rust fungi.

PubMed

Petre, Benjamin; Joly, David L; Duplessis, Sébastien

2014-01-01

Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.

Robotic end effector

DOEpatents

Minichan, R.L.

1993-10-05

An end effector is described for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gimbal with a probe, the gimbal holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gimbal and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion. 7 figures.

Robotic end effector

DOEpatents

Minichan, Richard L.

1993-01-01

An end effector for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gimbal with a probe, the gimbal holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gimbal and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion.

Hydrocarbon-soluble low-melting corrosion inhibitor TAL-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nesterenko, S.A.; Sorokin, V.I.; Naumenko, O.V.

1987-03-01

The inhibitor TAL-3 is intended for the corrosion protection of metals that come into contact with two-phase systems of the hydrocarbon-water type. It is applicable to the service conditions of equipment and pipelines of the petroleum and petroleum refining industries. The purpose of this paper was to electrochemically assess its solubility in such systems and its inhibitory properties on samples of 08kp steel toward the effects of refinery and oil field waste water and process emulsions both on the laboratory scale and in field tests.

Context influences on TALE–DNA binding revealed by quantitative profiling

PubMed Central

Rogers, Julia M.; Barrera, Luis A.; Reyon, Deepak; Sander, Jeffry D.; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L.

2015-01-01

Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE–DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000–20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE–DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design. PMID:26067805

Context influences on TALE-DNA binding revealed by quantitative profiling.

PubMed

Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

2015-06-11

Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

Advanced Aerodynamic Design of Passive Porosity Control Effectors

NASA Technical Reports Server (NTRS)

Hunter, Craig A.; Viken, Sally A.; Wood, Richard M.; Bauer, Steven X. S.

2001-01-01

This paper describes aerodynamic design work aimed at developing a passive porosity control effector system for a generic tailless fighter aircraft. As part of this work, a computational design tool was developed and used to layout passive porosity effector systems for longitudinal and lateral-directional control at a low-speed, high angle of attack condition. Aerodynamic analysis was conducted using the NASA Langley computational fluid dynamics code USM3D, in conjunction with a newly formulated surface boundary condition for passive porosity. Results indicate that passive porosity effectors can provide maneuver control increments that equal and exceed those of conventional aerodynamic effectors for low-speed, high-alpha flight, with control levels that are a linear function of porous area. This work demonstrates the tremendous potential of passive porosity to yield simple control effector systems that have no external moving parts and will preserve an aircraft's fixed outer mold line.

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions.

PubMed

Wallqvist, Anders; Wang, Hao; Zavaljevski, Nela; Memišević, Vesna; Kwon, Keehwan; Pieper, Rembert; Rajagopala, Seesandra V; Reifman, Jaques

2017-01-01

Coxiella burnetii is an obligate Gram-negative intracellular pathogen and the etiological agent of Q fever. Successful infection requires a functional Type IV secretion system, which translocates more than 100 effector proteins into the host cytosol to establish the infection, restructure the intracellular host environment, and create a parasitophorous vacuole where the replicating bacteria reside. We used yeast two-hybrid (Y2H) screening of 33 selected C. burnetii effectors against whole genome human and murine proteome libraries to generate a map of potential host-pathogen protein-protein interactions (PPIs). We detected 273 unique interactions between 20 pathogen and 247 human proteins, and 157 between 17 pathogen and 137 murine proteins. We used orthology to combine the data and create a single host-pathogen interaction network containing 415 unique interactions between 25 C. burnetii and 363 human proteins. We further performed complementary pairwise Y2H testing of 43 out of 91 C. burnetii-human interactions involving five pathogen proteins. We used the combined data to 1) perform enrichment analyses of target host cellular processes and pathways, 2) examine effectors with known infection phenotypes, and 3) infer potential mechanisms of action for four effectors with uncharacterized functions. The host-pathogen interaction profiles supported known Coxiella phenotypes, such as adapting cell morphology through cytoskeletal re-arrangements, protein processing and trafficking, organelle generation, cholesterol processing, innate immune modulation, and interactions with the ubiquitin and proteasome pathways. The generated dataset of PPIs-the largest collection of unbiased Coxiella host-pathogen interactions to date-represents a rich source of information with respect to secreted pathogen effector proteins and their interactions with human host proteins.

Plant immunity: a lesson from pathogenic bacterial effector proteins.

PubMed

Cui, Haitao; Xiang, Tingting; Zhou, Jian-Min

2009-10-01

Phytopathogenic bacteria inject an array of effector proteins into host cells to alter host physiology and assist the infection process. Some of these effectors can also trigger disease resistance as a result of recognition in the plant cell by cytoplasmic immune receptors. In addition to effector-triggered immunity, plants immunity can be triggered upon the detection of Pathogen/Microbe-Associated Molecular Patterns by surface-localized immune receptors. Recent progress indicates that many bacterial effector proteins use a variety of biochemical properties to directly attack key components of PAMP-triggered immunity and effector-triggered immunity, providing new insights into the molecular basis of plant innate immunity. Emerging evidence indicate that the evolution of disease resistance in plants is intimately linked to the mechanism by which bacterial effectors promote parasitism. This review focuses on how these studies have conceptually advanced our understanding of plant-pathogen interactions.

3'-NADP and 3'-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1.

PubMed

Schuebel, Felix; Rocker, Andrea; Edelmann, Daniel; Schessner, Julia; Brieke, Clara; Meinhart, Anton

2016-10-28

An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), are substantially different from the ubiquitous co-enzyme 2'-NADP and the calcium mobilizer 2'-NAADP. Interestingly, 3'-NADP and 3'-NAADP have previously been used as inhibitors or signaling molecules but were regarded as "artificial" compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3'-phosphorylated NAD derivatives. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

End-effector microprocessor

NASA Technical Reports Server (NTRS)

Doggett, William R.

1992-01-01

The topics are presented in viewgraph form and include: automated structures assembly facility current control hierarchy; automated structures assembly facility purposed control hierarchy; end-effector software state transition diagram; block diagram for ideal install composite; and conclusions.

Legionella and Coxiella effectors: strength in diversity and activity.

PubMed

Qiu, Jiazhang; Luo, Zhao-Qing

2017-10-01

Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular pathogens that use the Dot/Icm type IV secretion system to translocate effectors into host cells. These effectors are essential for the establishment of membrane-bound compartments known as replication vacuoles, which enable the survival and replication of bacteria inside host cells. The effectors interfere with diverse signalling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, to promote pathogen survival. In this Review, we explore Dot/Icm effectors from L. pneumophila and C. burnetii as key virulence factors, and we examine the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence.

Effector biology of plant-associated organisms: concepts and perspectives.

PubMed

Win, J; Chaparro-Garcia, A; Belhaj, K; Saunders, D G O; Yoshida, K; Dong, S; Schornack, S; Zipfel, C; Robatzek, S; Hogenhout, S A; Kamoun, S

2012-01-01

Every plant is closely associated with a variety of living organisms. Therefore, deciphering how plants interact with mutualistic and parasitic organisms is essential for a comprehensive understanding of the biology of plants. The field of plant-biotic interactions has recently coalesced around an integrated model. Major classes of molecular players both from plants and their associated organisms have been revealed. These include cell surface and intracellular immune receptors of plants as well as apoplastic and host-cell-translocated (cytoplasmic) effectors of the invading organism. This article focuses on effectors, molecules secreted by plant-associated organisms that alter plant processes. Effectors have emerged as a central class of molecules in our integrated view of plant-microbe interactions. Their study has significantly contributed to advancing our knowledge of plant hormones, plant development, plant receptors, and epigenetics. Many pathogen effectors are extraordinary examples of biological innovation; they include some of the most remarkable proteins known to function inside plant cells. Here, we review some of the key concepts that have emerged from the study of the effectors of plant-associated organisms. In particular, we focus on how effectors function in plant tissues and discuss future perspectives in the field of effector biology.

Mutational analysis of a predicted double β-propeller domain of the DspA/E effector of Erwinia amylovora.

PubMed

Siamer, Sabrina; Gaubert, Stéphane; Boureau, Tristan; Brisset, Marie-Noëlle; Barny, Marie-Anne

2013-05-01

The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double β-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted β-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

PubMed Central

Matsuda, Shigeaki; Okada, Natsumi; Kodama, Toshio; Honda, Takeshi; Iida, Tetsuya

2012-01-01

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H+-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death. PMID:22829766

Robotic end-effector for rewaterproofing shuttle tiles

NASA Astrophysics Data System (ADS)

Manouchehri, Davoud; Hansen, Joseph M.; Wu, Cheng M.; Yamamoto, Brian S.; Graham, Todd

1992-11-01

This paper summarizes work by Rockwell International's Space Systems Division's Robotics Group at Downey, California. The work is part of a NASA-led team effort to automate Space Shuttle rewaterproofing in the Orbiter Processing Facility at the Kennedy Space Center and the ferry facility at the Ames-Dryden Flight Research Facility. Rockwell's effort focuses on the rewaterproofing end-effector, whose function is to inject hazardous dimethylethyloxysilane into thousands of ceramic tiles on the underside of the orbiter after each flight. The paper has five sections. First, it presents background on the present manual process. Second, end-effector requirements are presented, including safety and interface control. Third, a design is presented for the five end-effector systems: positioning, delivery, containment, data management, and command and control. Fourth, end-effector testing and integrating to the total system are described. Lastly, future applications for this technology are discussed.

Protection by and maintenance of CD4 effector memory and effector T cell subsets in persistent malaria infection.

PubMed

Opata, Michael M; Ibitokou, Samad A; Carpio, Victor H; Marshall, Karis M; Dillon, Brian E; Carl, Jordan C; Wilson, Kyle D; Arcari, Christine M; Stephens, Robin

2018-04-01

Protection at the peak of Plasmodium chabaudi blood-stage malaria infection is provided by CD4 T cells. We have shown that an increase in Th1 cells also correlates with protection during the persistent phase of malaria; however, it is unclear how these T cells are maintained. Persistent malaria infection promotes protection and generates both effector T cells (Teff), and effector memory T cells (Tem). We have previously defined new CD4 Teff (IL-7Rα-) subsets from Early (TeffEarly, CD62LhiCD27+) to Late (TeffLate, CD62LloCD27-) activation states. Here, we tested these effector and memory T cell subsets for their ability to survive and protect in vivo. We found that both polyclonal and P. chabaudi Merozoite Surface Protein-1 (MSP-1)-specific B5 TCR transgenic Tem survive better than Teff. Surprisingly, as Tem are associated with antigen persistence, Tem survive well even after clearance of infection. As previously shown during T cell contraction, TeffEarly, which can generate Tem, also survive better than other Teff subsets in uninfected recipients. Two other Tem survival mechanisms identified here are that low-level chronic infection promotes Tem both by driving their proliferation, and by programming production of Tem from Tcm. Protective CD4 T cell phenotypes have not been precisely determined in malaria, or other persistent infections. Therefore, we tested purified memory (Tmem) and Teff subsets in protection from peak pathology and parasitemia in immunocompromised recipient mice. Strikingly, among Tmem (IL-7Rαhi) subsets, only TemLate (CD62LloCD27-) reduced peak parasitemia (19%), though the dominant memory subset is TemEarly, which is not protective. In contrast, all Teff subsets reduced peak parasitemia by more than half, and mature Teff can generate Tem, though less. In summary, we have elucidated four mechanisms of Tem maintenance, and identified two long-lived T cell subsets (TemLate, TeffEarly) that may represent correlates of protection or a target for

Robotic End Effectors for Hard-Rock Climbing

NASA Technical Reports Server (NTRS)

Kennedy, Brett; Leger, Patrick

2004-01-01

Special-purpose robot hands (end effectors) now under development are intended to enable robots to traverse cliffs much as human climbers do. Potential applications for robots having this capability include scientific exploration (both on Earth and other rocky bodies in space), military reconnaissance, and outdoor search and rescue operations. Until now, enabling robots to traverse cliffs has been considered too difficult a task because of the perceived need of prohibitively sophisticated planning algorithms as well as end effectors as dexterous as human hands. The present end effectors are being designed to enable robots to attach themselves to typical rock-face features with less planning and simpler end effectors. This advance is based on the emulation of the equipment used by human climbers rather than the emulation of the human hand. Climbing-aid equipment, specifically cams, aid hooks, and cam hooks, are used by sport climbers when a quick ascent of a cliff is desired (see Figure 1). Currently two different end-effector designs have been created. The first, denoted the simple hook emulator, consists of three "fingers" arranged around a central "palm." Each finger emulates the function of a particular type of climbing hook (aid hook, wide cam hook, and a narrow cam hook). These fingers are connected to the palm via a mechanical linkage actuated with a leadscrew/nut. This mechanism allows the fingers to be extended or retracted. The second design, denoted the advanced hook emulator (see Figure 2), shares these features, but it incorporates an aid hook and a cam hook into each finger. The spring-loading of the aid hook allows the passive selection of the type of hook used. The end effectors can be used in several different modes. In the aid-hook mode, the aid hook on one of the fingers locks onto a horizontal ledge while the other two fingers act to stabilize the end effector against the cliff face. In the cam-hook mode, the broad, flat tip of the cam hook is

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes†

PubMed Central

Alontaga, Aileen Y.; Fenton, Aron W.

2011-01-01

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM1-PYK). To detail similarities/differences in the allosteric function of these two homologs, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM1-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM1-PYK (i.e. the L-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs. rM1-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologs of a protein family. PMID:21261284

Genome-scale identification of Legionella pneumophila effectors using a machine learning approach.

PubMed

Burstein, David; Zusman, Tal; Degtyar, Elena; Viner, Ram; Segal, Gil; Pupko, Tal

2009-07-01

A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF) was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine learning algorithms for

Jet Engine Exhaust Nozzle Flow Effector

NASA Technical Reports Server (NTRS)

Turner, Travis L. (Inventor); Cano, Roberto J. (Inventor); Silox, Richard J. (Inventor); Buehrle, Ralph D. (Inventor); Cagle, Christopher M. (Inventor); Cabell, Randolph H. (Inventor); Hilton, George C. (Inventor)

2014-01-01

A jet engine exhaust nozzle flow effector is a chevron formed with a radius of curvature with surfaces of the flow effector being defined and opposing one another. At least one shape memory alloy (SMA) member is embedded in the chevron closer to one of the chevron's opposing surfaces and substantially spanning from at least a portion of the chevron's root to the chevron's tip.

Jet Engine Exhaust Nozzle Flow Effector

NASA Technical Reports Server (NTRS)

Turner, Travis L. (Inventor); Buehrle, Ralph D. (Inventor); Silcox, Richard J. (Inventor); Cagle, Christopher M. (Inventor); Cabell, Randolph H. (Inventor); Hilton, George C. (Inventor); Cano, Roberto J. (Inventor)

2011-01-01

A jet engine exhaust nozzle flow effector is a chevron formed with a radius of curvature with surfaces of the flow effector being defined and opposing one another. At least one shape memory alloy (SMA) member is embedded in the chevron closer to one of the chevron's opposing surfaces and substantially spanning from at least a portion of the chevron's root to the chevron's tip.

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

PubMed

Lewis, Laura A; Polanski, Krzysztof; de Torres-Zabala, Marta; Jayaraman, Siddharth; Bowden, Laura; Moore, Jonathan; Penfold, Christopher A; Jenkins, Dafyd J; Hill, Claire; Baxter, Laura; Kulasekaran, Satish; Truman, William; Littlejohn, George; Prusinska, Justyna; Mead, Andrew; Steinbrenner, Jens; Hickman, Richard; Rand, David; Wild, David L; Ott, Sascha; Buchanan-Wollaston, Vicky; Smirnoff, Nick; Beynon, Jim; Denby, Katherine; Grant, Murray

2015-11-01

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. © 2015 American Society of Plant Biologists. All rights reserved.

Regulation of Effector Treg Cells in Murine Lupus.

PubMed

Chandrasekaran, Uma; Yi, Woelsung; Gupta, Sanjay; Weng, Chien-Huan; Giannopoulou, Eugenia; Chinenov, Yurii; Jessberger, Rolf; Weaver, Casey T; Bhagat, Govind; Pernis, Alessandra B

2016-06-01

Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF-4) is an essential controller of effector Treg cells and regulates their ability to express interleukin-10 (IL-10). In non-Treg cells, IRF-4 activity is modulated by interactions with DEF-6 and its homolog switch-associated protein 70 (SWAP-70). Although mice lacking both DEF-6 and SWAP-70 (double-knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF-6 and SWAP-70 to restrain IRF-4 activity. Treg cells were evaluated by fluorescence-activated cell sorting. The B lymphocyte-induced maturation protein 1 (BLIMP-1)/IL-10 axis was assessed by crossing DKO mice with BLIMP-1-YFP-10BiT dual-reporter mice. Deletion of IRF-4 in Treg cells from DKO mice was achieved by generating FoxP3(Cre) IRF-4(fl/fl) DKO mice. The concomitant absence of DEF-6 and SWAP-70 led to increased numbers of Treg cells, which acquired an effector phenotype in a cell-intrinsic manner. In addition, Treg cells from DKO mice exhibited enhanced expression of the BLIMP-1/IL-10 axis. Notably, DKO effector Treg cells survived and expanded as disease progressed. The accumulation of Treg cells from DKO mice was associated with the up-regulation of genes controlling autophagy. IRF-4 was required for the expansion and function of effector Treg cells from DKO mice. This study revealed the existence of mechanisms that, by acting on IRF-4, can fine-tune the function and survival of effector Treg cells in lupus. These findings suggest that the existence of a powerful effector Treg cell compartment that successfully survives in an unfavorable inflammatory environment could

Differential Effector Engagement by Oncogenic KRAS. | Office of Cancer Genomics

Cancer.gov

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines.

Effector CD8+ T cell IFN-γ production and cytotoxicity are enhanced by mild hyperthermia

PubMed Central

Mace, Thomas A.; Zhong, Lingwen; Kokolus, Kathleen M.; Repasky, Elizabeth A.

2012-01-01

Purpose Clinical trials combining hyperthermia with radiation and/or chemotherapy for cancer treatment have resulted in improved overall survival and control of local recurrences. The contribution of thermally enhanced anti-immune function in these effects is of considerable interest, but not understood; studies on the fundamental effects of elevated temperature on immune effector cells are needed. The goal of this study is to investigate the potential of mild hyperthermia to impact tumor antigen-specific (Ag) effector CD8+ T cell functions. Method Pmel-1 Ag-specific CD8+ T cells were exposed to mild hyperthermia and tested for changes in IFN-γ production and cytotoxicity. Additionally, overall plasma membrane organization and the phosphorylation of signaling proteins were also investigated following heat treatment. Results Exposing effector Pmel-1 specific CD8+ T cells to mild hyperthermia (39.5°C) resulted in significantly enhanced Ag-specific IFN-γ production and tumor target cell killing compared to that seen using lower temperatures (33 and 37°C). Further, inhibition of protein synthesis during hyperthermia did not reduce subsequent Ag-induced IFN-γ production by CD8+ T cells. Correlated with these effects, we observed a distinct clustering of GM1+ lipid microdomains at the plasma membrane and enhanced phosphorylation of LAT and PKCθ which may be related to an observed enhancement of Ag-specific effector CD8+ T cell IFN-γ gene transcription following mild hyperthermia. However, mitogen–mediated production of IFN-γ, which bypasses T cell receptor activation with antigen, was not enhanced. Conclusions Antigen-dependent effector T cell activity is enhanced following mild hyperthermia. These effects could potentially occur in patients being treated with thermal therapies. These data also provide support for the use of thermal therapy as an adjuvant for immunotherapies to improve CD8+ effector cell function. PMID:22235780

Macrophages are critical effectors of antibody therapies for cancer.

PubMed

Weiskopf, Kipp; Weissman, Irving L

2015-01-01

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

A smart end-effector for assembly of space truss structures

NASA Technical Reports Server (NTRS)

Doggett, William R.; Rhodes, Marvin D.; Wise, Marion A.; Armistead, Maurice F.

1992-01-01

A unique facility, the Automated Structures Research Laboratory, is being used to investigate robotic assembly of truss structures. A special-purpose end-effector is used to assemble structural elements into an eight meter diameter structure. To expand the capabilities of the facility to include construction of structures with curved surfaces from straight structural elements of different lengths, a new end-effector has been designed and fabricated. This end-effector contains an integrated microprocessor to monitor actuator operations through sensor feedback. This paper provides an overview of the automated assembly tasks required by this end-effector and a description of the new end-effector's hardware and control software.

[The Impact of Electronic Monitoring on Healthcare Associated Infections: The Role of the HViTAL Platform].

PubMed

Oliveira, Rita Fontes; Castro, Lídia; Almeida, José Pedro; Alves, Carlos; Ferreira, António

2016-11-01

In Portugal, 9.8% of patients admitted were inflicted with healthcare associated infections, corresponding to a prevalence of 11.7%. The Hospital de São João has developed a business intelligence platform able to supervise (the patients), monitor (the clinical condition) and notify (the healthcare personnel): HViTAL. This study aims to assess the impact of electronic monitoring on healthcare associated infections since the year of HViTAL implementation. We evaluated data since January 2008 (moment from which computerized records exist) until December 2011, comparing them with subsequent data, those corresponding to January 2012 (implementation date of HViTAL) until 19 October 2015. There was an upward trend of infection parameters in the 2008 - 2011 period. Since January 2012 and October 2015, all parameters of the infection indicator showed a negative linear trend. The results are very suggestive that the HVITAL may have had an impact on improving parameters associated to healthcare associated infections. Basic measures of infection control were highlighted since 2005, with an increasing number of health professional awareness campaigns, a fact which, although not analyzed in this report, may also have contributed to the observed improvement. Our study did not include other variables such as investment in human capital. There was a clear improvement in all areas characterizing the healthcare associated infections, with obvious positive impact with the introduction of HViTAL.

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000[OPEN

PubMed Central

Lewis, Laura A.; Polanski, Krzysztof; de Torres-Zabala, Marta; Bowden, Laura; Jenkins, Dafyd J.; Hill, Claire; Baxter, Laura; Truman, William; Prusinska, Justyna; Hickman, Richard; Wild, David L.; Ott, Sascha; Buchanan-Wollaston, Vicky; Beynon, Jim

2015-01-01

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. PMID:26566919

Determination of Rab5 activity in the cell by effector pull-down assay.

PubMed

Qi, Yaoyao; Liang, Zhimin; Wang, Zonghua; Lu, Guodong; Li, Guangpu

2015-01-01

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

PubMed

Ramachandran, Sowmya R; Yin, Chuntao; Kud, Joanna; Tanaka, Kiwamu; Mahoney, Aaron K; Xiao, Fangming; Hulbert, Scot H

2017-01-01

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2, and GPA/RBP-1) and one mutated R gene-Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

Rack Insertion End Effector (RIEE) guidance

NASA Technical Reports Server (NTRS)

Malladi, Narasimha S.

1994-01-01

NASA-KSC has developed a mechanism to handle and insert Racks into the Space Station Logistic Modules. This mechanism consists of a Base with 3 motorized degrees of freedom, a 3 section motorized Boom that goes from 15 to 44 feet in length, and a Rack Insertion End Effector (RIEE) with 5 hand wheels for precise alignment. During the 1993 NASA-ASEE Summer Faculty Fellowship Program at KSC, I designed an Active Vision (Camera) Arrangement and developed an algorithm to determine (1) the displacements required by the Room for its initial positioning and (2) the rotations required at the five hand-wheels of the RIEE, for the insertion of the Rack, using the centroids fo the Camera Images of the Location Targets in the Logistic Module. Presently, during the summer of '94, I completed the preliminary design of an easily portable measuring instrument using encoders to obtain the 3-Dimensional Coordinates of Location Targets in the Logistics Module relative to the RIEE mechanism frame. The algorithm developed in '93 can use the output of this instrument also. Simplification of the '93 work and suggestions for the future work are discussed.

Spiral lead platen robotic end effector

NASA Technical Reports Server (NTRS)

Beals, David C. (Inventor)

1990-01-01

A robotic end effector is disclosed which makes use of a rotating platen with spiral leads used to impact lateral motion to gripping fingers. Actuation is provided by the contact of rolling pins with the walls of the leads. The use of the disclosed method of actuation avoids jamming and provides excellent mechanical advantage while remaining light in weight and durable. The entire end effector is compact and easily adapted for attachment to robotic arms currently in use.

Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs.

PubMed

Ham, Jong Hyun; Majerczak, Doris R; Nomura, Kinya; Mecey, Christy; Uribe, Francisco; He, Sheng-Yang; Mackey, David; Coplin, David L

2009-06-01

The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well.

The Genome Biology of Effector Gene Evolution in Filamentous Plant Pathogens.

PubMed

Sánchez-Vallet, Andrea; Fouché, Simone; Fudal, Isabelle; Hartmann, Fanny E; Soyer, Jessica L; Tellier, Aurélien; Croll, Daniel

2018-05-16

Filamentous pathogens, including fungi and oomycetes, pose major threats to global food security. Crop pathogens cause damage by secreting effectors that manipulate the host to the pathogen's advantage. Genes encoding such effectors are among the most rapidly evolving genes in pathogen genomes. Here, we review how the major characteristics of the emergence, function, and regulation of effector genes are tightly linked to the genomic compartments where these genes are located in pathogen genomes. The presence of repetitive elements in these compartments is associated with elevated rates of point mutations and sequence rearrangements with a major impact on effector diversification. The expression of many effectors converges on an epigenetic control mediated by the presence of repetitive elements. Population genomics analyses showed that rapidly evolving pathogens show high rates of turnover at effector loci and display a mosaic in effector presence-absence polymorphism among strains. We conclude that effective pathogen containment strategies require a thorough understanding of the effector genome biology and the pathogen's potential for rapid adaptation. Expected final online publication date for the Annual Review of Phytopathology Volume 56 is August 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex.

PubMed

Hiruta, Chizue; Ogino, Yukiko; Sakuma, Tetsushi; Toyota, Kenji; Miyagawa, Shinichi; Yamamoto, Takashi; Iguchi, Taisen

2014-11-18

The cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have its complete genome sequenced, an unprecedented ca. 36% of which has no known homologs with any other species. Moreover, D. pulex is ideally suited for experimental manipulation because of its short reproductive cycle, large numbers of offspring, synchronization of oocyte maturation, and other life history characteristics. However, existing gene manipulation techniques are insufficient to accurately define gene functions. Although our previous investigations developed an RNA interference (RNAi) system in D. pulex, the possible time period of functional analysis was limited because the effectiveness of RNAi is transient. Thus, in this study, we developed a genome editing system for D. pulex by first microinjecting transcription activator-like effector nuclease (TALEN) mRNAs into early embryos and then evaluating TALEN activity and mutation phenotypes. We assembled a TALEN construct specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for distal limb development in invertebrates and vertebrates, and evaluated its activity in vitro by single-strand annealing assay. Then, we injected TALEN mRNAs into eggs within 1 hour post-ovulation. Injected embryos presented with defects in the second antenna and altered appendage development, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene. We succeeded, for the first time in D. pulex, in targeted mutagenesis by use of Platinum TALENs. This genome editing technique makes it possible to conduct reverse genetic analysis in D. pulex, making this species an even more appropriate model organism for environmental, evolutionary, and developmental genomics.

Functional heterogeneity of human effector CD8+ T cells.

PubMed

Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

2012-02-09

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.

PubMed

Sharma, Rahul; Xia, Xiaojuan; Cano, Liliana M; Evangelisti, Edouard; Kemen, Eric; Judelson, Howard; Oome, Stan; Sambles, Christine; van den Hoogen, D Johan; Kitner, Miloslav; Klein, Joël; Meijer, Harold J G; Spring, Otmar; Win, Joe; Zipper, Reinhard; Bode, Helge B; Govers, Francine; Kamoun, Sophien; Schornack, Sebastian; Studholme, David J; Van den Ackerveken, Guido; Thines, Marco

2015-10-05

Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.

The rise of the undead:Pseudokinases as mediators of effector-triggered immunity

USDA-ARS?s Scientific Manuscript database

Pathogens use effector proteins to suppress host immunity and promote infection. However, plants can recognize specific effectors and mount an effector-triggered immune response that suppresses pathogen growth. The YopJ/HopZ family of type III secreted effector proteins is broadly distributed in bac...

A transcription activator-like effector induction system mediated by proteolysis

PubMed Central

Copeland, Matthew F.; Politz, Mark C.; Johnson, Charles B.; Markley, Andrew L.; Pfleger, Brian F.

2016-01-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications due to their customizable DNA binding specificity. In this work we expand the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded following the induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator agnostic. PMID:26854666

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen

PubMed Central

Lu, Xunli; Kracher, Barbara; Saur, Isabel M. L.; Bauer, Saskia; Ellwood, Simon R.; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

2016-01-01

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1. Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation. PMID:27702901

Transposons to toxins: the provenance, architecture and diversification of a widespread class of eukaryotic effectors

PubMed Central

Zhang, Dapeng; Burroughs, A. Maxwell; Vidal, Newton D.; Iyer, Lakshminarayan M.; Aravind, L.

2016-01-01

Enzymatic effectors targeting nucleic acids, proteins and other cellular components are the mainstay of conflicts across life forms. Using comparative genomics we identify a large class of eukaryotic proteins, which include effectors from oomycetes, fungi and other parasites. The majority of these proteins have a characteristic domain architecture with one of several N-terminal ‘Header’ domains, which are predicted to play a role in trafficking of these effectors, including a novel version of the Ubiquitin fold. The Headers are followed by one or more diverse C-terminal domains, such as restriction endonuclease (REase), protein kinase, HNH endonuclease, LK-nuclease (a RNase) and multiple distinct peptidase domains, which are predicted to carry their toxicity determinants. The most common types of these proteins appear to have originated from prokaryotic transposases (e.g. TN7 and Mu) and combine a CDC6/ORC1-STAND clade NTPase domain with a C-terminal REase domain. Other than the so-called Crinkler effectors of oomycetes and fungi, these effectors are encoded by other eukaryotic parasites such as trypanosomatids (the RHS proteins) and the rhizarian Plasmodiophora, and symbionts like Capsaspora. Remarkably, we also find these proteins in free-living eukaryotes, including several viridiplantae, fungi, amoebozoans and animals. These versions might either still be transposons or function in other poorly understood eukaryote-specific inter-organismal and inter-genomic conflicts. These include the Medea1 selfish element of Tribolium that spreads via post-zygotic killing. We present a unified mechanism for the recombination-dependent diversification and action of this widespread class of molecular weaponry deployed across diverse conflicts ranging from parasitic to free-living forms. PMID:27060143

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

PubMed

Lu, Xunli; Kracher, Barbara; Saur, Isabel M L; Bauer, Saskia; Ellwood, Simon R; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

2016-10-18

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVR a gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVR a genes and identified AVR a1 and AVR a13 , encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVR a1 and AVR a13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVR A1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVR A1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVR A1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

Emerging concepts in effector biology of plant-associated organisms.

PubMed

Hogenhout, Saskia A; Van der Hoorn, Renier A L; Terauchi, Ryohei; Kamoun, Sophien

2009-02-01

Plant-associated organisms secrete proteins and other molecules to modulate plant defense circuitry and enable colonization of plant tissue. Understanding the molecular function of these secreted molecules, collectively known as effectors, became widely accepted as essential for a mechanistic understanding of the processes underlying plant colonization. This review summarizes recent findings in the field of effector biology and highlights the common concepts that have emerged from the study of cellular plant pathogen effectors.

Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

PubMed Central

Drube, Sebastian; Beyer, Mandy; Rothe, Mandy; Rabenhorst, Anja; Göpfert, Christiane; Meininger, Isabel; Diamanti, Michaela A.; Stegner, David; Häfner, Norman; Böttcher, Martin; Reinecke, Kirstin; Herdegen, Thomas; Greten, Florian R.; Nieswandt, Bernhard; Hartmann, Karin; Krämer, Oliver H.; Kamradt, Thomas

2015-01-01

Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca2+-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term “subthreshold IKK activation”. This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33. We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo. Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure. PMID:25749030

The hematopoietic regulator TAL1 is required for chromatin looping between the β-globin LCR and human γ-globin genes to activate transcription.

PubMed

Yun, Won Ju; Kim, Yea Woon; Kang, Yujin; Lee, Jungbae; Dean, Ann; Kim, AeRi

2014-04-01

TAL1 is a key hematopoietic transcription factor that binds to regulatory regions of a large cohort of erythroid genes as part of a complex with GATA-1, LMO2 and Ldb1. The complex mediates long-range interaction between the β-globin locus control region (LCR) and active globin genes, and although TAL1 is one of the two DNA-binding complex members, its role is unclear. To explore the role of TAL1 in transcription activation of the human γ-globin genes, we reduced the expression of TAL1 in erythroid K562 cells using lentiviral short hairpin RNA, compromising its association in the β-globin locus. In the TAL1 knockdown cells, the γ-globin transcription was reduced to 35% and chromatin looping of the (G)γ-globin gene with the LCR was disrupted with decreased occupancy of the complex member Ldb1 and LMO2 in the locus. However, GATA-1 binding, DNase I hypersensitive site formation and several histone modifications were largely maintained across the β-globin locus. In addition, overexpression of TAL1 increased the γ-globin transcription and increased interaction frequency between the (G)γ-globin gene and LCR. These results indicate that TAL1 plays a critical role in chromatin loop formation between the γ-globin genes and LCR, which is a critical step for the transcription of the γ-globin genes.

The hematopoietic regulator TAL1 is required for chromatin looping between the β-globin LCR and human γ-globin genes to activate transcription

PubMed Central

Yun, Won Ju; Kim, Yea Woon; Kang, Yujin; Lee, Jungbae; Dean, Ann; Kim, AeRi

2014-01-01

TAL1 is a key hematopoietic transcription factor that binds to regulatory regions of a large cohort of erythroid genes as part of a complex with GATA-1, LMO2 and Ldb1. The complex mediates long-range interaction between the β-globin locus control region (LCR) and active globin genes, and although TAL1 is one of the two DNA-binding complex members, its role is unclear. To explore the role of TAL1 in transcription activation of the human γ-globin genes, we reduced the expression of TAL1 in erythroid K562 cells using lentiviral short hairpin RNA, compromising its association in the β-globin locus. In the TAL1 knockdown cells, the γ-globin transcription was reduced to 35% and chromatin looping of the Gγ-globin gene with the LCR was disrupted with decreased occupancy of the complex member Ldb1 and LMO2 in the locus. However, GATA-1 binding, DNase I hypersensitive site formation and several histone modifications were largely maintained across the β-globin locus. In addition, overexpression of TAL1 increased the γ-globin transcription and increased interaction frequency between the Gγ-globin gene and LCR. These results indicate that TAL1 plays a critical role in chromatin loop formation between the γ-globin genes and LCR, which is a critical step for the transcription of the γ-globin genes. PMID:24470145

IL-15 induces CD4 effector memory T cell production and tissue emigration in nonhuman primates.

PubMed

Picker, Louis J; Reed-Inderbitzin, Edward F; Hagen, Shoko I; Edgar, John B; Hansen, Scott G; Legasse, Alfred; Planer, Shannon; Piatak, Michael; Lifson, Jeffrey D; Maino, Vernon C; Axthelm, Michael K; Villinger, Francois

2006-06-01

HIV infection selectively targets CD4+ effector memory T (T EM) cells, resulting in dramatic depletion of CD4+ T cells in mucosal effector sites in early infection. Regeneration of the T EM cell compartment is slow and incomplete, even when viral replication is controlled by antiretroviral therapy (ART). Here, we demonstrate that IL-15 dramatically increases in vivo proliferation of rhesus macaque (RM) CD4+ and CD8+ T EM cells with little effect on the naive or central memory T (T CM) cell subsets, a response pattern that is quite distinct from that of either IL-2 or IL-7. T EM cells produced in response to IL-15 did not accumulate in blood. Rather, 5-bromo-2'-deoxyuridine (BrdU) labeling studies suggest that many of these cells rapidly disperse to extralymphoid effector sites, where they manifest (slow) decay kinetics indistinguishable from that of untreated controls. In RMs with uncontrolled SIV infection and highly activated immune systems, IL-15 did not significantly increase CD4+ T EM cell proliferation, but with virologic control and concomitant reduction in immune activation by ART, IL-15 responsiveness was again observed. These data suggest that therapeutic use of IL-15 in the setting of ART might facilitate specific restoration of the CD4 + T cell compartment that is the primary target of HIV with less risk of exhausting precursor T cell compartments or generating potentially deleterious regulatory subsets.

IL-15 induces CD4+ effector memory T cell production and tissue emigration in nonhuman primates

PubMed Central

Picker, Louis J.; Reed-Inderbitzin, Edward F.; Hagen, Shoko I.; Edgar, John B.; Hansen, Scott G.; Legasse, Alfred; Planer, Shannon; Piatak, Michael; Lifson, Jeffrey D.; Maino, Vernon C.; Axthelm, Michael K.; Villinger, Francois

2006-01-01

HIV infection selectively targets CD4+ effector memory T (TEM) cells, resulting in dramatic depletion of CD4+ T cells in mucosal effector sites in early infection. Regeneration of the TEM cell compartment is slow and incomplete, even when viral replication is controlled by antiretroviral therapy (ART). Here, we demonstrate that IL-15 dramatically increases in vivo proliferation of rhesus macaque (RM) CD4+ and CD8+ TEM cells with little effect on the naive or central memory T (TCM) cell subsets, a response pattern that is quite distinct from that of either IL-2 or IL-7. TEM cells produced in response to IL-15 did not accumulate in blood. Rather, 5-bromo-2′-deoxyuridine (BrdU) labeling studies suggest that many of these cells rapidly disperse to extralymphoid effector sites, where they manifest (slow) decay kinetics indistinguishable from that of untreated controls. In RMs with uncontrolled SIV infection and highly activated immune systems, IL-15 did not significantly increase CD4+ TEM cell proliferation, but with virologic control and concomitant reduction in immune activation by ART, IL-15 responsiveness was again observed. These data suggest that therapeutic use of IL-15 in the setting of ART might facilitate specific restoration of the CD4+ T cell compartment that is the primary target of HIV with less risk of exhausting precursor T cell compartments or generating potentially deleterious regulatory subsets. PMID:16691294

A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery.

PubMed

Flaugnatti, Nicolas; Le, Thi Thu Hang; Canaan, Stéphane; Aschtgen, Marie-Stéphanie; Nguyen, Van Son; Blangy, Stéphanie; Kellenberger, Christine; Roussel, Alain; Cambillau, Christian; Cascales, Eric; Journet, Laure

2016-03-01

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins. © 2015 John Wiley & Sons Ltd.

Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity.

PubMed

Popov, Georgy; Fraiture, Malou; Brunner, Frederic; Sessa, Guido

2016-08-01

Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast-based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid-inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.

An assay for entry of secreted fungal effectors into plant cells.

PubMed

Lo Presti, Libera; Zechmann, Bernd; Kumlehn, Jochen; Liang, Liang; Lanver, Daniel; Tanaka, Shigeyuki; Bock, Ralph; Kahmann, Regine

2017-01-01

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors. We demonstrate translocation of a number of effectors in the U. maydis-maize system and show data that suggest that the uptake mechanism could be rather nonspecific The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Tool use and the distalization of the end-effector

PubMed Central

Bonaiuto, James B.; Jacobs, Stéphane; Frey, Scott H.

2009-01-01

We review recent neurophysiological data from macaques and humans suggesting that the use of tools extends the internal representation of the actor’s hand, and relate it to our modeling of the visual control of grasping. We introduce the idea that, in addition to extending the body schema to incorporate the tool, tool use involves distalization of the end-effector from hand to tool. Different tools extend the body schema in different ways, with a displaced visual target and a novel, task-specific processing of haptic feedback to the hand. This distalization is critical in order to exploit the unique functional capacities engendered by complex tools. PMID:19347356

Concurrent simulation of a parallel jaw end effector

NASA Technical Reports Server (NTRS)

Bynum, Bill

1985-01-01

A system of programs developed to aid in the design and development of the command/response protocol between a parallel jaw end effector and the strategic planner program controlling it are presented. The system executes concurrently with the LISP controlling program to generate a graphical image of the end effector that moves in approximately real time in response to commands sent from the controlling program. Concurrent execution of the simulation program is useful for revealing flaws in the communication command structure arising from the asynchronous nature of the message traffic between the end effector and the strategic planner. Software simulation helps to minimize the number of hardware changes necessary to the microprocessor driving the end effector because of changes in the communication protocol. The simulation of other actuator devices can be easily incorporated into the system of programs by using the underlying support that was developed for the concurrent execution of the simulation process and the communication between it and the controlling program.

3′-NADP and 3′-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1*♦

PubMed Central

Schuebel, Felix; Rocker, Andrea; Edelmann, Daniel; Schessner, Julia; Brieke, Clara; Meinhart, Anton

2016-01-01

An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3′-hydroxyl group. Both products of AvrRxo1, 3′-NADP and 3′-nicotinic acid adenine dinucleotide phosphate (3′-NAADP), are substantially different from the ubiquitous co-enzyme 2′-NADP and the calcium mobilizer 2′-NAADP. Interestingly, 3′-NADP and 3′-NAADP have previously been used as inhibitors or signaling molecules but were regarded as “artificial” compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3′-phosphorylated NAD derivatives. PMID:27621317

Effector-triggered defence against apoplastic fungal pathogens

PubMed Central

Stotz, Henrik U.; Mitrousia, Georgia K.; de Wit, Pierre J.G.M.; Fitt, Bruce D.L.

2014-01-01

R gene-mediated host resistance against apoplastic fungal pathogens is not adequately explained by the terms pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or effector-triggered immunity (ETI). Therefore, it is proposed that this type of resistance is termed ‘effector-triggered defence’ (ETD). Unlike PTI and ETI, ETD is mediated by R genes encoding cell surface-localised receptor-like proteins (RLPs) that engage the receptor-like kinase SOBIR1. In contrast to this extracellular recognition, ETI is initiated by intracellular detection of pathogen effectors. ETI is usually associated with fast, hypersensitive host cell death, whereas ETD often triggers host cell death only after an elapsed period of endophytic pathogen growth. In this opinion, we focus on ETD responses against foliar fungal pathogens of crops. PMID:24856287

Effector CD8 T cells dedifferentiate into long-lived memory cells.

PubMed

Youngblood, Ben; Hale, J Scott; Kissick, Haydn T; Ahn, Eunseon; Xu, Xiaojin; Wieland, Andreas; Araki, Koichi; West, Erin E; Ghoneim, Hazem E; Fan, Yiping; Dogra, Pranay; Davis, Carl W; Konieczny, Bogumila T; Antia, Rustom; Cheng, Xiaodong; Ahmed, Rafi

2017-12-21

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long

Nanorobotic end-effectors: Design, fabrication, and in situ characterization

NASA Astrophysics Data System (ADS)

Fan, Zheng

Nano-robotic end-effectors have promising applications for nano-fabrication, nano-manufacturing, nano-optics, nano-medical, and nano-sensing; however, low performances of the conventional end-effectors have prevented the widespread utilization of them in various fields. There are two major difficulties in developing the end-effectors: their nano-fabrication and their advanced characterization in the nanoscale. Here we introduce six types of end-effectors: the nanotube fountain pen (NFP), the super-fine nanoprobe, the metal-filled carbon nanotube (m CNT)-based sphere-on-pillar (SOP) nanoantennas, the tunneling nanosensor, and the nanowire-based memristor. The investigations on the NFP are focused on nano-fluidics and nano-fabrications. The NFP could direct write metallic "inks" and fabricating complex metal nanostructures from 0D to 3D with a position servo control, which is critically important to future large-scale, high-throughput nanodevice production. With the help of NFP, we could fabricate the end-effectors such as super-fine nanoprobe and m CNT-based SOP nanoantennas. Those end-effectors are able to detect local flaws or characterize the electrical/mechanical properties of the nanostructure. Moreover, using electron-energy-loss-spectroscopy (EELS) technique during the operation of the SOP optical antenna opens a new basis for the application of nano-robotic end-effectors. The technique allows advanced characterization of the physical changes, such as carrier diffusion, that are directly responsible for the device's properties. As the device was coupled with characterization techniques of scanning-trasmission-electron-microscopy (STEM), the development of tunneling nanosensor advances this field of science into quantum world. Furthermore, the combined STEM-EELS technique plays an important role in our understanding of the memristive switching performance in the nanowire-based memristor. The developments of those nano-robotic end-effectors expend the study

p53 as an Effector or Inhibitor of Therapy Response

PubMed Central

Ablain, Julien; Poirot, Brigitte; Esnault, Cécile; Lehmann-Che, Jacqueline; de Thé, Hugues

2016-01-01

Although integrity of the p53 signaling pathway in a given tumor was expected to be a critical determinant of response to therapies, most clinical studies failed to link p53 status and treatment outcome. Here, we present two opposite situations: one in which p53 is an essential effector of cure by targeted leukemia therapies and another one in advanced breast cancers in which p53 inactivation is required for the clinical efficacy of dose-dense chemotherapy. If p53 promotes or blocks therapy response, therapies must be tailored on its status in individual tumors. PMID:26637438

Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

PubMed Central

Selin, Carrie; de Kievit, Teresa R.; Belmonte, Mark F.; Fernando, W. G. Dilantha

2016-01-01

Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function. PMID:27199930

Long-distance endosome trafficking drives fungal effector production during plant infection

PubMed Central

Bielska, Ewa; Higuchi, Yujiro; Schuster, Martin; Steinberg, Natascha; Kilaru, Sreedhar; Talbot, Nicholas J.; Steinberg, Gero

2014-01-01

To cause plant disease, pathogenic fungi can secrete effector proteins into plant cells to suppress plant immunity and facilitate fungal infection. Most fungal pathogens infect plants using very long strand-like cells, called hyphae, that secrete effectors from their tips into host tissue. How fungi undergo long-distance cell signalling to regulate effector production during infection is not known. Here we show that long-distance retrograde motility of early endosomes (EEs) is necessary to trigger transcription of effector-encoding genes during plant infection by the pathogenic fungus Ustilago maydis. We demonstrate that motor-dependent retrograde EE motility is necessary for regulation of effector production and secretion during host cell invasion. We further show that retrograde signalling involves the mitogen-activated kinase Crk1 that travels on EEs and participates in control of effector production. Fungal pathogens therefore undergo long-range signalling to orchestrate host invasion. PMID:25283249

Long-distance endosome trafficking drives fungal effector production during plant infection.

PubMed

Bielska, Ewa; Higuchi, Yujiro; Schuster, Martin; Steinberg, Natascha; Kilaru, Sreedhar; Talbot, Nicholas J; Steinberg, Gero

2014-10-06

To cause plant disease, pathogenic fungi can secrete effector proteins into plant cells to suppress plant immunity and facilitate fungal infection. Most fungal pathogens infect plants using very long strand-like cells, called hyphae, that secrete effectors from their tips into host tissue. How fungi undergo long-distance cell signalling to regulate effector production during infection is not known. Here we show that long-distance retrograde motility of early endosomes (EEs) is necessary to trigger transcription of effector-encoding genes during plant infection by the pathogenic fungus Ustilago maydis. We demonstrate that motor-dependent retrograde EE motility is necessary for regulation of effector production and secretion during host cell invasion. We further show that retrograde signalling involves the mitogen-activated kinase Crk1 that travels on EEs and participates in control of effector production. Fungal pathogens therefore undergo long-range signalling to orchestrate host invasion.

Investigation of a bio-inspired lift-enhancing effector on a 2D airfoil.

PubMed

Johnston, Joe; Gopalarathnam, Ashok

2012-09-01

A flap mounted on the upper surface of an airfoil, called a 'lift-enhancing effector', has been shown in wind tunnel tests to have a similar function to a bird's covert feathers, which rise off the wing's surface in response to separated flows. The effector, fabricated from a thin Mylar sheet, is allowed to rotate freely about its leading edge. The tests were performed in the NCSU subsonic wind tunnel at a chord Reynolds number of 4 × 10(5). The maximum lift coefficient with the effector was the same as that for the clean airfoil, but was maintained over an angle-of-attack range from 12° to almost 20°, resulting in a very gentle stall behavior. To better understand the aerodynamics and to estimate the deployment angle of the free-moving effector, fixed-angle effectors fabricated out of stiff wood were also tested. A progressive increase in the stall angle of attack with increasing effector angle was observed, with diminishing returns beyond the effector angle of 60°. Drag tests on both the free-moving and fixed effectors showed a marked improvement in drag at high angles of attack. Oil flow visualization on the airfoil with and without the fixed-angle effectors proved that the effector causes the separation point to move aft on the airfoil, as compared to the clean airfoil. This is thought to be the main mechanism by which an effector improves both lift and drag. A comparison of the fixed-effector results with those from the free-effector tests shows that the free effector's deployment angle is between 30° and 45°. When operating at and beyond the clean airfoil's stall angle, the free effector automatically deploys to progressively higher angles with increasing angles of attack. This slows down the rapid upstream movement of the separation point and avoids the severe reduction in the lift coefficient and an increase in the drag coefficient that are seen on the clean airfoil at the onset of stall. Thus, the effector postpones the stall by 4-8° and makes the

Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila.

PubMed

Fontana, Mary F; Banga, Simran; Barry, Kevin C; Shen, Xihui; Tan, Yunhao; Luo, Zhao-Qing; Vance, Russell E

2011-02-01

The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also

Potential for reducing the numbers of SiPM readout surfaces of laser-processed X'tal cube PET detectors.

PubMed

Hirano, Yoshiyuki; Inadama, Naoko; Yoshida, Eiji; Nishikido, Fumihiko; Murayama, Hideo; Watanabe, Mitsuo; Yamaya, Taiga

2013-03-07

We are developing a three-dimensional (3D) position-sensitive detector with isotropic spatial resolution, the X'tal cube. Originally, our design consisted of a crystal block for which all six surfaces were covered with arrays of multi-pixel photon counters (MPPCs). In this paper, we examined the feasibility of reducing the number of surfaces on which a MPPC array must be connected with the aim of reducing the complexity of the system. We evaluated two kinds of laser-processed X'tal cubes of 3 mm and 2 mm pitch segments while varying the numbers of the 4 × 4 MPPC arrays down to two surfaces. The sub-surface laser engraving technique was used to fabricate 3D grids into a monolithic crystal block. The 3D flood histograms were obtained by the Anger-type calculation. Two figures of merit, peak-to-valley ratios and distance-to-width ratios, were used to evaluate crystal identification performance. Clear separation was obtained even in the 2-surface configuration for the 3 mm X'tal cube, and the average peak-to-valley ratios and the distance-to-width ratios were 6.7 and 2.6, respectively. Meanwhile, in the 2 mm X'tal cube, the 6-surface configuration could separate all crystals and even the 2-surface case could also, but the flood histograms were relatively shrunk in the 2-surface case, especially on planes parallel to the sensitive surfaces. However, the minimum peak-to-valley ratio did not fall below 3.9. We concluded that reducing the numbers of MPPC readout surfaces was feasible for both the 3 mm and the 2 mm X'tal cubes.

Design and force analysis of end-effector for plug seedling transplanter.

PubMed

Jiang, Zhuohua; Hu, Yang; Jiang, Huanyu; Tong, Junhua

2017-01-01

Automatic transplanters have been very important in greenhouses since the popularization of seedling nurseries. End-effector development is a key technology for transplanting plug seedlings. Most existing end-effectors have problems with holding root plugs or releasing plugs. An efficient end-effector driven by a linear pneumatic cylinder was designed in this study, which could hold root plugs firmly and release plugs easily. This end-effector with four needles could clamp the plug simultaneously while the needles penetrate into the substrate. The depth and verticality of the needles could be adjusted conveniently for different seedling trays. The effectiveness of this end-effector was tested by a combinational trial examining three seedling nursery factors (the moisture content of the substrate, substrate bulk density and the volume proportion of substrate ingredients). Results showed that the total transplanting success rate for the end-effector was 100%, and the root plug harm rate was below 17%. A force measure system with tension and pressure transducers was installed on the designed end-effector. The adhesive force FL between the root plug and the cell of seedling trays and the extrusion force FK on the root plug were measured and analyzed. The results showed that all three variable factors and their interactions had significant effects on the extrusion force. Each factor had a significant effect on adhesive force. Additionally, it was found that the end-effector did not perform very well when the value of FK/FL was beyond the range of 5.99~8.67. This could provide a scientific basis for end-effector application in transplanting.

Apparatus for adapting an end effector device remotely controlled manipulator arm

NASA Technical Reports Server (NTRS)

Clark, K. H. (Inventor)

1985-01-01

Apparatus for adapting a general purpose and effector device to a special purpose and effector is disclosed which includes an adapter bracket assembly which provides a mechanical and electrical interface between the end effector devices. The adapter bracket assembly includes an adapter connector post which interlocks with a diamond shaped gripping channel formed in closed jaws of the general purpose end effector. The angularly intersecting surfaces of the connector post and gripping channel prevent any relative movement there between. Containment webs constrain the outer finger plates of the general purpose jaws to prevent pitch motion. Electrical interface is provided by conical, self aligning electrical connector components carried by respective ones of said end effectors.

Immune Effector Recovery in Chronic Myeloid Leukemia and Treatment-Free Remission

PubMed Central

Hughes, Amy; Yong, Agnes S. M.

2017-01-01

Chronic myeloid leukemia (CML) is a hematological cancer, characterized by a reciprocal chromosomal translocation between chromosomes 9 and 22 [t(9;22)], producing the Bcr-Abl oncogene. Tyrosine kinase inhibitors (TKIs) represent the standard of care for CML patients and exert a dual mode of action: direct oncokinase inhibition and restoration of effector-mediated immune surveillance, which is rendered dysfunctional in CML patients at diagnosis, prior to TKI therapy. TKIs such as imatinib, and more potent second-generation nilotinib and dasatinib induce a high rate of deep molecular response (DMR, BCR-ABL1 ≤ 0.01%) in CML patients. As a result, the more recent goal of therapy in CML treatment is to induce a durable DMR as a prelude to successful treatment-free remission (TFR), which occurs in approximately half of all CML patients who cease TKI therapy. The lack of overt relapse in such patients has been attributed to immunological control of CML. In this review, we discuss an immunological timeline to successful TFR, focusing on the immunology of CML during TKI treatment; an initial period of immune suppression, limiting antitumor immune effector responses in newly diagnosed CML patients, linked to an expansion of immature myeloid-derived suppressor cells and regulatory T cells and aberrant expression of immune checkpoint signaling pathways, including programmed death-1/programmed death ligand-1. Commencement of TKI treatment is associated with immune system re-activation and restoration of effector-mediated [natural killer (NK) cell and T cell] immune surveillance in CML patients, albeit with differing frequencies in concert with differing levels of molecular response achieved on TKI. DMR is associated with maximal restoration of immune recovery in CML patients on TKI. Current data suggest a net balance between both the effector and suppressor arms of the immune system, at a minimum involving mature, cytotoxic CD56dim NK cells may be important in mediating

Generation mechanism of RANKL(+) effector memory B cells: relevance to the pathogenesis of rheumatoid arthritis.

PubMed

Ota, Yuri; Niiro, Hiroaki; Ota, Shun-Ichiro; Ueki, Naoko; Tsuzuki, Hirofumi; Nakayama, Tsuyoshi; Mishima, Koji; Higashioka, Kazuhiko; Jabbarzadeh-Tabrizi, Siamak; Mitoma, Hiroki; Akahoshi, Mitsuteru; Arinobu, Yojiro; Kukita, Akiko; Yamada, Hisakata; Tsukamoto, Hiroshi; Akashi, Koichi

2016-03-16

The efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T-B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL(+) effector B cells and their impacts on osteoclast differentiation. Peripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells. RANKL expression was accentuated in CD80(+)CD86(+) B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21. Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3(+)RANKL(+) effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL(+) effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro. Our current findings have shed light on the generation mechanism of pathogenic RANKL(+) effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future.

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering

PubMed Central

Blaževitš, Olga; Mideksa, Yonatan G.; Šolman, Maja; Ligabue, Alessio; Ariotti, Nicholas; Nakhaeizadeh, Hossein; Fansa, Eyad K.; Papageorgiou, Anastassios C.; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Abankwa, Daniel

2016-01-01

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling. PMID:27087647

Design and force analysis of end-effector for plug seedling transplanter

PubMed Central

Hu, Yang; Jiang, Huanyu; Tong, Junhua

2017-01-01

Automatic transplanters have been very important in greenhouses since the popularization of seedling nurseries. End-effector development is a key technology for transplanting plug seedlings. Most existing end-effectors have problems with holding root plugs or releasing plugs. An efficient end-effector driven by a linear pneumatic cylinder was designed in this study, which could hold root plugs firmly and release plugs easily. This end-effector with four needles could clamp the plug simultaneously while the needles penetrate into the substrate. The depth and verticality of the needles could be adjusted conveniently for different seedling trays. The effectiveness of this end-effector was tested by a combinational trial examining three seedling nursery factors (the moisture content of the substrate, substrate bulk density and the volume proportion of substrate ingredients). Results showed that the total transplanting success rate for the end-effector was 100%, and the root plug harm rate was below 17%. A force measure system with tension and pressure transducers was installed on the designed end-effector. The adhesive force FL between the root plug and the cell of seedling trays and the extrusion force FK on the root plug were measured and analyzed. The results showed that all three variable factors and their interactions had significant effects on the extrusion force. Each factor had a significant effect on adhesive force. Additionally, it was found that the end-effector did not perform very well when the value of FK/FL was beyond the range of 5.99~8.67. This could provide a scientific basis for end-effector application in transplanting. PMID:28678858

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

PubMed

Tyler, Brett M; Kale, Shiv D; Wang, Qunqing; Tao, Kai; Clark, Helen R; Drews, Kelly; Antignani, Vincenzo; Rumore, Amanda; Hayes, Tristan; Plett, Jonathan M; Fudal, Isabelle; Gu, Biao; Chen, Qinghe; Affeldt, Katharyn J; Berthier, Erwin; Fischer, Gregory J; Dou, Daolong; Shan, Weixing; Keller, Nancy P; Martin, Francis; Rouxel, Thierry; Lawrence, Christopher B

2013-06-01

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

Functional differences between PD-1+ and PD-1- CD4+ effector T cells in healthy donors and patients with glioblastoma multiforme

PubMed Central

Lucca, Liliana E.; Lerner, Benjamin A.; Gunel, Murat; Raddassi, Khadir; Coric, Vlad; Hafler, David A.; Love, J. Christopher

2017-01-01

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) have been highly successful in the treatment of cancer. While PD-1 expression has been widely investigated, its role in CD4+ effector T cells in the setting of health and cancer remains unclear, particularly in the setting of glioblastoma multiforme (GBM), the most aggressive and common form of brain cancer. We examined the functional and molecular features of PD-1+CD4+CD25—CD127+Foxp3—effector cells in healthy subjects and in patients with GBM. In healthy subjects, we found that PD-1+CD4+ effector cells are dysfunctional: they do not proliferate but can secrete large quantities of IFNγ. Strikingly, blocking antibodies against PD-1 did not rescue proliferation. RNA-sequencing revealed features of exhaustion in PD-1+ CD4 effectors. In the context of GBM, tumors were enriched in PD-1+ CD4+ effectors that were similarly dysfunctional and unable to proliferate. Furthermore, we found enrichment of PD-1+TIM-3+ CD4+ effectors in tumors, suggesting that co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. RNA-sequencing of blood and tumors from GBM patients revealed distinct differences between CD4+ effectors from both compartments with enrichment in multiple gene sets from tumor infiltrating PD-1—CD4+ effectors cells. Enrichment of these gene sets in tumor suggests a more metabolically active cell state with signaling through other co-receptors. PD-1 expression on CD4 cells identifies a dysfunctional subset refractory to rescue with PD-1 blocking antibodies, suggesting that the influence of immune checkpoint inhibitors may involve recovery of function in the PD-1—CD4+ T cell compartment. Additionally, co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. PMID:28880903

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

PubMed

Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

2016-04-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

Intrinsic spatial resolution evaluation of the X'tal cube PET detector based on a 3D crystal block segmented by laser processing.

PubMed

Yoshida, Eiji; Tashima, Hideaki; Inadama, Naoko; Nishikido, Fumihiko; Moriya, Takahiro; Omura, Tomohide; Watanabe, Mitsuo; Murayama, Hideo; Yamaya, Taiga

2013-01-01

The X'tal cube is a depth-of-interaction (DOI)-PET detector which is aimed at obtaining isotropic resolution by effective readout of scintillation photons from the six sides of a crystal block. The X'tal cube is composed of the 3D crystal block with isotropic resolution and arrays of multi-pixel photon counters (MPPCs). In this study, to fabricate the 3D crystal block efficiently and precisely, we applied a sub-surface laser engraving (SSLE) technique to a monolithic crystal block instead of gluing segmented small crystals. The SSLE technique provided micro-crack walls which carve a groove into a monolithic scintillator block. Using the fabricated X'tal cube, we evaluated its intrinsic spatial resolution to show a proof of concept of isotropic resolution. The 3D grids of 2 mm pitch were fabricated into an 18 × 18 × 18 mm(3) monolithic lutetium yttrium orthosilicate (LYSO) crystal by the SSLE technique. 4 × 4 MPPCs were optically coupled to each surface of the crystal block. The X'tal cube was uniformly irradiated by (22)Na gamma rays, and all of the 3D grids on the 3D position histogram were separated clearly by an Anger-type calculation from the 96-channel MPPC signals. Response functions of the X'tal cube were measured by scanning with a (22)Na point source. The gamma-ray beam with a 1.0 mm slit was scanned in 0.25 mm steps by positioning of the X'tal cube at vertical and 45° incident angles. The average FWHM resolution at both incident angles was 2.1 mm. Therefore, we confirmed the isotropic spatial resolution performance of the X'tal cube.

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

PubMed

Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

2017-01-01

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

PubMed

Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

2015-01-01

The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery. Copyright © 2015 Elsevier GmbH. All rights reserved.

From Tumor Immunosuppression to Eradication: Targeting Homing and Activity of Immune Effector Cells to Tumors

PubMed Central

Draghiciu, Oana; Nijman, Hans W.; Daemen, Toos

2011-01-01

Unraveling the mechanisms used by the immune system to fight cancer development is one of the most ambitious undertakings in immunology. Detailed knowledge regarding the mechanisms of induction of tolerance and immunosuppression within the tumor microenvironment will contribute to the development of highly effective tumor eradication strategies. Research within the last few decades has shed more light on the matter. This paper aims to give an overview on the current knowledge of the main tolerance and immunosuppression mechanisms elicited within the tumor microenvironment, with the focus on development of effective immunotherapeutic strategies to improve homing and activity of immune effector cells to tumors. PMID:22190971

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

PubMed Central

Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R.; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A.; Barry, Kerrie W.; Spatafora, Joseph; Grigoriev, Igor V.; Martin, Francis M.; Pujade-Renaud, Valérie

2018-01-01

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

PubMed

Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A; Barry, Kerrie W; Spatafora, Joseph; Grigoriev, Igor V; Martin, Francis M; Pujade-Renaud, Valérie

2018-01-01

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species ( Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca , and Botrosphaeria dothidea ) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

Space-based multifunctional end effector systems functional requirements and proposed designs

NASA Technical Reports Server (NTRS)

Mishkin, A. H.; Jau, B. M.

1988-01-01

The end effector is an essential element of teleoperator and telerobot systems to be employed in space in the next decade. The report defines functional requirements for end effector systems to perform operations that are currently only feasible through Extra-Vehicular Activity (EVA). Specific tasks and functions that the end effectors must be capable of performing are delineated. Required capabilities for forces and torques, clearances, compliance, and sensing are described, using current EVA requirements as guidelines where feasible. The implications of these functional requirements on the elements of potential end effector systems are discussed. The systems issues that must be considered in the design of space-based manipulator systems are identified; including impacts on subsystems tightly coupled to the end effector, i.e., control station, information processing, manipulator arm, tool and equipment stowage. Possible end effector designs are divided into three categories: single degree-of-freedom end effectors, multiple degree of freedom end effectors, and anthropomorphic hands. Specific design alternatives are suggested and analyzed within the individual categories. Two evaluations are performed: the first considers how well the individual end effectors could substitute for EVA; the second compares how manipulator systems composed of the top performers from the first evaluation would improve the space shuttle Remote Manipulator System (RMS) capabilities. The analysis concludes that the anthropomorphic hand is best-suited for EVA tasks. A left- and right-handed anthropomorphic manipulator arm configuration is suggested as appropriate to be affixed to the RMS, but could also be used as part of the Smart Front End for the Orbital Maneuvering Vehicle (OMV). The technical feasibility of the anthropomorphic hand and its control are demonstrated. An evolutionary development approach is proposed and approximate scheduling provided for implementing the suggested

Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

PubMed Central

2014-01-01

Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. PMID:24997498

MIX and match: mobile T6SS MIX-effectors enhance bacterial fitness

PubMed Central

Salomon, Dor

2016-01-01

ABSTRACT Protein secretion systems that mediate interbacterial competition secret a wide repertoire of antibacterial toxins. A major player in these competitions is the newly discovered bacterial type VI secretion system (T6SS). We recently found that a subset of polymorphic MIX-effectors, which are a widespread class of effectors secreted by T6SSs, are horizontally shared between marine bacteria and are used to diversify their T6SS effector repertoires, thus enhancing their environmental fitness. In this commentary, I expand on the ideas that were introduced in the previous report, and further speculate on the possible mobility of other MIX-effectors. In addition, I discuss the possible role of horizontal gene transfer in the dissemination of MIX-effectors through bacterial genomes, as well as its possible role in diversifying the T6SS effector repertoire. PMID:27066305

p53 as an Effector or Inhibitor of Therapy Response.

PubMed

Ablain, Julien; Poirot, Brigitte; Esnault, Cécile; Lehmann-Che, Jacqueline; de Thé, Hugues

2015-12-04

Although integrity of the p53 signaling pathway in a given tumor was expected to be a critical determinant of response to therapies, most clinical studies failed to link p53 status and treatment outcome. Here, we present two opposite situations: one in which p53 is an essential effector of cure by targeted leukemia therapies and another one in advanced breast cancers in which p53 inactivation is required for the clinical efficacy of dose-dense chemotherapy. If p53 promotes or blocks therapy response, therapies must be tailored on its status in individual tumors. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

Effector profiles distinguish formae speciales of Fusarium oxysporum.

PubMed

van Dam, Peter; Fokkens, Like; Schmidt, Sarah M; Linmans, Jasper H J; Kistler, H Corby; Ma, Li-Jun; Rep, Martijn

2016-11-01

Formae speciales (ff.spp.) of the fungus Fusarium oxysporum are often polyphyletic within the species complex, making it impossible to identify them on the basis of conserved genes. However, sequences that determine host-specific pathogenicity may be expected to be similar between strains within the same forma specialis. Whole genome sequencing was performed on strains from five different ff.spp. (cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici). In each genome, genes for putative effectors were identified based on small size, secretion signal, and vicinity to a "miniature impala" transposable element. The candidate effector genes of all genomes were collected and the presence/absence patterns in each individual genome were clustered. Members of the same forma specialis turned out to group together, with cucurbit-infecting strains forming a supercluster separate from other ff.spp. Moreover, strains from different clonal lineages within the same forma specialis harbour identical effector gene sequences, supporting horizontal transfer of genetic material. These data offer new insight into the genetic basis of host specificity in the F. oxysporum species complex and show that (putative) effectors can be used to predict host specificity in F. oxysporum. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Effector-triggered immunity: from pathogen perception to robust defense.

PubMed

Cui, Haitao; Tsuda, Kenichi; Parker, Jane E

2015-01-01

In plant innate immunity, individual cells have the capacity to sense and respond to pathogen attack. Intracellular recognition mechanisms have evolved to intercept perturbations by pathogen virulence factors (effectors) early in host infection and convert it to rapid defense. One key to resistance success is a polymorphic family of intracellular nucleotide-binding/leucine-rich-repeat (NLR) receptors that detect effector interference in different parts of the cell. Effector-activated NLRs connect, in various ways, to a conserved basal resistance network in order to transcriptionally boost defense programs. Effector-triggered immunity displays remarkable robustness against pathogen disturbance, in part by employing compensatory mechanisms within the defense network. Also, the mobility of some NLRs and coordination of resistance pathways across cell compartments provides flexibility to fine-tune immune outputs. Furthermore, a number of NLRs function close to the nuclear chromatin by balancing actions of defense-repressing and defense-activating transcription factors to program cells dynamically for effective disease resistance.

A Virulence Essential CRN Effector of Phytophthora capsici Suppresses Host Defense and Induces Cell Death in Plant Nucleus.

PubMed

Mafurah, Joseph Juma; Ma, Huifei; Zhang, Meixiang; Xu, Jing; He, Feng; Ye, Tingyue; Shen, Danyu; Chen, Yanyu; Rajput, Nasir Ahmed; Dou, Daolong

2015-01-01

Phytophthora capsici is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in P. capsici. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of PcCRN4 gene induced cell death in N. benthamiana, N. tabacum and Solanum lycopersicum. Overexpression of the gene in N. benthamiana enhanced susceptibility to P. capsici. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing PcCRN4 gene in P. capsici significantly reduced pathogen virulence. The expression of the pathogenesis-related gene PR1b in N. benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P. capsici transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with PcCRN4-silenced transformant, indicating that silencing of PcCRN4 in P. capsici reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when PcCRN4-silenced line were inoculated on Arabidopsis thaliana, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses.

[Transcription activator-like effectors(TALEs)based genome engineering].

PubMed

Zhao, Mei-Wei; Duan, Cheng-Li; Liu, Jiang

2013-10-01

Systematic reverse-engineering of functional genome architecture requires precise modifications of gene sequences and transcription levels. The development and application of transcription activator-like effectors(TALEs) has created a wealth of genome engineering possibilities. TALEs are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas species. The DNA-binding domain of each TALE typically consists of tandem 34-amino acid repeat modules rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Such "genome engineering" has now been established in human cells and a number of model organisms, thus opening the door to better understanding gene function in model organisms, improving traits in crop plants and treating human genetic disorders.

The Research Team Approach to Learning (ReTAL): A Structure for Open-Endedness.

ERIC Educational Resources Information Center

Del Giorno, Bette J.

The ReTAL technique is described by using role sheets for the teacher and the student members of the research team; the "Researcher" defines the problem and searches the literature, the "Technician" plans and executes the experiment, and the "Recorder-Discussion Leader" coordinates records, interprets, evaluates, and reports the study. Each team…

A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize

PubMed Central

Tanaka, Shigeyuki; Brefort, Thomas; Neidig, Nina; Djamei, Armin; Kahnt, Jörg; Vermerris, Wilfred; Koenig, Stefanie; Feussner, Kirstin; Feussner, Ivo; Kahmann, Regine

2014-01-01

The biotrophic fungus Ustilago maydis causes smut disease in maize with characteristic tumor formation and anthocyanin induction. Here, we show that anthocyanin biosynthesis is induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with maize protein kinase ZmTTK1. Tin2 masks a ubiquitin–proteasome degradation motif in ZmTTK1, thus stabilizing the active kinase. Active ZmTTK1 controls activation of genes in the anthocyanin biosynthesis pathway. Without Tin2, enhanced lignin biosynthesis is observed in infected tissue and vascular bundles show strong lignification. This is presumably limiting access of fungal hyphae to nutrients needed for massive proliferation. Consistent with this assertion, we observe that maize brown midrib mutants affected in lignin biosynthesis are hypersensitive to U. maydis infection. We speculate that Tin2 rewires metabolites into the anthocyanin pathway to lower their availability for other defense responses. DOI: http://dx.doi.org/10.7554/eLife.01355.001 PMID:24473076

Genome editing comes of age.

PubMed

Kim, Jin-Soo

2016-09-01

Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster of regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) RNA-guided endonucleases (RGENs). I specifically highlight the key advances that set the foundation for the rapid and widespread implementation of CRISPR-Cas9 genome editing approaches that has revolutionized the field.

Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

USDA-ARS?s Scientific Manuscript database

Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

The effector candidate repertoire of the arbuscular mycorrhizal fungus Rhizophagus clarus.

PubMed

Sędzielewska Toro, Kinga; Brachmann, Andreas

2016-02-09

Arbuscular mycorrhizal fungi (AMF) form an ecologically important symbiosis with more than two thirds of studied land plants. Recent studies of plant-pathogen interactions showed that effector proteins play a key role in host colonization by controlling the plant immune system. We hypothesise that also for symbiotic-plant interactions the secreted effectome of the fungus is a major component of communication and the conservation level of effector proteins between AMF species may be indicative whether they play a fundamental role. In this study, we used a bioinformatics pipeline to predict and compare the effector candidate repertoire of the two AMF species, Rhizophagus irregularis and Rhizophagus clarus. Our in silico pipeline revealed a list of 220 R. irregularis candidate effector genes that create a valuable information source to elucidate the mechanism of plant infection and colonization by fungi during AMF symbiotic interaction. While most of the candidate effectors show no homologies to known domains or proteins, the candidates with homologies point to potential roles in signal transduction, cell wall modification or transcription regulation. A remarkable aspect of our work is presence of a large portion of the effector proteins involved in symbiosis, which are not unique to each fungi or plant species, but shared along the Glomeromycota phylum. For 95% of R. irregularis candidates we found homologs in a R. clarus genome draft generated by Illumina high-throughput sequencing. Interestingly, 9% of the predicted effectors are at least as conserved between the two Rhizophagus species as proteins with housekeeping functions (similarity > 90%). Therefore, we state that this group of highly conserved effector proteins between AMF species may play a fundamental role during fungus-plant interaction. We hypothesise that in symbiotic interactions the secreted effectome of the fungus might be an important component of communication. Identification and functional

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

PubMed

Ve, Thomas; Williams, Simon J; Catanzariti, Ann-Maree; Rafiqi, Maryam; Rahman, Motiur; Ellis, Jeffrey G; Hardham, Adrienne R; Jones, David A; Anderson, Peter A; Dodds, Peter N; Kobe, Bostjan

2013-10-22

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells

PubMed Central

Wells, Alexandria C; Daniels, Keith A; Angelou, Constance C; Fagerberg, Eric; Burnside, Amy S; Markstein, Michele; Alfandari, Dominique; Welsh, Raymond M; Pobezinskaya, Elena L; Pobezinsky, Leonid A

2017-01-01

The differentiation of naive CD8 T cells into effector cytotoxic T lymphocytes upon antigen stimulation is necessary for successful antiviral, and antitumor immune responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs in the regulation of CD8 T cell responses, where maintenance of the naive phenotype in CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T lymphocytes depends upon T cell receptor-mediated let-7 downregulation. Decrease of let-7 expression in activated T cells enhances clonal expansion and the acquisition of effector function through derepression of the let-7 targets, including Myc and Eomesodermin. Ultimately, we have identified a novel let-7-mediated mechanism, which acts as a molecular brake controlling the magnitude of CD8 T cell responses. DOI: http://dx.doi.org/10.7554/eLife.26398.001 PMID:28737488

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita

PubMed Central

Rutter, William B.; Hewezi, Tarek; Abubucker, Sahar; Maier, Tom R.; Huang, Guozhong; Mitreva, Makedonka; Hussey, Richard S.; Baum, Thomas J.

2014-01-01

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins, which M. incognita secretes into its host plants during infection, is an important step towards finding new ways to manage this pest. In this study we have identified the cDNAs for 18 putative effectors, i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants. These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically up-regulated during different stages of the nematode’s life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of Meloidogyne hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed, and reproduce on their host plants. Future studies investigating the roles these proteins play in planta will help mitigate the effects of this damaging pest. PMID:24875667

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita.

PubMed

Rutter, William B; Hewezi, Tarek; Abubucker, Sahar; Maier, Tom R; Huang, Guozhong; Mitreva, Makedonka; Hussey, Richard S; Baum, Thomas J

2014-09-01

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins which M. incognita secretes into its host plants during infection is an important step toward finding new ways to manage this pest. In this study, we have identified the cDNAs for 18 putative effectors (i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants). These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that, in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated during different stages of the nematode's life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of M. hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors, we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest.

Co-expression of TAL1 and ADH1 in recombinant xylose-fermenting Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural.

PubMed

Hasunuma, Tomohisa; Ismail, Ku Syahidah Ku; Nambu, Yumiko; Kondo, Akihiko

2014-02-01

Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL-ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Structural Evolution of Differential Amino Acid Effector Regulation in Plant Chorismate Mutases*

PubMed Central

Westfall, Corey S.; Xu, Ang; Jez, Joseph M.

2014-01-01

Chorismate mutase converts chorismate into prephenate for aromatic amino acid biosynthesis. To understand the molecular basis of allosteric regulation in the plant chorismate mutases, we analyzed the three Arabidopsis thaliana chorismate mutase isoforms (AtCM1–3) and determined the x-ray crystal structures of AtCM1 in complex with phenylalanine and tyrosine. Functional analyses show a wider range of effector control in the Arabidopsis chorismate mutases than previously reported. AtCM1 is activated by tryptophan with phenylalanine and tyrosine acting as negative effectors; however, tryptophan, cysteine, and histidine activate AtCM3. AtCM2 is a nonallosteric form. The crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. Site-directed mutagenesis of residues in the effector site reveals key features leading to differential effector regulation in these enzymes. In AtCM1, mutations of Gly-213 abolish allosteric regulation, as observed in AtCM2. A second effector site position, Gly-149 in AtCM1 and Asp-132 in AtCM3, controls amino acid effector specificity in AtCM1 and AtCM3. Comparisons of chorismate mutases from multiple plants suggest that subtle differences in the effector site are conserved in different lineages and may lead to specialized regulation of this branch point enzyme. PMID:25160622

Active Flow Effectors for Noise and Separation Control

NASA Technical Reports Server (NTRS)

Turner, Travis L.

2011-01-01

New flow effector technology for separation control and enhanced mixing is based upon shape memory alloy hybrid composite (SMAHC) technology. The technology allows for variable shape control of aircraft structures through actively deformable surfaces. The flow effectors are made by embedding shape memory alloy actuator material in a composite structure. When thermally actuated, the flow effector def1ects into or out of the flow in a prescribed manner to enhance mixing or induce separation for a variety of applications, including aeroacoustic noise reduction, drag reduction, and f1ight control. The active flow effectors were developed for noise reduction as an alternative to fixed-configuration effectors, such as static chevrons, that cannot be optimized for airframe installation effects or variable operating conditions and cannot be retracted for off-design or fail-safe conditions. Benefits include: Increased vehicle control, overall efficiency, and reduced noise throughout all f1ight regimes, Reduced flow noise, Reduced drag, Simplicity of design and fabrication, Simplicity of control through direct current stimulation, autonomous re sponse to environmental heating, fast re sponse, and a high degree of geometric stability. The concept involves embedding prestrained SMA actuators on one side of the chevron neutral axis in order to generate a thermal moment and def1ect the structure out of plane when heated. The force developed in the host structure during def1ection and the aerodynamic load is used for returning the structure to the retracted position. The chevron design is highly scalable and versatile, and easily affords active and/or autonomous (environmental) control. The technology offers wide-ranging market applications, including aerospace, automotive, and any application that requires flow separation or noise control.

CUP promotes deadenylation and inhibits decapping of mRNA targets

PubMed Central

Igreja, Catia; Izaurralde, Elisa

2011-01-01

CUP is an eIF4E-binding protein (4E-BP) that represses the expression of specific maternal mRNAs prior to their posterior localization. Here, we show that CUP employs multiple mechanisms to repress the expression of target mRNAs. In addition to inducing translational repression, CUP maintains mRNA targets in a repressed state by promoting their deadenylation and protects deadenylated mRNAs from further degradation. Translational repression and deadenylation are independent of eIF4E binding and require both the middle and C-terminal regions of CUP, which collectively we termed the effector domain. This domain associates with the deadenylase complex CAF1–CCR4–NOT and decapping activators. Accordingly, in isolation, the effector domain is a potent trigger of mRNA degradation and promotes deadenylation, decapping and decay. However, in the context of the full-length CUP protein, the decapping and decay mediated by the effector domain are inhibited, and target mRNAs are maintained in a deadenylated, repressed form. Remarkably, an N-terminal regulatory domain containing a noncanonical eIF4E-binding motif is required to protect CUP-associated mRNAs from decapping and further degradation, suggesting that this domain counteracts the activity of the effector domain. Our findings indicate that the mode of action of CUP is more complex than previously thought and provide mechanistic insight into the regulation of mRNA expression by 4E-BPs. PMID:21937713

A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

PubMed Central

Sanjana, Neville E.; Cong, Le; Zhou, Yang; Cunniff, Margaret M.; Feng, Guoping; Zhang, Feng

2013-01-01

Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA binding proteins found in the plant pathogen Xanthomonas sp. The DNA binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within one week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using, respectively, qRT-PCR and Surveyor nuclease. The TALE toolbox described here will enable a broad range of biological applications. PMID:22222791

Cereal powdery mildew effectors: a complex toolbox for an obligate pathogen.

PubMed

Bourras, Salim; Praz, Coraline R; Spanu, Pietro D; Keller, Beat

2018-02-15

Cereal powdery mildews are major pathogens of cultivated monocot crops, and all are obligate biotrophic fungi that can only grow and reproduce on living hosts. This lifestyle is combined with extreme host specialization where every mildew subspecies (referred to as forma specialis) can only infect one plant species. Recently there has been much progress in our understanding of the possible roles effectors play in this complex host-pathogen interaction. Here, we review current knowledge on the origin, evolution, and mode of action of cereal mildew effectors, with a particular focus on recent advances in the identification of bona fide effectors and avirulence effector proteins from wheat and barley powdery mildews. Copyright © 2018 Elsevier Ltd. All rights reserved.

Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

PubMed

Rajput, Nasir Ahmed; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Zhang, Qimeng; Ru, Yanyan; Sun, Peng; Dou, Daolong

2015-12-01

The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector.

PubMed

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis -specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

PubMed Central

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria. PMID:28638803

Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors

PubMed Central

Sperschneider, Jana; Ying, Hua; Dodds, Peter N.; Gardiner, Donald M.; Upadhyaya, Narayana M.; Singh, Karam B.; Manners, John M.; Taylor, Jennifer M.

2014-01-01

Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control. PMID:25225496

Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8+ T lymphocytes

PubMed Central

Singh, Shailbala; Toro, Haroldo; Tang, De-Chu; Briles, Worthie E.; Yates, Linda M.; Kopulos, Renee T.; Collisson, Ellen W.

2010-01-01

Avian influenza virus (AIV) specific CD8+ T lymphocyte responses stimulated by intramuscular administration of an adenovirus (Ad) vector expressing either HA or NP were evaluated in chickens following ex vivo stimulation by non-professional antigen presenting cells. The CD8+ T lymphocyte responses were AIV specific, MHC-I restricted, and cross-reacted with heterologousH7N2 AIV strain. Specific effector responses, at 10 days post-inoculation (p.i.), were undetectable at 2 weeks p.i., and memory responses were detected from 3 to 8 weeks p.i. Effector memory responses, detected 1 week following a booster inoculation, were significantly greater than the primary responses and, within 7 days, declined to undetectable levels. Inoculation of an Ad-vector expressing human NP resulted in significantly greater MHC restricted, activation of CD8+ T cell responses specific for AIV. Decreases in all responses with time were most dramatic with maximum activation of T cells as observed following effector and effector memory responses. PMID:20557918

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

PubMed Central

Moore, Gregory L; Chen, Hsing; Karki, Sher

2010-01-01

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complementenhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens. PMID:20150767

Transfer of Motor Learning Is More Pronounced in Proximal Compared to Distal Effectors in Upper Extremities

PubMed Central

Aune, Tore K.; Aune, Morten A.; Ingvaldsen, Rolf P.; Vereijken, Beatrix

2017-01-01

The current experiment investigated generalizability of motor learning in proximal versus distal effectors in upper extremities. Twenty-eight participants were divided into three groups: training proximal effectors, training distal effectors, and no training control group (CG). Performance was tested pre- and post-training for specific learning and three learning transfer conditions: (1) bilateral learning transfer between homologous effectors, (2) lateral learning transfer between non-homologous effectors, and (3) bilateral learning transfer between non-homologous effectors. With respect to specific learning, both training groups showed significant, similar improvement for the trained proximal and distal effectors, respectively. In addition, there was significant learning transfer to all three transfer conditions, except for bilateral learning transfer between non-homologous effectors for the distal training group. Interestingly, the proximal training group showed significantly larger learning transfer to other effectors compared to the distal training group. The CG did not show significant improvements from pre- to post-test. These results show that learning is partly effector independent and generalizable to different effectors, even though transfer is suboptimal compared to specific learning. Furthermore, there is a proximal-distal gradient in generalizability, in that learning transfer from trained proximal effectors is larger than from trained distal effectors, which is consistent with neuroanatomical differences in activation of proximal and distal muscles. PMID:28943857

In vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. III. Spontaneous and natural cytotoxic effector cells.

PubMed Central

Herbert, A G; Le Gros, G S; Bidawid, S; Watson, J D

1984-01-01

Cytotoxic effector cell populations in murine spleen can be characterized by the phenotype of the cytotoxic cells or the nature of target cells. Lytic events can be antigen-specific, MHC-restricted and clonal, or target cell-specific but apparently non-MHC-restricted. Two cytotoxic effectors of this latter category are spontaneous and natural killers. Normal spleen cells from (BALB/c X DBA/2J)F1 mice (CDF1) cultured without added antigen develop a population of Thy-1+ spontaneous cytotoxic lymphocytes (SCTL) that lyse the DBA/2J mastocytoma P815, as well as the BALB/c-derived plasmacytomas MOPC-11 and SP2/0. Cold target competition experiments reveal the BALB/c-derived plasmacytomas MOPC-11, SP2/0, J558 and the A strain-derived T cell lymphoma YAC-1, but not normal lymphoblasts, block the lysis of P815 target cells. Thus, while these tumour cells appear to express common antigens which are recognized by SCTL cells, plasmacytomas such as J558 are not susceptible to lysis by SCTL. The relationship of SCTL to natural killer (NK) cells was examined. In-vivo treatment of mice with monoclonal anti-Thy-1 antibody leads to a rapid loss of SCTL and precursors from the spleen, but there is a concomitant increase in NK cell activity. PMID:6607213

Identification and characterization of LysM effectors in Penicillium expansum.

PubMed

Levin, Elena; Ballester, Ana Rosa; Raphael, Ginat; Feigenberg, Oleg; Liu, Yongsheng; Norelli, John; Gonzalez-Candelas, Luis; Ma, Jing; Dardick, Christopher; Wisniewski, Michael; Droby, Samir

2017-01-01

P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4) were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

'Drugs from bugs': bacterial effector proteins as promising biological (immune-) therapeutics.

PubMed

Rüter, Christian; Hardwidge, Philip R

2014-02-01

Immune system malfunctions cause many of the most severe human diseases. The immune system has evolved primarily to control bacterial, viral, fungal, and parasitic infections. In turn, over millions of years of coevolution, microbial pathogens have evolved various mechanisms to control and modulate the host immune system for their own benefit and survival. For example, many bacterial pathogens use virulence proteins to modulate and exploit target cell mechanisms. Our understanding of these bacterial strategies opens novel possibilities to exploit 'microbial knowledge' to control excessive immune reactions. Gaining access to strategies of microbial pathogens could lead to potentially huge benefits for the therapy of inflammatory diseases. Most work on bacterial pathogen effector proteins has the long-term aim of neutralizing the infectious capabilities of the pathogen. However, attenuated pathogens and microbial products have been used for over a century with overwhelming success in the form of vaccines to induce specific immune responses that protect against the respective infectious diseases. In this review, we focus on bacterial effector and virulence proteins capable of modulating and suppressing distinct signaling pathways with potentially desirable immune-modulating effects for treating unrelated inflammatory diseases. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

System for exchanging tools and end effectors on a robot

DOEpatents

Burry, David B.; Williams, Paul M.

1991-02-19

A system and method for exchanging tools and end effectors on a robot permits exchange during a programmed task. The exchange mechanism is located off the robot, thus reducing the mass of the robot arm and permitting smaller robots to perform designated tasks. A simple spring/collet mechanism mounted on the robot is used which permits the engagement and disengagement of the tool or end effector without the need for a rotational orientation of the tool to the end effector/collet interface. As the tool changing system is not located on the robot arm no umbilical cords are located on robot.

System for exchanging tools and end effectors on a robot

DOEpatents

Burry, D.B.; Williams, P.M.

1991-02-19

A system and method for exchanging tools and end effectors on a robot permits exchange during a programmed task. The exchange mechanism is located off the robot, thus reducing the mass of the robot arm and permitting smaller robots to perform designated tasks. A simple spring/collet mechanism mounted on the robot is used which permits the engagement and disengagement of the tool or end effector without the need for a rotational orientation of the tool to the end effector/collet interface. As the tool changing system is not located on the robot arm no umbilical cords are located on robot. 12 figures.

Diverse secreted effectors are required for Salmonella persistence in a mouse infection model.

PubMed

Kidwai, Afshan S; Mushamiri, Ivy; Niemann, George S; Brown, Roslyn N; Adkins, Joshua N; Heffron, Fred

2013-01-01

Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.

Diverse Secreted Effectors Are Required for Salmonella Persistence in a Mouse Infection Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kidwai, Afshan S.; Mushamiri, Ivy T.; Niemann, George

Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 moremore » strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.« less

The genome sequence and effector complement of the flax rust pathogen Melampsora lini.

PubMed

Nemri, Adnane; Saunders, Diane G O; Anderson, Claire; Upadhyaya, Narayana M; Win, Joe; Lawrence, Gregory J; Jones, David A; Kamoun, Sophien; Ellis, Jeffrey G; Dodds, Peter N

2014-01-01

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

PubMed

Pombo, Marina A; Zheng, Yi; Fernandez-Pozo, Noe; Dunham, Diane M; Fei, Zhangjun; Martin, Gregory B

2014-01-01

Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types during Infection[OPEN

PubMed Central

Henry, Elizabeth; Jauneau, Alain; Deslandes, Laurent

2017-01-01

To cause disease, diverse pathogens deliver effector proteins into host cells. Pathogen effectors can inhibit defense responses, alter host physiology, and represent important cellular probes to investigate plant biology. However, effector function and localization have primarily been investigated after overexpression in planta. Visualizing effector delivery during infection is challenging due to the plant cell wall, autofluorescence, and low effector abundance. Here, we used a GFP strand system to directly visualize bacterial effectors delivered into plant cells through the type III secretion system. GFP is a beta barrel that can be divided into 11 strands. We generated transgenic Arabidopsis thaliana plants expressing GFP1-10 (strands 1 to 10). Multiple bacterial effectors tagged with the complementary strand 11 epitope retained their biological function in Arabidopsis and tomato (Solanum lycopersicum). Infection of plants expressing GFP1-10 with bacteria delivering GFP11-tagged effectors enabled direct effector detection in planta. We investigated the temporal and spatial delivery of GFP11-tagged effectors during infection with the foliar pathogen Pseudomonas syringae and the vascular pathogen Ralstonia solanacearum. Thus, the GFP strand system can be broadly used to investigate effector biology in planta. PMID:28600390

The Shigella flexneri OspB effector: an early immunomodulator.

PubMed

Ambrosi, Cecilia; Pompili, Monica; Scribano, Daniela; Limongi, Dolores; Petrucca, Andrea; Cannavacciuolo, Sonia; Schippa, Serena; Zagaglia, Carlo; Grossi, Milena; Nicoletti, Mauro

2015-01-01

Through the action of the type three secretion system (T3SS) Shigella flexneri delivers several effectors into host cells to promote cellular invasion, multiplication and to exploit host-cell signaling pathways to modulate the host innate immune response. Although much progress has been made in the understanding of many type III effectors, the molecular and cellular mechanism of the OspB effector is still poorly characterized. In this study we present new evidence that better elucidates the role of OspB as pro-inflammatory factor at very early stages of infection. Indeed, we demonstrate that, during the first hour of infection, OspB is required for full activation of ERK1/2 and p38 MAPKs and the cytosolic phospholipase A(2) (cPLA(2)). Activation of cPLA(2) ultimately leads to the production and secretion of PMN chemoattractant metabolite(s) uncoupled with release of IL-8. Moreover, we also present evidence that OspB is required for the development of the full and promptly inflammatory reaction characteristic of S. flexneri wild-type infection in vivo. Based on OspB and OspF similarity (both effectors share similar transcription regulation, temporal secretion into host cells and nuclear localization) we hypothesized that OspB and OspF effectors may form a pair aimed at modulating the host cell response throughout the infection process, with opposite effects. A model is presented to illustrate how OspB activity would promote S. flexneri invasion and bacterial dissemination at early critical phases of infection. Copyright © 2014 Elsevier GmbH. All rights reserved.

Novel Control Effectors for Truss Braced Wing

NASA Technical Reports Server (NTRS)

White, Edward V.; Kapania, Rakesh K.; Joshi, Shiv

2015-01-01

At cruise flight conditions very high aspect ratio/low sweep truss braced wings (TBW) may be subject to design requirements that distinguish them from more highly swept cantilevered wings. High aspect ratio, short chord length and relative thinness of the airfoil sections all contribute to relatively low wing torsional stiffness. This may lead to aeroelastic issues such as aileron reversal and low flutter margins. In order to counteract these issues, high aspect ratio/low sweep wings may need to carry additional high speed control effectors to operate when outboard ailerons are in reversal and/or must carry additional structural weight to enhance torsional stiffness. The novel control effector evaluated in this study is a variable sweep raked wing tip with an aileron control surface. Forward sweep of the tip allows the aileron to align closely with the torsional axis of the wing and operate in a conventional fashion. Aft sweep of the tip creates a large moment arm from the aileron to the wing torsional axis greatly enhancing aileron reversal. The novelty comes from using this enhanced and controllable aileron reversal effect to provide roll control authority by acting as a servo tab and providing roll control through intentional twist of the wing. In this case the reduced torsional stiffness of the wing becomes an advantage to be exploited. The study results show that the novel control effector concept does provide roll control as described, but only for a restricted class of TBW aircraft configurations. For the configuration studied (long range, dual aisle, Mach 0.85 cruise) the novel control effector provides significant benefits including up to 12% reduction in fuel burn.

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

PubMed

Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko

2015-05-26

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

PubMed

Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W

2018-01-01

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .

The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems

PubMed Central

Ma, Jiale; Pan, Zihao; Huang, Jinhu; Sun, Min; Lu, Chengping; Yao, Huochun

2017-01-01

ABSTRACT The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3–4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors. PMID:28060574

Independently evolved virulence effectors converge onto hubs in a plant immune system network.

PubMed

Mukhtar, M Shahid; Carvunis, Anne-Ruxandra; Dreze, Matija; Epple, Petra; Steinbrenner, Jens; Moore, Jonathan; Tasan, Murat; Galli, Mary; Hao, Tong; Nishimura, Marc T; Pevzner, Samuel J; Donovan, Susan E; Ghamsari, Lila; Santhanam, Balaji; Romero, Viviana; Poulin, Matthew M; Gebreab, Fana; Gutierrez, Bryan J; Tam, Stanley; Monachello, Dario; Boxem, Mike; Harbort, Christopher J; McDonald, Nathan; Gai, Lantian; Chen, Huaming; He, Yijian; Vandenhaute, Jean; Roth, Frederick P; Hill, David E; Ecker, Joseph R; Vidal, Marc; Beynon, Jim; Braun, Pascal; Dangl, Jeffery L

2011-07-29

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.

The bacterial type III-secreted protein AvrRps4 is a bipartite effector

PubMed Central

Spears, Benjamin J.; Garner, Christopher M.; Rogan, Conner J.; Su, Jianbin; Bhattacharjee, Saikat

2018-01-01

Bacterial effector proteins secreted into host plant cells manipulate those cells to the benefit of the pathogen, but effector-triggered immunity (ETI) occurs when effectors are recognized by host resistance proteins. The RPS4/RRS1 pair recognizes the Pseudomonas syringae pv. pisi effector AvrRps4. AvrRps4 is processed in planta into AvrRps4N (133 amino acids), homologous to the N-termini of other effectors including the native P. syringae pv. tomato strain DC3000 effector HopK1, and AvrRps4C (88 amino acids). Previous data suggested that AvrRps4C alone is necessary and sufficient for resistance when overexpressed in heterologous systems. We show that delivering AvrRps4C from DC3000, but not from a DC3000 hopK1- strain, triggers resistance in the Arabidopsis accession Col-0. Delivering AvrRps4C in tandem with AvrRps4N, or as a chimera with HopK1N, fully complements AvrRps4-triggered immunity. AvrRps4N in the absence of AvrRps4C enhances virulence in Col-0. In addition, AvrRps4N triggers a hypersensitive response in lettuce that is attenuated by coexpression of AvrRps4C, further supporting the role of AvrRps4N as a bona fide effector domain. Based on these results we propose that evolutionarily, fusion of AvrRps4C to AvrRps4N may have counteracted recognition of AvrRps4N, and that the plant RPS4/RRS1 resistance gene pair was selected as a countermeasure. We conclude that AvrRps4 represents an unusual chimeric effector, with recognition in Arabidopsis by RPS4/RRS1 requiring the presence of both processed effector moieties. PMID:29601603

The bacterial type III-secreted protein AvrRps4 is a bipartite effector.

PubMed

Halane, Morgan K; Kim, Sang Hee; Spears, Benjamin J; Garner, Christopher M; Rogan, Conner J; Okafor, Elizabeth C; Su, Jianbin; Bhattacharjee, Saikat; Gassmann, Walter

2018-03-01

Bacterial effector proteins secreted into host plant cells manipulate those cells to the benefit of the pathogen, but effector-triggered immunity (ETI) occurs when effectors are recognized by host resistance proteins. The RPS4/RRS1 pair recognizes the Pseudomonas syringae pv. pisi effector AvrRps4. AvrRps4 is processed in planta into AvrRps4N (133 amino acids), homologous to the N-termini of other effectors including the native P. syringae pv. tomato strain DC3000 effector HopK1, and AvrRps4C (88 amino acids). Previous data suggested that AvrRps4C alone is necessary and sufficient for resistance when overexpressed in heterologous systems. We show that delivering AvrRps4C from DC3000, but not from a DC3000 hopK1- strain, triggers resistance in the Arabidopsis accession Col-0. Delivering AvrRps4C in tandem with AvrRps4N, or as a chimera with HopK1N, fully complements AvrRps4-triggered immunity. AvrRps4N in the absence of AvrRps4C enhances virulence in Col-0. In addition, AvrRps4N triggers a hypersensitive response in lettuce that is attenuated by coexpression of AvrRps4C, further supporting the role of AvrRps4N as a bona fide effector domain. Based on these results we propose that evolutionarily, fusion of AvrRps4C to AvrRps4N may have counteracted recognition of AvrRps4N, and that the plant RPS4/RRS1 resistance gene pair was selected as a countermeasure. We conclude that AvrRps4 represents an unusual chimeric effector, with recognition in Arabidopsis by RPS4/RRS1 requiring the presence of both processed effector moieties.

Modulation of hemoglobin dynamics by an allosteric effector

DOE PAGES

Lal, Jyotsana; Maccarini, Marco; Fouquet, Peter; ...

2016-12-15

Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure-function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy-Hb andmore » HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo (NSE) measurements accompanied by wideangle x-ray scattering (WAXS) to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large-scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. Furthermore, these observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.« less

Identification and functional analysis of secreted effectors from phytoparasitic nematodes.

PubMed

Rehman, Sajid; Gupta, Vijai K; Goyal, Aakash K

2016-03-21

Plant parasitic nematodes develop an intimate and long-term feeding relationship with their host plants. They induce a multi-nucleate feeding site close to the vascular bundle in the roots of their host plant and remain sessile for the rest of their life. Nematode secretions, produced in the oesophageal glands and secreted through a hollow stylet into the host plant cytoplasm, are believed to play key role in pathogenesis. To combat these persistent pathogens, the identity and functional analysis of secreted effectors can serve as a key to devise durable control measures. In this review, we will recapitulate the knowledge over the identification and functional characterization of secreted nematode effector repertoire from phytoparasitic nematodes. Despite considerable efforts, the identity of genes encoding nematode secreted proteins has long been severely hampered because of their microscopic size, long generation time and obligate biotrophic nature. The methodologies such as bioinformatics, protein structure modeling, in situ hybridization microscopy, and protein-protein interaction have been used to identify and to attribute functions to the effectors. In addition, RNA interference (RNAi) has been instrumental to decipher the role of the genes encoding secreted effectors necessary for parasitism and genes attributed to normal development. Recent comparative and functional genomic approaches have accelerated the identification of effectors from phytoparasitic nematodes and offers opportunities to control these pathogens. Plant parasitic nematodes pose a serious threat to global food security of various economically important crops. There is a wealth of genomic and transcriptomic information available on plant parasitic nematodes and comparative genomics has identified many effectors. Bioengineering crops with dsRNA of phytonematode genes can disrupt the life cycle of parasitic nematodes and therefore holds great promise to develop resistant crops against plant

A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism

PubMed Central

Huang, Qiuling; Hu, Lili; Zhuo, Kan

2017-01-01

Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM) and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode Meloidogyne graminicola has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of M. graminicola. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to M. graminicola infection than wild-type control plants, and conversely, in planta, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M. graminicola. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the N-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, N-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections. PMID:28403192

A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism.

PubMed

Chen, Jiansong; Lin, Borong; Huang, Qiuling; Hu, Lili; Zhuo, Kan; Liao, Jinling

2017-04-01

Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM) and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode Meloidogyne graminicola has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of M. graminicola. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to M. graminicola infection than wild-type control plants, and conversely, in planta, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M. graminicola. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the N-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, N-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.

Effector T cells require fatty acid metabolism during murine graft-versus-host disease

PubMed Central

Byersdorfer, Craig A.; Tkachev, Victor; Opipari, Anthony W.; Goodell, Stefanie; Swanson, Jacob; Sandquist, Stacy; Glick, Gary D.; Ferrara, James L. M.

2013-01-01

Activated T cells require increased energy to proliferate and mediate effector functions, but the metabolic changes that occur in T cells following stimulation in vivo are poorly understood, particularly in the context of inflammation. We have previously shown that T cells activated during graft-versus-host disease (GVHD) primarily rely on oxidative phosphorylation to synthesize adenosine 5′-triphosphate. Here, we demonstrate that alloreactive effector T cells (Teff) use fatty acids (FAs) as a fuel source to support their in vivo activation. Alloreactive T cells increased FA transport, elevated levels of FA oxidation enzymes, up-regulated transcriptional coactivators to drive oxidative metabolism, and increased their rates of FA oxidation. Importantly, increases in FA transport and up-regulation of FA oxidation machinery occurred specifically in T cells during GVHD and were not seen in Teff following acute activation. Pharmacological blockade of FA oxidation decreased the survival of alloreactive T cells but did not influence the survival of T cells during normal immune reconstitution. These studies suggest that pathways controlling FA metabolism might serve as therapeutic targets to treat GVHD and other T-cell–mediated immune diseases. PMID:24046012

Chronic Dry Eye Disease is Principally Mediated by Effector Memory Th17 Cells

PubMed Central

Chen, Yihe; Chauhan, Sunil K.; Lee, Hyun Soo; Saban, Daniel R.; Dana, Reza

2013-01-01

Recent experimental and clinical data suggest that there is a link between dry eye disease (DED) and T cell-mediated immunity. However, whether these immune responses are a consequence or cause of ocular surface inflammation remains to be determined. Thus far, only models of acute DED have been used to derive experimental data. This is in contrast to clinical DED which usually presents as a chronic disease. In the present study, using a murine model of chronic DED, it was established that the chronic phase of the disease is accompanied by Th17 responses at the ocular surface, and that a significant memory T cell population can be recovered from chronic DED. This memory response is predominantly mediated by Th17 cells. Moreover, adoptive transfer of this memory T cell population was shown to induce more severe and rapidly progressing DED than did the adoptive transfer of its effector or naïve counterparts. Not only do these results clearly demonstrate that effector memory Th17 cells are primarily responsible for maintaining the chronic and relapsing course of DED, but they also highlight a potentially novel therapeutic strategy for targeting memory immune responses in patients with DED. PMID:23571503

Disruption or Excision of Provirus as an Approach to HIV Cure.

PubMed

Jerome, Keith R

2016-12-01

An effective approach to HIV cure will almost certainly require a combination of strategies, including some means of reducing the latent HIV reservoir. Because the integrated HIV provirus represents the major source of viral persistence and reactivation, one attractive approach is the direct targeting of provirus for disruption or excision using targeted endonucleases, such as CRISPR/Cas9, zinc finger nucleases, TAL effector nucleases, or meganucleases (homing endonucleases). This article highlights some of the challenges for successful endonuclease therapy for HIV, including optimization of enzyme activity and specificity, the possible emergence of viral resistance, and most importantly, efficient in vivo delivery of the enzymes to a sufficient portion of the latent reservoir.

End effector on orbiter's RMS arm

NASA Image and Video Library

2001-03-13

STS102-E-5201 (13 March 2001) --- A view of the interior of the end effector apparatus on the end of the Canadian-built remote manipulator system (RMS) arm. The photograph was taken with a digital still camera.

IQGAP1 is important for activation of caspase-1 in macrophages and is targeted by Yersinia pestis type III effector YopM.

PubMed

Chung, Lawton K; Philip, Naomi H; Schmidt, Valentina A; Koller, Antonius; Strowig, Till; Flavell, Richard A; Brodsky, Igor E; Bliska, James B

2014-07-01

YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM(-) YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopM(YPIII). To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM(32777)) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopM(KIM)) or inactivated (YopM(KIM) D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM(32777), YopM(KIM), and YopM(KIM) D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopM(KIM) deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM(32777), YopM(KIM), and YopM(KIM) deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopM(KIM) but not YopM(32777). Additionally, YopM(KIM) bound IQGAP1 and the use of Iqgap1(-/-) macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM(-) Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopM(KIM) target IQGAP1, a novel regulator of caspase-1, in macrophages. Importance: Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert

Direct observation of transcription activator-like effector (TALE) protein dynamics

NASA Astrophysics Data System (ADS)

Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

2014-03-01

In this work, we describe a single molecule assay to probe the site-search dynamics of transcription activator-like effector (TALE) proteins along DNA. In modern genetics, the ability to selectively edit the human genome is an unprecedented development, driven by recent advances in targeted nuclease proteins. Specific gene editing can be accomplished using TALE proteins, which are programmable DNA-binding proteins that can be fused to a nuclease domain. In this way, TALENs are a leading technology that has shown great success in the genomic editing of pluripotent stem cells. A major hurdle facing clinical implementation, however, is the potential for deleterious off-target binding events. For these reasons, a molecular-level understanding of TALE binding and target sequence search on DNA is essential. To this end, we developed a single-molecule fluorescence imaging assay that provides a first-of-its-kind view of the 1-D diffusion of TALE proteins along stretched DNA. Taken together with co-crystal structures of DNA-bound TALEs, our results suggest a rotationally-coupled, major groove tracking model for diffusion. We further report diffusion constants for TALE proteins as a function of salt concentration, consistent with previously described models of 1-D protein diffusion.

IL-15 signaling promotes adoptive effector T-cell survival and memory formation in irradiation-induced lymphopenia.

PubMed

Xu, Aizhang; Bhanumathy, Kalpana Kalyanasundaram; Wu, Jie; Ye, Zhenmin; Freywald, Andrew; Leary, Scot C; Li, Rongxiu; Xiang, Jim

2016-01-01

Lymphopenia promotes naïve T-cell homeostatic proliferation and adoptive effector T-cell survival and memory formation. IL-7 plays a critical role in homeostatic proliferation, survival and memory formation of naïve T-cells in lymphopenia, and its underlying molecular mechanism has also been well studied. However, the mechanism for adoptively transferred effector T-cell survival and memory formation is not fully understood. Here, we transferred in vitro-activated transgenic OT-I CD8(+) effector T-cells into irradiation (600 rads)-induced lymphopenic C57BL/6, IL-7 knockout (KO) and IL-15 KO mice, and investigated the survival and memory formation of transferred T-cells in lymphopenia. We demonstrate that transferred T-cells prolong their survival and enhance their memory in lymphopenic mice, in a manner that depends on IL-15 signaling, but not IL-7. We determine that in vitro stimulation of naïve or effector T-cells with IL-7 and IL-15 reduces IL-7Rα, and increases and/or maintains IL-15Rβ expression, respectively. Consistent with these findings, the expression of IL-7Rα and IL-15Rβ is down- and up-regulated, respectively, in vivo on transferred T-cells in an early phase post T-cell transfer in lymphopenia. We further show that in vitro IL-15 restimulation-induced memory T-cells (compared to IL-2 restimulation-induced effector T-cells) and in vivo transferred T-cells in irradiated IL-15-sufficient C57BL/6 mice (compared to IL-15-deficient IL-15 KO mice) have increased mitochondrial content, but less NADH and lower mitochondrial potential (ΔΨm), and demonstrate greater phosphorylation of signal transducers and activators of transcription-5 (STAT5) and Unc-51-like kinase-1 (ULK1), and higher expression of B-cell leukemia/lymphoma-2 (Bcl2) and memory-, autophagy- and mitochondrial biogenesis-related molecules. Irradiation-induced lymphopenia promotes effector T-cell survival via IL-15 signaling the STAT5/Bcl2 pathway, enhances T-cell memory formation via IL

Putative rust fungal effector proteins in infected bean and soybean leaves

USDA-ARS?s Scientific Manuscript database

The plant pathogenic fungi Uromyces appendiculatus and Phakopsora pachyrhizi cause debilitating rust diseases on common bean and soybean. These rust fungi secrete effector proteins that allow them to infect plants, but the effector repertoire for U. appendiculatus and P. pachyrhizi is not fully def...

B7-H1 limits the entry of effector CD8(+) T cells to the memory pool by upregulating Bim.

PubMed

Gibbons, Rachel M; Liu, Xin; Pulko, Vesna; Harrington, Susan M; Krco, Christopher J; Kwon, Eugene D; Dong, Haidong

2012-10-01

Protective T‑cell immunity against cancer and infections is dependent on the generation of a durable effector and memory T‑cell pool. Studies from cancer and chronic infections reveal that B7-H1 (PD-L1) engagement with its receptor PD-1 promotes apoptosis of effector T cells. It is not clear how B7-H1 regulates T‑cell apoptosis and the subsequent impact of B7-H1 on the generation of memory T cells. In immunized B7-H1-deficient mice, we detected an increased expansion of effector CD8(+) T cells and a delayed T‑cell contraction followed by the emergence of a protective CD8(+) T‑cell memory capable of completely rejecting tumor metastases in the lung. Intracellular staining revealed that antigen-primed CD8(+) T cells in B7-H1-deficient mice express lower levels of the pro-apoptotic molecule Bim. The engagement of activated CD8(+) T cells by a plate-bound B7-H1 fusion protein led to the upregulation of Bim and increased cell death. Assays based on blocking antibodies determined that both PD-1 and CD80 are involved in the B7-H1-mediated regulation of Bim in activated CD8(+) T cells. Our results suggest that B7-H1 may negatively regulate CD8(+) T‑cell memory by enhancing the depletion of effector CD8(+) T cells through the upregulation of Bim. Our findings may provide a new strategy for targeting B7-H1 signaling in effector CD8(+) T cells to achieve protective antitumor memory responses.

T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System.

PubMed

Tay, Daniel Ming Ming; Govindarajan, Kunde Ramamoorthy; Khan, Asif M; Ong, Terenze Yao Rui; Samad, Hanif M; Soh, Wei Wei; Tong, Minyan; Zhang, Fan; Tan, Tin Wee

2010-10-15

Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of

New type III effectors from Xanthomonas campestris pv. vesicatoria trigger plant reactions dependent on a conserved N-myristoylation motif.

PubMed

Thieme, Frank; Szczesny, Robert; Urban, Alexander; Kirchner, Oliver; Hause, Gerd; Bonas, Ulla

2007-10-01

Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion system, which translocates bacterial effector proteins into the plant cell. In this study, we identified two novel type III effectors, XopE1 and XopE2 (Xanthomonas outer proteins), using the AvrBs3 effector domain as reporter. XopE1 and XopE2 belong to the HopX family and possess a conserved putative N-myristoylation motif that is also present in the effector XopJ from X. campestris pv. vesicatoria 85-10. XopJ is a member of the YopJ/AvrRxv family of acetyltransferases. Confocal laser scanning microscopy and immunocytochemistry revealed that green fluorescent protein fusions of XopE1, XopE2, and XopJ localized to the plant cell plasma membrane. Targeting to the membrane is probably due to N-myristoylation, because a point mutation in the putative myristoylated glycine residue G2 in XopE1, XopE2, and XopJ resulted in cytoplasmic localization of the mutant proteins. Results of hydroxylamine treatments of XopE2 protein extracts suggest that the proteins are additionally anchored in the host cell plasma membrane by palmitoylation. The membrane localization of the effectors strongly influences the phenotypes they trigger in the plant. Agrobacterium-mediated expression of xopE1 and xopJ in Nicotiana benthamiana led to cell-death reactions that, for xopJ, were dependent on the N-myristoylation motif. In the case of xopE1(G2A), cell death was more pronounced with the mutant than with the wild-type protein. In addition, XopE2 has an avirulence activity in Solanum pseudocapsicum.

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

PubMed

Teper, Doron; Girija, Anil Madhusoodana; Bosis, Eran; Popov, Georgy; Savidor, Alon; Sessa, Guido

2018-01-01

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis.

PubMed

Sugio, Akiko; Kingdom, Heather N; MacLean, Allyson M; Grieve, Victoria M; Hogenhout, Saskia A

2011-11-29

Phytoplasmas are insect-transmitted phytopathogenic bacteria that can alter plant morphology and the longevity and reproduction rates and behavior of their insect vectors. There are various examples of animal and plant parasites that alter the host phenotype to attract insect vectors, but it is unclear how these parasites accomplish this. We hypothesized that phytoplasmas produce effectors that modulate specific targets in their hosts leading to the changes in plant development and insect performance. Previously, we sequenced and mined the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) and identified 56 candidate effectors. Here, we report that the secreted AY-WB protein 11 (SAP11) effector modulates plant defense responses to the advantage of the AY-WB insect vector Macrosteles quadrilineatus. SAP11 binds and destabilizes Arabidopsis CINCINNATA (CIN)-related TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors, which control plant development and promote the expression of lipoxygenase (LOX) genes involved in jasmonate (JA) synthesis. Both the Arabidopsis SAP11 lines and AY-WB-infected plants produce less JA on wounding. Furthermore, the AY-WB insect vector produces more offspring on AY-WB-infected plants, SAP11 transgenic lines, and plants impaired in CIN-TCP and JA synthesis. Thus, SAP11-mediated destabilization of CIN-TCPs leads to the down-regulation of LOX2 expression and JA synthesis and an increase in M. quadrilineatus progeny. Phytoplasmas are obligate inhabitants of their plant host and insect vectors, in which the latter transmits AY-WB to a diverse range of plant species. This finding demonstrates that pathogen effectors can reach beyond the pathogen-host interface to modulate a third organism in the biological interaction.

Antipsychotics activate the TGFβ pathway effector SMAD3

PubMed Central

Cohen, T.; Sundaresh, S.; Levine, F.

2014-01-01

Although effective in treating an array of neurological disorders, antipsychotics are associated with deleterious metabolic side effects. Through high-throughput screening, we previously identified phenothiazine antipsychotics as modulators of the human insulin promoter. Here, we extended our initial finding to structurally diverse typical and atypical antipsychotics. We then identified the TGFβ pathway as being involved in the effect of antipsychotics on the insulin promoter, finding that antipsychotics activated SMAD3, a downstream effector of the TGFβ pathway, through a receptor distinct from the TGFβ receptor family and known neurotransmitter receptor targets of antipsychotics. Of note, antipsychotics that do not cause metabolic side effects did not activate SMAD3. In vivo relevance was demonstrated by reanalysis of gene expression data from human brains treated with antipsychotics, which showed altered expression of SMAD3 responsive genes. This work raises the possibility that antipsychotics could be designed that retain beneficial CNS activity while lacking deleterious metabolic side effects. PMID:22290122

Intervention of Phytohormone Pathways by Pathogen Effectors[OPEN

PubMed Central

Kazan, Kemal; Lyons, Rebecca

2014-01-01

The constant struggle between plants and microbes has driven the evolution of multiple defense strategies in the host as well as offense strategies in the pathogen. To defend themselves from pathogen attack, plants often rely on elaborate signaling networks regulated by phytohormones. In turn, pathogens have adopted innovative strategies to manipulate phytohormone-regulated defenses. Tactics frequently employed by plant pathogens involve hijacking, evading, or disrupting hormone signaling pathways and/or crosstalk. As reviewed here, this is achieved mechanistically via pathogen-derived molecules known as effectors, which target phytohormone receptors, transcriptional activators and repressors, and other components of phytohormone signaling in the host plant. Herbivores and sap-sucking insects employ obligate pathogens such as viruses, phytoplasma, or symbiotic bacteria to intervene with phytohormone-regulated defenses. Overall, an improved understanding of phytohormone intervention strategies employed by pests and pathogens during their interactions with plants will ultimately lead to the development of new crop protection strategies. PMID:24920334

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

PubMed

Rodríguez-Herva, José J; González-Melendi, Pablo; Cuartas-Lanza, Raquel; Antúnez-Lamas, María; Río-Alvarez, Isabel; Li, Ziduo; López-Torrejón, Gema; Díaz, Isabel; Del Pozo, Juan C; Chakravarthy, Suma; Collmer, Alan; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

2012-05-01

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression. © 2012 Blackwell Publishing Ltd.

Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae

PubMed Central

Erkes, Annett; Reschke, Maik; Boch, Jens

2017-01-01

Abstract Transcription activator-like effectors (TALEs) are secreted by plant–pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity. PMID:28637323

Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.

PubMed

Reyon, Deepak; Khayter, Cyd; Regan, Maureen R; Joung, J Keith; Sander, Jeffry D

2012-10-01

Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)-based protocols can be practiced using plasmid libraries and user-friendly, Web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques. 2012 by John Wiley & Sons, Inc.

Role of effector cells (CCR7(-)CD27(-)) and effector-memory cells (CCR7(-)CD27(+)) in drug-induced maculopapular exanthema.

PubMed

Fernandez, T D; Torres, M J; Lopez, S; Antunez, C; Gomez, E; Del Prado, M F; Canto, G; Blanca, M; Mayorga, C

2010-01-01

Maculopapular exanthema (MPE) induced by drugs is a T-cell mediated reaction and effector cells may play an important role in its development. We assessed the effector and cutaneous homing phenotype in peripheral blood cells from allergic patients after drug stimulation. This study included 10 patients and 10 controls. The effector phenotype (CCR7(-)CD27(+/-)), chemokine receptors (CCR4 and CCR10), and activation (CD25(low)) and regulatory markers (CD25(high)) were measured by flow cytometry in both peripheral blood mononuclear cells (PBMCs) and CD4-T-lymphocytes. Proliferation was determined by 5-(-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay and the migratory capacity by a chemotaxis assay using CCL17 and CCL27. Compared to controls, CCR7(-)CD27(-) cells were increased in patients without (p=0.003) and with drug stimulation (p less than 0.001) and had significantly higher proliferation (p=0.010). CCR10 expression was increased in patients after drug stimulation in total and memory CD27(+) T-cells. Lymphocyte migration with CCL27 was higher in patients with drug stimulation (p=0.048), with a decrease in CCR7(-)CD27(-) (p less than 0.0001) and an increase in CCR7(-)CD27(+) (p=0.017). In patients, CD4-T-lymphocytes were significantly activated after drug stimulation (p less than 0.001). In conclusion, we show that effector memory CD4(+) T-cells (CCR7(-)CD27(+)) respond specifically to the drug responsible for MPE and confirm previous data about the involvement of CCR10 in cell trafficking to the skin.

Development of a DOI PET Detector Having the Structure of the X'tal Cube Extended in One Direction

NASA Astrophysics Data System (ADS)

Inadama, Naoko; Hirano, Yoshiyuki; Nishikido, Fumihiko; Murayama, Hideo; Yamaya, Taiga

2016-10-01

X'tal cube is the cubic depth of interaction (DOI) PET detector which our research group developed. In this work, aiming to get higher sensitivity, we developed the long rectangular shape X'tal cube (long-XC) by extending the cubic X'tal cube structure in one direction. We verified performance of this long-XC and also studied detector parameters for optimization. The same as the X'tal cube, the crystal block of the long-XC is composed of a 3D array of cubic scintillation crystal elements. Reflectors are not inserted between these crystal elements. The scintillation light then spreads without being obstructed by reflectors and is detected by multiple numbers of the multi-pixel photon counters (MPPCs) coupled on all six sides of the crystal block. For crystal element identification, a simple Anger-type calculation is used. In this study, we arranged 3.0 mm×3.0 mm×3.0 mm LGSO crystal elements in a 6 × 6 × 14 array for the long-XC. In a previous study, we had already confirmed that for the X'tal cube consisting of a 6 × 6 × 6 array of the same crystal elements and 54 MPPCs, identification of all 216 crystal elements was possible and the average energy resolution for all the elements was about 11 %. The long-XC contains more than twice the number of the crystal elements but less than twice the number of the MPPCs compared to the previous X'tal cube. The detector parameters investigated with the long-XC were: the number of MPPCs on both sides in the extended direction (edge MPPCs); the MPPC type, the MPPCs of 25 μm × 25 μm or 50 μm × 50 μm pixel sizes; the material between the crystal elements, an air gap or an optical glue having a closer refractive index to that of LGSO than air has; and the MPPC signals used in the Anger-type calculation. Results of the crystal element identification performance showed that reducing the number of the edge MPPCs caused performance degradation only at the part near the edge. For the MPPC type, the 50 μm type was better than

pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

PubMed Central

Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

2016-01-01

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

PubMed Central

Chen, Changlong; Chen, Yongpan; Jian, Heng; Yang, Dan; Dai, Yiran; Pan, Lingling; Shi, Fengwei; Yang, Shanshan; Liu, Qian

2018-01-01

Heterodera avenae is one of the most important plant pathogens and causes vast losses in cereal crops. As a sedentary endoparasitic nematode, H. avenae secretes effectors that modify plant defenses and promote its biotrophic infection of its hosts. However, the number of effectors involved in the interaction between H. avenae and host defenses remains unclear. Here, we report the identification of putative effectors in H. avenae that regulate plant defenses on a large scale. Our results showed that 78 of the 95 putative effectors suppressed programmed cell death (PCD) triggered by BAX and that 7 of the putative effectors themselves caused cell death in Nicotiana benthamiana. Among the cell-death-inducing effectors, three were found to be dependent on their specific domains to trigger cell death and to be expressed in esophageal gland cells by in situ hybridization. Ten candidate effectors that suppressed BAX-triggered PCD also suppressed PCD triggered by the elicitor PsojNIP and at least one R-protein/cognate effector pair, suggesting that they are active in suppressing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Notably, with the exception of isotig16060, these putative effectors could also suppress PCD triggered by cell-death-inducing effectors from H. avenae, indicating that those effectors may cooperate to promote nematode parasitism. Collectively, our results indicate that the majority of the tested effectors of H. avenae may play important roles in suppressing cell death induced by different elicitors in N. benthamiana. PMID:29379510

TNF-induced target cell killing by CTL activated through cross-presentation.

PubMed

Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

2012-09-27

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of a streptococcal preparation (OK432) on natural killer activity of tumour-associated lymphoid cells in human ovarian carcinoma and on lysis of fresh ovarian tumour cells.

PubMed Central

Colotta, F.; Rambaldi, A.; Colombo, N.; Tabacchi, L.; Introna, M.; Mantovani, A.

1983-01-01

The streptococcal preparation OK432 was studied for its effects on natural killer (NK) activity of peripheral blood lymphocytes (PBL) from normal donors and from ovarian cancer patients, and of tumour-associated lymphocytes (TAL) from peritoneal effusions. OK432 augmented NK activity against the susceptible K562 line and induced killing of the relatively resistant Raji line. Freshly isolated ovarian carcinoma cells were relatively resistant to killing by unstimulated PBL and TAL. OK432 induced significant, though low, levels of cytotoxicity against 51Cr-labelled ovarian carcinoma cells. Augmentation of killing of fresh tumour cells by OK432 was best observed in a 20 h assay and both autologous and allogeneic targets were lysed. PBL were separated on discontinuous Percoll gradients. Unstimulated and OK432-boosted activity were enriched in the lower density fractions where large granular lymphocytes (LGL) and activity against K562 were found. Thus, OK432 augments NK activity of PBL and TAL in human ovarian carcinomas and induces low, but significant, levels of killing of fresh tumour cells. Effector cells involved in killing of fresh ovarian tumours copurify with LGL on discontinuous gradients of Percoll. PMID:6626452

Methods and Systems for Authorizing an Effector Command in an Integrated Modular Environment

NASA Technical Reports Server (NTRS)

Sunderland, Dean E. (Inventor); Ahrendt, Terry J. (Inventor); Moore, Tim (Inventor)

2013-01-01

Methods and systems are provided for authorizing a command of an integrated modular environment in which a plurality of partitions control actions of a plurality of effectors is provided. A first identifier, a second identifier, and a third identifier are determined. The first identifier identifies a first partition of the plurality of partitions from which the command originated. The second identifier identifies a first effector of the plurality of effectors for which the command is intended. The third identifier identifies a second partition of the plurality of partitions that is responsible for controlling the first effector. The first identifier and the third identifier are compared to determine whether the first partition is the same as the second partition for authorization of the command.

PD-1 inhibits antiviral immunity at the effector phase in the liver.

PubMed

Iwai, Yoshiko; Terawaki, Seigo; Ikegawa, Masaya; Okazaki, Taku; Honjo, Tasuku

2003-07-07

Unlike naive T cells, effector T cells can be activated by either T cell receptor signal or costimulatory signal alone and therefore the absence of costimulatory molecules on tissue cells cannot explain the tolerance mechanism at the effector phase. Here we report that PD-L1, the ligand for the immunoinhibitory receptor PD-1, was expressed on vascular endothelium in peripheral tissues. Liver nonparenchymal cells including sinusoidal endothelial cells and Kupffer cells constitutively expressed PD-L1 and inhibited proliferation and cell division of activated T cells expressing PD-1. The absence of PD-1 induced proliferation of effector T cells in the adenovirus-infected liver and resulted in rapid clearance of the virus. These results indicate that PD-1 plays an important role in T cell tolerance at the effector phase and the blockade of the PD-1 pathway can augment antiviral immunity.

The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

PubMed

Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

2016-10-01

Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effector prediction in host-pathogen interaction based on a Markov model of a ubiquitous EPIYA motif

PubMed Central

2010-01-01

Background Effector secretion is a common strategy of pathogen in mediating host-pathogen interaction. Eight EPIYA-motif containing effectors have recently been discovered in six pathogens. Once these effectors enter host cells through type III/IV secretion systems (T3SS/T4SS), tyrosine in the EPIYA motif is phosphorylated, which triggers effectors binding other proteins to manipulate host-cell functions. The objectives of this study are to evaluate the distribution pattern of EPIYA motif in broad biological species, to predict potential effectors with EPIYA motif, and to suggest roles and biological functions of potential effectors in host-pathogen interactions. Results A hidden Markov model (HMM) of five amino acids was built for the EPIYA-motif based on the eight known effectors. Using this HMM to search the non-redundant protein database containing 9,216,047 sequences, we obtained 107,231 sequences with at least one EPIYA motif occurrence and 3115 sequences with multiple repeats of the EPIYA motif. Although the EPIYA motif exists among broad species, it is significantly over-represented in some particular groups of species. For those proteins containing at least four copies of EPIYA motif, most of them are from intracellular bacteria, extracellular bacteria with T3SS or T4SS or intracellular protozoan parasites. By combining the EPIYA motif and the adjacent SH2 binding motifs (KK, R4, Tarp and Tir), we built HMMs of nine amino acids and predicted many potential effectors in bacteria and protista by the HMMs. Some potential effectors for pathogens (such as Lawsonia intracellularis, Plasmodium falciparum and Leishmania major) are suggested. Conclusions Our study indicates that the EPIYA motif may be a ubiquitous functional site for effectors that play an important pathogenicity role in mediating host-pathogen interactions. We suggest that some intracellular protozoan parasites could secrete EPIYA-motif containing effectors through secretion systems similar to the

Computational prediction of type III and IV secreted effectors in Gram-negative bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDermott, Jason E.; Corrigan, Abigail L.; Peterson, Elena S.

2011-01-01

In this review, we provide an overview of the methods employed by four recent papers that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. The results of the studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVEserver (http://www.biopilot.org). Finally, we assess the accuracy of the three type III effector prediction methods onmore » a small set of proteins not known prior to the development of these tools that we have recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif, and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.« less

A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta.

PubMed

Zhang, Lei; Davies, Laura J; Elling, Axel A

2015-01-01

Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-β-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Delivery of cytoplasmic and apoplastic effectors from Phytophthora infestans haustoria by distinct secretion pathways.

PubMed

Wang, Shumei; Boevink, Petra C; Welsh, Lydia; Zhang, Ruofang; Whisson, Stephen C; Birch, Paul R J

2017-10-01

The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease. We used transient Agrobacterium tumefaciens-mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection. The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively re-enter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well-characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells. Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Interferon-inducible effector mechanisms in cell-autonomous immunity

PubMed Central

MacMicking, John D.

2014-01-01

Interferons (IFNs) induce the expression of hundreds of genes as part of an elaborate antimicrobial programme designed to combat infection in all nucleated cells — a process termed cell-autonomous immunity. As described in this Review, recent genomic and subgenomic analyses have begun to assign functional properties to novel IFN-inducible effector proteins that restrict bacteria, protozoa and viruses in different subcellular compartments and at different stages of the pathogen life cycle. Several newly described host defence factors also participate in canonical oxidative and autophagic pathways by spatially coordinating their activities to enhance microbial killing. Together, these IFN-induced effector networks help to confer vertebrate host resistance to a vast and complex microbial world. PMID:22531325

Instability of Helios-deficient Tregs is associated with conversion to a T-effector phenotype and enhanced antitumor immunity.

PubMed

Nakagawa, Hidetoshi; Sido, Jessica M; Reyes, Edwin E; Kiers, Valerie; Cantor, Harvey; Kim, Hye-Jung

2016-05-31

Expression of the transcription factor Helios by Tregs ensures stable expression of a suppressive and anergic phenotype in the face of intense inflammatory responses, whereas Helios-deficient Tregs display diminished lineage stability, reduced FoxP3 expression, and production of proinflammatory cytokines. Here we report that selective Helios deficiency within CD4 Tregs leads to enhanced antitumor immunity through induction of an unstable phenotype and conversion of intratumoral Tregs into T effector cells within the tumor microenvironment. Induction of an unstable Treg phenotype is associated with enhanced production of proinflammatory cytokines by tumor-infiltrating but not systemic Tregs and significantly delayed tumor growth. Ab-dependent engagement of Treg surface receptors that result in Helios down-regulation also promotes conversion of intratumoral but not systemic Tregs into T effector cells and leads to enhanced antitumor immunity. These findings suggest that selective instability and conversion of intratumoral CD4 Tregs through genetic or Ab-based targeting of Helios may represent an effective approach to immunotherapy.

Pseudomonas syringae Type III Effector HopBB1 Promotes Host Transcriptional Repressor Degradation to Regulate Phytohormone Responses and Virulence.

PubMed

Yang, Li; Teixeira, Paulo José Pereira Lima; Biswas, Surojit; Finkel, Omri M; He, Yijian; Salas-Gonzalez, Isai; English, Marie E; Epple, Petra; Mieczkowski, Piotr; Dangl, Jeffery L

2017-02-08

Independently evolved pathogen effectors from three branches of life (ascomycete, eubacteria, and oomycete) converge onto the Arabidopsis TCP14 transcription factor to manipulate host defense. However, the mechanistic basis for defense control via TCP14 regulation is unknown. We demonstrate that TCP14 regulates the plant immune system by transcriptionally repressing a subset of the jasmonic acid (JA) hormone signaling outputs. A previously unstudied Pseudomonas syringae (Psy) type III effector, HopBB1, interacts with TCP14 and targets it to the SCF COI1 degradation complex by connecting it to the JA signaling repressor JAZ3. Consequently, HopBB1 de-represses the TCP14-regulated subset of JA response genes and promotes pathogen virulence. Thus, HopBB1 fine-tunes host phytohormone crosstalk by precisely manipulating part of the JA regulon to avoid pleiotropic host responses while promoting pathogen proliferation. Copyright © 2017 Elsevier Inc. All rights reserved.

Effector-independent motor sequence representations exist in extrinsic and intrinsic reference frames.

PubMed

Wiestler, Tobias; Waters-Metenier, Sheena; Diedrichsen, Jörn

2014-04-02

Many daily activities rely on the ability to produce meaningful sequences of movements. Motor sequences can be learned in an effector-specific fashion (such that benefits of training are restricted to the trained hand) or an effector-independent manner (meaning that learning also facilitates performance with the untrained hand). Effector-independent knowledge can be represented in extrinsic/world-centered or in intrinsic/body-centered coordinates. Here, we used functional magnetic resonance imaging (fMRI) and multivoxel pattern analysis to determine the distribution of intrinsic and extrinsic finger sequence representations across the human neocortex. Participants practiced four sequences with one hand for 4 d, and then performed these sequences during fMRI with both left and right hand. Between hands, these sequences were equivalent in extrinsic or intrinsic space, or were unrelated. In dorsal premotor cortex (PMd), we found that sequence-specific activity patterns correlated higher for extrinsic than for unrelated pairs, providing evidence for an extrinsic sequence representation. In contrast, primary sensory and motor cortices showed effector-independent representations in intrinsic space, with considerable overlap of the two reference frames in caudal PMd. These results suggest that effector-independent representations exist not only in world-centered, but also in body-centered coordinates, and that PMd may be involved in transforming sequential knowledge between the two. Moreover, although effector-independent sequence representations were found bilaterally, they were stronger in the hemisphere contralateral to the trained hand. This indicates that intermanual transfer relies on motor memories that are laid down during training in both hemispheres, but preferentially draws upon sequential knowledge represented in the trained hemisphere.

Effector-Independent Motor Sequence Representations Exist in Extrinsic and Intrinsic Reference Frames

PubMed Central

Wiestler, Tobias; Waters-Metenier, Sheena

2014-01-01

Many daily activities rely on the ability to produce meaningful sequences of movements. Motor sequences can be learned in an effector-specific fashion (such that benefits of training are restricted to the trained hand) or an effector-independent manner (meaning that learning also facilitates performance with the untrained hand). Effector-independent knowledge can be represented in extrinsic/world-centered or in intrinsic/body-centered coordinates. Here, we used functional magnetic resonance imaging (fMRI) and multivoxel pattern analysis to determine the distribution of intrinsic and extrinsic finger sequence representations across the human neocortex. Participants practiced four sequences with one hand for 4 d, and then performed these sequences during fMRI with both left and right hand. Between hands, these sequences were equivalent in extrinsic or intrinsic space, or were unrelated. In dorsal premotor cortex (PMd), we found that sequence-specific activity patterns correlated higher for extrinsic than for unrelated pairs, providing evidence for an extrinsic sequence representation. In contrast, primary sensory and motor cortices showed effector-independent representations in intrinsic space, with considerable overlap of the two reference frames in caudal PMd. These results suggest that effector-independent representations exist not only in world-centered, but also in body-centered coordinates, and that PMd may be involved in transforming sequential knowledge between the two. Moreover, although effector-independent sequence representations were found bilaterally, they were stronger in the hemisphere contralateral to the trained hand. This indicates that intermanual transfer relies on motor memories that are laid down during training in both hemispheres, but preferentially draws upon sequential knowledge represented in the trained hemisphere. PMID:24695723

Shallow subsurface structures and geotechnical characteristics of Tal El-Amarna area, middle Egypt

NASA Astrophysics Data System (ADS)

Toni, Mostafa; Hosny, Ahmed; Attia, Mohsen M.; Hassoup, Awad; El-Sharkawy, Amr

2013-12-01

The shallow seismic refraction profiling was carried out at 18 sites in Tal El-Amarna, which is a flat area on the eastern bank of the Nile River, 50 km south of El Minia Governorate, middle Egypt. The collected data are used to estimate the P-wave velocity and to delineate the near-surface ground model beneath the study area. This study is supported by the National Research Institute of Astronomy and Geophysics due to the historical interest of the Tal El-Amarna area as a famous tourist place where there exist many Pharaoh temples and tombs. This area is low seismically active, but it is probably of high vulnerability due to the influence of the local geological conditions on earthquake ground motion, as well as the presence of poor constructions in the absence of various issues such as building designs, quality of building materials, etc. Another dataset at the study area is obtained by multi-channel passive source (microtremor) measurements, which have been recorded at four arrays. The frequency-wavenumber (f-k) method was used to derive the dispersion curves from the raw signals at each array. The resulted dispersion curves were inverted using the neighborhood algorithm to obtain the shear and P-wave velocity models. The concluded Vs and Vp values provide a preliminary estimation of the geotechnical parameters and site classification for the shallow soil as they are of great interest in civil engineering applications.

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria.

PubMed

Ashida, Hiroshi; Sasakawa, Chihiro

2015-01-01

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria

PubMed Central

Ashida, Hiroshi; Sasakawa, Chihiro

2016-01-01

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections. PMID:26779450

Bcl-2 Allows Effector and Memory CD8+ T Cells To Tolerate Higher Expression of Bim

PubMed Central

Kurtulus, Sema; Tripathi, Pulak; Moreno-Fernandez, Maria E.; Sholl, Allyson; Katz, Jonathan D.; Grimes, H. Leighton; Hildeman, David A.

2014-01-01

As acute infections resolve, most effector CD8+ T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8+ T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8+ T cells reported to have a longer lifespan (i.e., KLRG1lowCD127high) have increased levels of Bcl-2 compared with their shorter-lived KLRG1highCD127low counterparts. Surprisingly, we found that these effector KLRG1lowCD127high CD8+ T cells also had increased levels of Bim compared with KLRG1highCD127low cells. Similar effects were observed in memory cells, in which CD8+ central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8+ effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8+ T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8+ T cells. Finally, we found that Bim levels were significantly increased in effector CD8+ T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate. PMID:21451108

An integrated control scheme for space robot after capturing non-cooperative target

NASA Astrophysics Data System (ADS)

Wang, Mingming; Luo, Jianjun; Yuan, Jianping; Walter, Ulrich

2018-06-01

How to identify the mass properties and eliminate the unknown angular momentum of space robotic system after capturing a non-cooperative target is of great challenge. This paper focuses on designing an integrated control framework which includes detumbling strategy, coordination control and parameter identification. Firstly, inverted and forward chain approaches are synthesized for space robot to obtain dynamic equation in operational space. Secondly, a detumbling strategy is introduced using elementary functions with normalized time, while the imposed end-effector constraints are considered. Next, a coordination control scheme for stabilizing both base and end-effector based on impedance control is implemented with the target's parameter uncertainty. With the measurements of the forces and torques exerted on the target, its mass properties are estimated during the detumbling process accordingly. Simulation results are presented using a 7 degree-of-freedom kinematically redundant space manipulator, which verifies the performance and effectiveness of the proposed method.

Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis.

PubMed

Abdelsamed, Hossam A; Moustaki, Ardiana; Fan, Yiping; Dogra, Pranay; Ghoneim, Hazem E; Zebley, Caitlin C; Triplett, Brandon M; Sekaly, Rafick-Pierre; Youngblood, Ben

2017-06-05

Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell-mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (T EM ), and longer-lived central memory (T CM ) and stem cell memory (T SCM ) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7- and IL-15-mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of T CM and T SCM memory cells resulted in phenotypic conversion into T EM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired T EM -associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells. © 2017 Abdelsamed et al.

Desirable cytolytic immune effector cell recruitment by interleukin-15 dendritic cells.

PubMed

Van Acker, Heleen H; Beretta, Ottavio; Anguille, Sébastien; De Caluwé, Lien; Papagna, Angela; Van den Bergh, Johan M; Willemen, Yannick; Goossens, Herman; Berneman, Zwi N; Van Tendeloo, Viggo F; Smits, Evelien L; Foti, Maria; Lion, Eva

2017-02-21

Success of dendritic cell (DC) therapy in treating malignancies is depending on the DC capacity to attract immune effector cells, considering their reciprocal crosstalk is partially regulated by cell-contact-dependent mechanisms. Although critical for therapeutic efficacy, immune cell recruitment is a largely overlooked aspect regarding optimization of DC vaccination. In this paper we have made a head-to-head comparison of interleukin (IL)-15-cultured DCs and conventional IL-4-cultured DCs with regard to their proficiency in the recruitment of (innate) immune effector cells. Here, we demonstrate that IL-4 DCs are suboptimal in attracting effector lymphocytes, while IL15 DCs provide a favorable chemokine milieu for recruiting CD8+ T cells, natural killer (NK) cells and gamma delta (γδ) T cells. Gene expression analysis revealed that IL-15 DCs exhibit a high expression of chemokines involved in antitumor immune effector cell attraction, while IL-4 DCs display a more immunoregulatory profile characterized by the expression of Th2 and regulatory T cell-attracting chemokines. This is confirmed by functional data indicating an enhanced recruitment of granzyme B+ effector lymphocytes by IL-15 DCs, as compared to IL-4 DCs, and subsequent superior killing of tumor cells by the migrated lymphocytes. Elevated CCL4 gene expression in IL-15 DCs and lowered CCR5 expression on both migrated γδ T cells and NK cells, led to validation of increased CCL4 secretion by IL15 DCs. Moreover, neutralization of CCR5 prior to migration resulted in an important inhibition of γδ T cell and NK cell recruitment by IL-15 DCs. These findings further underscore the strong immunotherapeutic potential of IL-15 DCs.

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

PubMed

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

[Genome-editing: focus on the off-target effects].

PubMed

He, Xiubin; Gu, Feng

2017-10-25

Breakthroughs of genome-editing in recent years have paved the way to develop new therapeutic strategies. These genome-editing tools mainly include Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases. However, off-target effects are still the major issue in genome editing, and limit the application in gene therapy. Here, we summarized the cause and compared different detection methods of off-targets.

Intrinsic disorder in pathogen effectors: protein flexibility as an evolutionary hallmark in a molecular arms race.

PubMed

Marín, Macarena; Uversky, Vladimir N; Ott, Thomas

2013-09-01

Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants' innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in long-disordered regions (>50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein-protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors.

Targeting Tumor Vasculature with TNF Leads Effector T Cells to the Tumor and Enhances Therapeutic Efficacy of Immune Checkpoint Blockers in Combination with Adoptive Cell Therapy.

PubMed

Elia, Angela Rita; Grioni, Matteo; Basso, Veronica; Curnis, Flavio; Freschi, Massimo; Corti, Angelo; Mondino, Anna; Bellone, Matteo

2018-05-01

Purpose: Irregular blood flow and endothelial cell anergy, which characterize many solid tumors, hinder tumor infiltration by cytotoxic T lymphocytes (CTL). This confers resistance to cancer immunotherapy with monoclonal antibodies directed against regulatory pathways in T lymphocytes (i.e., immune checkpoint blockade, ICB). We investigated whether NGR-TNF, a TNF derivative capable of targeting the tumor vasculature, and improving intratumor infiltration by activated CTLs, could sensitize tumors to ICB with antibodies specific for the PD-1 and CTLA-4 receptors. Experimental Design: Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with autochthonous prostate cancer and C57BL/6 mice with orthotopic B16 melanoma were treated with NGR-TNF, adoptive T-cell therapy (ACT), and ICB, and monitored for immune surveillance and disease progression. Results: The combination of ACT, NGR-TNF, and ICB was the most effective in delaying disease progression, and in improving overall survival of mice bearing ICB-resistant prostate cancer or melanoma. Mechanistically, the therapeutic effects were associated with potent tumor infiltration, especially by endogenous but also by adoptively transferred PD-1 + , granzyme B + , and interferon-γ + CTLs. The therapeutic effects were also associated with favorable T-effector/regulatory T cell ratios. Conclusions: Targeting the tumor vasculature with low-dose TNF in association with ACT may represent a novel strategy for enhancing T-cell infiltration in tumors and overcoming resistance to immune checkpoint blockers. Clin Cancer Res; 24(9); 2171-81. ©2018 AACR . ©2018 American Association for Cancer Research.

Testing promotes effector transfer.

PubMed

Boutin, Arnaud; Panzer, Stefan; Salesse, Robin N; Blandin, Yannick

2012-11-01

The retrieval of information from memory during testing has recently been shown to promote transfer in the verbal domain. Motor-related research, however, has ignored testing as a relevant method to enhance motor transfer. We thus investigated whether testing has the potential to induce generalised motor memories by favouring effector transfer. Participants were required to reproduce a spatial-temporal pattern of elbow extensions and flexions with their dominant right arm. We tested the ability of participants to transfer the original pattern (extrinsic transformation; i.e., goal-based configuration) or the mirrored pattern (intrinsic transformation; i.e., movement-based configuration) to the unpractised non-dominant left arm. To evaluate how testing affects motor transfer at 24-h testing, participants were either administered an initial testing session during early practice (early testing group) or shortly after the end of practice (late testing group; i.e., no alternation between practice and testing sessions). No initial testing session was completed for the control group. We found better effector transfer at 24-h testing for the early testing group for both extrinsic and intrinsic transformations of the movement pattern when compared with the control group, while no testing benefit was observed for the late testing group. This indicates that testing positively affects motor learning, yielding enhanced long-term transfer capabilities. We thus demonstrate the critical role of retrieval practice via testing during the process of motor memory encoding, and provide the conditions under which testing effectively contributes to the generalisation of motor memories. Copyright © 2012 Elsevier B.V. All rights reserved.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

PubMed

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Exact positioning of the robotic arm end effector

NASA Astrophysics Data System (ADS)

Korepanov, Valery; Dudkin, Fedir

2016-07-01

Orbital service becomes a new challenge of space exploration. The necessity to introduce it is connected first of all with an attractive opportunity to prolong the exploitation terms of expensive commercial satellites by, e.g., refilling of fuel or changing batteries. Other application area is a fight with permanently increasing amount of space litter - defunct satellites, burnt-out rocket stages, discarded trash and other debris. Now more than few tens of thousands orbiting objects larger than 5-10 cm (or about 1 million junks larger than 1 cm) are a huge problem for crucial and costly satellites and manned vehicles. For example, in 2014 the International Space Station had to change three times its orbit to avoid collision with space debris. So the development of the concepts and actions related to removal of space debris or non-operational satellites with use of robotic arm of a servicing satellite is very actual. Such a technology is also applicable for unmanned exploratory missions in solar system, for example for collecting a variety of samples from a celestial body surface. Naturally, the robotic arm movements should be controlled with great accuracy at influence of its non-rigidity, thermal and other factors. In these circumstances often the position of the arm end effector has to be controlled with high accuracy. The possibility of coordinate determination for the robotic arm end effector with use of a low frequency active electromagnetic system has been considered in the presented report. The proposed design of such a system consists of a small magnetic dipole source, which is mounted inside of the arm end effector and two or three 3-component magnetic field sensors mounted on a servicing satellite body. The data from this set of 3-component magnetic field sensors, which are fixed relatively to the satellite body, allows use of the mathematical approach for determination of position and orientation of the magnetic dipole source. The theoretical

Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector

PubMed Central

Caplan, Jeffrey L.; Mamillapalli, Padmavathi; Burch-Smith, Tessa M.; Czymmek, Kirk; Dinesh-Kumar, S.P.

2008-01-01

Summary Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50-kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a pre-recognition complex containing the p50 effector and NRIP1. PMID:18267075

TSC1 regulates the balance between effector and regulatory T cells.

PubMed

Park, Yoon; Jin, Hyung-Seung; Lopez, Justine; Elly, Chris; Kim, Gisen; Murai, Masako; Kronenberg, Mitchell; Liu, Yun-Cai

2013-12-01

Mammalian target of rapamycin (mTOR) plays a crucial role in the control of T cell fate determination; however, the precise regulatory mechanism of the mTOR pathway is not fully understood. We found that T cell-specific deletion of the gene encoding tuberous sclerosis 1 (TSC1), an upstream negative regulator of mTOR, resulted in augmented Th1 and Th17 differentiation and led to severe intestinal inflammation in a colitis model. Conditional Tsc1 deletion in Tregs impaired their suppressive activity and expression of the Treg marker Foxp3 and resulted in increased IL-17 production under inflammatory conditions. A fate-mapping study revealed that Tsc1-null Tregs that lost Foxp3 expression gained a stronger effector-like phenotype compared with Tsc1-/- Foxp3+ Tregs. Elevated IL-17 production in Tsc1-/- Treg cells was reversed by in vivo knockdown of the mTOR target S6K1. Moreover, IL-17 production was enhanced by Treg-specific double deletion of Tsc1 and Foxo3a. Collectively, these studies suggest that TSC1 acts as an important checkpoint for maintaining immune homeostasis by regulating cell fate determination.

Go in for the kill: How plants deploy effector-triggered immunity to combat pathogens. [Corrected].

PubMed

Wu, Liang; Chen, Huan; Curtis, Chad; Fu, Zheng Qing

2014-01-01

Plant resistance (R) proteins perceive specific pathogen effectors from diverse plant pathogens to initiate defense responses, designated effector-triggered immunity (ETI). Plant R proteins are mostly nucleotide binding-leucine rich repeat (NB-LRR) proteins, which recognize pathogen effectors directly or indirectly through sophisticated mechanisms. Upon activation by effector proteins, R proteins elicit robust defense responses, including a rapid burst of reactive oxygen species (ROS), induced biosynthesis and accumulation of salicylic acid (SA), a rapid programmed cell death (PCD) called hypersensitive response (HR) at the infection sites, and increased expression of pathogenesis-related (PR) genes. Initiation of ETI is correlated with a complex network of defense signaling pathways, resulting in defensive cellular responses and large-scale transcriptional reprogramming events. In this review, we highlight important recent advances on the recognition of effectors, regulation and activation of plant R proteins, dynamic intracellular trafficking of R proteins, induction of cell death, and transcriptional reprogramming associated with ETI. Current knowledge gaps and future research directions are also discussed in this review.

Genome editing and the next generation of antiviral therapy

PubMed Central

Stone, Daniel; Niyonzima, Nixon

2016-01-01

Engineered endonucleases such as homing endonucleases (HEs), zinc finger nucleases (ZFNs), Tal-effector nucleases (TALENS) and the RNA-guided engineered nucleases (RGENs or CRISPR/Cas9) can target specific DNA sequences for cleavage, and are proving to be valuable tools for gene editing. Recently engineered endonucleases have shown great promise as therapeutics for the treatment of genetic disease and infectious pathogens. In this review, we discuss recent efforts to use the HE, ZFN, TALEN and CRISPR/Cas9 gene-editing platforms as antiviral therapeutics. We also discuss the obstacles facing gene-editing antiviral therapeutics as they are tested in animal models of disease and transition towards human application. PMID:27272125

Joint-space adaptive control of a 6 DOF end-effector with closed-kinematic chain mechanism

NASA Technical Reports Server (NTRS)

Nguyen, Charles C.; Zhou, Zhen-Lei

1989-01-01

The development is presented for a joint-space adaptive scheme that controls the joint position of a six-degree-of-freedom (DOF) robot end-effector performing fine and precise motion within a very limited workspace. The end-effector was built to study autonomous assembly of NASA hardware in space. The design of the adaptive controller is based on the concept of model reference adaptive control (MRAC) and Lyapunov direct method. In the development, it is assumed that the end-effector performs slowly varying motion. Computer simulation is performed to investigate the performance of the developed control scheme on position control of the end-effector. Simulation results manifest that the adaptive control scheme provides excellent tracking of several test paths.

Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine-learning approach.

PubMed

Teper, Doron; Burstein, David; Salomon, Dor; Gershovitz, Michael; Pupko, Tal; Sessa, Guido

2016-04-01

The Gram-negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85-10 strain by various experimental and computational techniques. To identify additional Xcv 85-10 effectors, we applied a genome-wide machine-learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)-dependent expression, 5' regulatory sequences, amino acid composition bias and GC content. Thirty-six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)-inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild-type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild-type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth. © 2015 BSPP and John Wiley & Sons Ltd.

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene

PubMed Central

Lilley, Catherine J.; Maqbool, Abbas; Wu, Duqing; Yusup, Hazijah B.; Jones, Laura M.; Birch, Paul R. J.; Urwin, Peter E.

2018-01-01

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function. PMID:29641602

In-flight adaptive performance optimization (APO) control using redundant control effectors of an aircraft

NASA Technical Reports Server (NTRS)

Gilyard, Glenn B. (Inventor)

1999-01-01

Practical application of real-time (or near real-time) Adaptive Performance Optimization (APO) is provided for a transport aircraft in steady climb, cruise, turn descent or other flight conditions based on measurements and calculations of incremental drag from a forced response maneuver of one or more redundant control effectors defined as those in excess of the minimum set of control effectors required to maintain the steady flight condition in progress. The method comprises the steps of applying excitation in a raised-cosine form over an interval of from 100 to 500 sec. at the rate of 1 to 10 sets/sec of excitation, and data for analysis is gathered in sets of measurements made during the excitation to calculate lift and drag coefficients C.sub.L and C.sub.D from two equations, one for each coefficient. A third equation is an expansion of C.sub.D as a function of parasitic drag, induced drag, Mach and altitude drag effects, and control effector drag, and assumes a quadratic variation of drag with positions .delta..sub.i of redundant control effectors i=1 to n. The third equation is then solved for .delta..sub.iopt the optimal position of redundant control effector i, which is then used to set the control effector i for optimum performance during the remainder of said steady flight or until monitored flight conditions change by some predetermined amount as determined automatically or a predetermined minimum flight time has elapsed.

Wind Tunnel Test of an RPV with Shape-Change Control Effector and Sensor Arrays

NASA Technical Reports Server (NTRS)

Raney, David L.; Cabell, Randolph H.; Sloan, Adam R.; Barnwell, William G.; Lion, S. Todd; Hautamaki, Bret A.

2004-01-01

A variety of novel control effector concepts have recently emerged that may enable new approaches to flight control. In particular, the potential exists to shift the composition of the typical aircraft control effector suite from a small number of high authority, specialized devices (rudder, aileron, elevator, flaps), toward larger numbers of smaller, less specialized, distributed device arrays. The concept envisions effector and sensor networks composed of relatively small high-bandwidth devices able to simultaneously perform a variety of control functions using feedback from disparate data sources. To investigate this concept, a remotely piloted flight vehicle has been equipped with an array of 24 trailing edge shape-change effectors and associated pressure measurements. The vehicle, called the Multifunctional Effector and Sensor Array (MESA) testbed, was recently tested in NASA Langley's 12-ft Low Speed wind tunnel to characterize its stability properties, control authorities, and distributed pressure sensitivities for use in a dynamic simulation prior to flight testing. Another objective was to implement and evaluate a scheme for actively controlling the spanwise pressure distribution using the shape-change array. This report describes the MESA testbed, design of the pressure distribution controller, and results of the wind tunnel test.

The role of effectors of biotrophic and hemibiotrophic fungi in infection.

PubMed

Koeck, Markus; Hardham, Adrienne R; Dodds, Peter N

2011-12-01

Biotrophic and hemibiotrophic fungi are successful groups of plant pathogens that require living plant tissue to survive and complete their life cycle. Members of these groups include the rust fungi and powdery mildews and species in the Ustilago, Cladosporium and Magnaporthe genera. Collectively, they represent some of the most destructive plant parasites, causing huge economic losses and threatening global food security. During plant infection, pathogens synthesize and secrete effector proteins, some of which are translocated into the plant cytosol where they can alter the host's response to the invading pathogen. In a successful infection, pathogen effectors facilitate suppression of the plant's immune system and orchestrate the reprogramming of the infected tissue so that it becomes a source of nutrients that are required by the pathogen to support its growth and development. This review summarizes our current understanding of the function of fungal effectors in infection. © 2011 Blackwell Publishing Ltd.

Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

PubMed Central

Catz, Sergio Daniel

2013-01-01

The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

Prediction of Ras-effector interactions using position energy matrices.

PubMed

Kiel, Christina; Serrano, Luis

2007-09-01

One of the more challenging problems in biology is to determine the cellular protein interaction network. Progress has been made to predict protein-protein interactions based on structural information, assuming that structural similar proteins interact in a similar way. In a previous publication, we have determined a genome-wide Ras-effector interaction network based on homology models, with a high accuracy of predicting binding and non-binding domains. However, for a prediction on a genome-wide scale, homology modelling is a time-consuming process. Therefore, we here successfully developed a faster method using position energy matrices, where based on different Ras-effector X-ray template structures, all amino acids in the effector binding domain are sequentially mutated to all other amino acid residues and the effect on binding energy is calculated. Those pre-calculated matrices can then be used to score for binding any Ras or effector sequences. Based on position energy matrices, the sequences of putative Ras-binding domains can be scanned quickly to calculate an energy sum value. By calibrating energy sum values using quantitative experimental binding data, thresholds can be defined and thus non-binding domains can be excluded quickly. Sequences which have energy sum values above this threshold are considered to be potential binding domains, and could be further analysed using homology modelling. This prediction method could be applied to other protein families sharing conserved interaction types, in order to determine in a fast way large scale cellular protein interaction networks. Thus, it could have an important impact on future in silico structural genomics approaches, in particular with regard to increasing structural proteomics efforts, aiming to determine all possible domain folds and interaction types. All matrices are deposited in the ADAN database (http://adan-embl.ibmc.umh.es/). Supplementary data are available at Bioinformatics online.

Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants.

PubMed

Anderson, Ryan G; Casady, Megan S; Fee, Rachel A; Vaughan, Martha M; Deb, Devdutta; Fedkenheuer, Kevin; Huffaker, Alisa; Schmelz, Eric A; Tyler, Brett M; McDowell, John M

2012-12-01

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR-like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non-pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's host. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Julie Anne Roden, Branids Belt, Jason Barzel Ross, Thomas Tachibana, Joe Vargas, Mary Beth Mudgett

2004-11-23

The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resultedmore » in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.« less

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

PubMed

Kale, Shiv D; Gu, Biao; Capelluto, Daniel G S; Dou, Daolong; Feldman, Emily; Rumore, Amanda; Arredondo, Felipe D; Hanlon, Regina; Fudal, Isabelle; Rouxel, Thierry; Lawrence, Christopher B; Shan, Weixing; Tyler, Brett M

2010-07-23

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues. Copyright 2010 Elsevier Inc. All rights reserved.

Factors determining electrostatic fields in molecular dynamics simulations of the Ras/effector interface.

PubMed

Ensign, Daniel L; Webb, Lauren J

2011-12-01

Using molecular dynamics simulations, we explore geometric and physical factors contributing to calculated electrostatic fields at the binding surface of the GTPase Ras with a spectroscopically labeled variant of a downstream effector, the Ras-binding domain of Ral guanine nucleotide dissociation stimulator (RalGDS). A related system (differing by mutation of one amino acid) has been studied in our group using vibrational Stark effect spectroscopy, a technique sensitive to electrostatic fields. Electrostatic fields were computed using the AMBER 2003 force field and averaged over snapshots from molecular dynamics simulation. We investigate geometric factors by exploring how the orientation of the spectroscopic probe changes on Ras-effector binding. In addition, we explore the physical origin of electrostatic fields at our spectroscopic probe by comparing contributions to the field from discrete components of the system, such as explicit solvent, residues on the Ras surface, and residues on the RalGDS surface. These models support our experimental hypothesis that vibrational Stark shifts are caused by Ras binding to its effector and not the structural rearrangements of the effector surface or probe reorientation on Ras-effector binding, for at least some of our experimental probes. These calculations provide physical insight into the origin, magnitude, and importance of electrostatic fields in protein-protein interactions and suggest new experiments to probe the field's role in protein docking. Copyright © 2011 Wiley-Liss, Inc.

Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

PubMed

Joseph, Thomas T; Osman, Roman

2012-01-01

In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of

High immunosuppressive burden in advanced hepatocellular carcinoma patients: Can effector functions be restored?

PubMed

Lugade, Amit A; Kalathil, Suresh; Miller, Austin; Iyer, Renuka; Thanavala, Yasmin

2013-07-01

The accumulation of immunosuppressive cells and exhausted effector T cells highlight an important immune dysfunction in advanced stage hepatocellular carcinoma (HCC) patients. These cells significantly hamper the efficacy immunotherapies and facilitate HCC progression. We have recently demonstrated that the multipronged depletion of immunosuppressive cells potentially restores effector T-cell function in HCC.

The pore-forming bacterial effector, VopQ, halts autophagic turnover.

PubMed

Sreelatha, Anju; Orth, Kim; Starai, Vincent J

2013-12-01

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the Vo domain of the conserved vacuolar H(+)-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents. These studies show how a bacterial pathogen can compromise host ion potentials using a gated pore-forming effector to equilibrate levels of small molecules found in endolysosomal compartments and disrupt cellular processes such as autophagy.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells*

PubMed Central

Ali, Ramadan A.; Camick, Christina; Wiles, Katherine; Walseth, Timothy F.; Slama, James T.; Bhattacharya, Sumit; Giovannucci, David R.; Wall, Katherine A.

2016-01-01

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca2+ mobilizing second messenger discovered to date, has been implicated in Ca2+ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca2+ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca2+ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca2+ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca2+ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. PMID:26728458

Effector stage CC chemokine receptor-1 selective antagonism reduces multiple sclerosis-like rat disease.

PubMed

Eltayeb, Sana; Sunnemark, Dan; Berg, Anna-Lena; Nordvall, Gunnar; Malmberg, Asa; Lassmann, Hans; Wallström, Erik; Olsson, Tomas; Ericsson-Dahlstrand, Anders

2003-09-01

We have studied the role of the chemokine receptor CCR1 during the effector stage of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in DA rats. In situ hybridization histochemistry revealed local production of the CCR1 ligands CCL3 (MIP-1 alpha) and CCL5 (RANTES), as well as large numbers of CCR1 and CCR5 expressing cells within inflammatory brain lesions. A low-molecular weight CCR1 selective antagonist potently abrogated both clinical and histopathological disease signs during a 5-day treatment period, without signs of peripheral immune compromise. Thus, we demonstrate therapeutic targeting of CCR1-dependent leukocyte recruitment to the central nervous system in a multiple sclerosis (MS)-like rat model.

A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

PubMed

Zhang, Meixiang; Ahmed Rajput, Nasir; Shen, Danyu; Sun, Peng; Zeng, Wentao; Liu, Tingli; Juma Mafurah, Joseph; Dou, Daolong

2015-06-03

Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

PubMed Central

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-01-01

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

Molecular pathways: targeting RAC-p21-activated serine-threonine kinase signaling in RAS-driven cancers.

PubMed

Baker, Nicole M; Yee Chow, Hoi; Chernoff, Jonathan; Der, Channing J

2014-09-15

Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers. ©2014 American Association for Cancer Research.

Role of Rab family GTPases and their effectors in melanosomal logistics.

PubMed

Ohbayashi, Norihiko; Fukuda, Mitsunori

2012-04-01

Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

PubMed

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Sideritis romana L. subsp. purpurea (Tal. ex Benth.) Heywood, a new chemotype from Montenegro.

PubMed

Garzoli, Stefania; Božović, Mijat; Baldisserotto, Anna; Andreotti, Elisa; Pepi, Federico; Tadić, Vanja; Manfredini, Stefano; Ragno, Rino

2018-05-01

A study on essential oil fractions of the Western Balkan endemic Sideritis romana L. subsp. purpurea (Tal. ex Benth.) Heywood collected in Montenegro is reported. The 24-h systematic steam distillation extraction procedure was performed. The gas chromatographic/mass spectrometric (GC/MS) analysis of the fractions showed γ-elemene and spathulenol as two main constituents, revealing a new chemotype of this plant species. Although varying in the content of these two main compounds, which makes the fractions quite different between each other, evaluation of the anti-Candida activity showed the lack of any significant efficacy.

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

PubMed Central

Redkar, Amey; Hoser, Rafal; Schilling, Lena; Zechmann, Bernd; Krzymowska, Magdalena; Walbot, Virginia; Doehlemann, Gunther

2015-01-01

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. PMID:25888589

Silicon protects soybean plants against Phytophthora sojae by interfering with effector-receptor expression.

PubMed

Rasoolizadeh, Aliyeh; Labbé, Caroline; Sonah, Humira; Deshmukh, Rupesh K; Belzile, François; Menzies, James G; Bélanger, Richard R

2018-05-30

Silicon (Si) is known to protect against biotrophic and hemibiotrophic plant pathogens; however, the mechanisms by which it exerts its prophylactic role remain unknown. In an attempt to obtain unique insights into the mode of action of Si, we conducted a full comparative transcriptomic analysis of soybean (Glycine max) plants and Phytophthora sojae, a hemibiotroph that relies heavily on effectors for its virulence. Supplying Si to inoculated plants provided a strong protection against P. sojae over the course of the experiment (21 day). Our results showed that the response of Si-free (Si - ) plants to inoculation was characterized early (4 dpi) by a high expression of defense-related genes, including plant receptors, which receded over time as the pathogen progressed into the roots. The infection was synchronized with a high expression of effectors by P. sojae, the nature of which changed over time. By contrast, the transcriptomic response of Si-fed (Si + ) plants was remarkably unaffected by the presence of P. sojae, and the expression of effector-coding genes by the pathogen was significantly reduced. Given that the apoplast is a key site of interaction between effectors and plant defenses and receptors in the soybean-P. sojae complex, as well as the site of amorphous-Si accumulation, our results indicate that Si likely interferes with the signaling network between P. sojae and the plant, preventing or decreasing the release of effectors reaching plant receptors, thus creating a form of incompatible interaction.

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen

Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or asmore » enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.« less

Learning-based position control of a closed-kinematic chain robot end-effector

NASA Technical Reports Server (NTRS)

Nguyen, Charles C.; Zhou, Zhen-Lei

1990-01-01

A trajectory control scheme whose design is based on learning theory, for a six-degree-of-freedom (DOF) robot end-effector built to study robotic assembly of NASA hardwares in space is presented. The control scheme consists of two control systems: the feedback control system and the learning control system. The feedback control system is designed using the concept of linearization about a selected operating point, and the method of pole placement so that the closed-loop linearized system is stabilized. The learning control scheme consisting of PD-type learning controllers, provides additional inputs to improve the end-effector performance after each trial. Experimental studies performed on a 2 DOF end-effector built at CUA, for three tracking cases show that actual trajectories approach desired trajectories as the number of trials increases. The tracking errors are substantially reduced after only five trials.

Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens.

PubMed

Vleeshouwers, Vivianne G A A; Oliver, Richard P

2014-03-01

One of most important challenges in plant breeding is improving resistance to the plethora of pathogens that threaten our crops. The ever-growing world population, changing pathogen populations, and fungicide resistance issues have increased the urgency of this task. In addition to a vital inflow of novel resistance sources into breeding programs, the functional characterization and deployment of resistance also needs improvement. Therefore, plant breeders need to adopt new strategies and techniques. In modern resistance breeding, effectors are emerging as tools to accelerate and improve the identification, functional characterization, and deployment of resistance genes. Since genome-wide catalogues of effectors have become available for various pathogens, including biotrophs as well as necrotrophs, effector-assisted breeding has been shown to be successful for various crops. "Effectoromics" has contributed to classical resistance breeding as well as for genetically modified approaches. Here, we present an overview of how effector-assisted breeding and deployment is being exploited for various pathosystems.

Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors.

PubMed

Kast, David J; Yang, Changsong; Disanza, Andrea; Boczkowska, Malgorzata; Madasu, Yadaiah; Scita, Giorgio; Svitkina, Tatyana; Dominguez, Roberto

2014-04-01

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

Bacterial effector HopF2 interacts with AvrPto and suppresses Arabidopsis innate immunity at the plasma membrane

USDA-ARS?s Scientific Manuscript database

Plant pathogenic bacteria inject a cocktail of effector proteins into host plant cells to modulate the host immune response, thereby promoting pathogenicity. How or whether these effectors work cooperatively is largely unknown. The Pseudomonas syringae DC3000 effector HopF2 suppresses the host plan...

Vaccinating for natural killer cell effector functions.

PubMed

Wagstaffe, Helen R; Mooney, Jason P; Riley, Eleanor M; Goodier, Martin R

2018-01-01

Vaccination has proved to be highly effective in reducing global mortality and eliminating infectious diseases. Building on this success will depend on the development of new and improved vaccines, new methods to determine efficacy and optimum dosing and new or refined adjuvant systems. NK cells are innate lymphoid cells that respond rapidly during primary infection but also have adaptive characteristics enabling them to integrate innate and acquired immune responses. NK cells are activated after vaccination against pathogens including influenza, yellow fever and tuberculosis, and their subsequent maturation, proliferation and effector function is dependent on myeloid accessory cell-derived cytokines such as IL-12, IL-18 and type I interferons. Activation of antigen-presenting cells by live attenuated or whole inactivated vaccines, or by the use of adjuvants, leads to enhanced and sustained NK cell activity, which in turn contributes to T cell recruitment and memory cell formation. This review explores the role of cytokine-activated NK cells as vaccine-induced effector cells and in recall responses and their potential contribution to vaccine and adjuvant development.

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

PubMed

Jefferis, R; Lund, J; Pound, J D

1998-06-01

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3.

PubMed

Esposito, Diego; Günster, Regina A; Martino, Luigi; El Omari, Kamel; Wagner, Armin; Thurston, Teresa L M; Rittinger, Katrin

2018-04-06

The Salmonella -secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N -acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating D X D motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N -acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N -glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N -glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors. © 2018 Esposito et al.

The Xanthomonas campestris type III effector XopJ proteolytically degrades proteasome subunit RPT6.

PubMed

Üstün, Suayib; Börnke, Frederik

2015-05-01

Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. © 2015 American Society of Plant Biologists. All Rights Reserved.

Effector Gene Suites in Some Soil Isolates of Fusarium oxysporum Are Not Sufficient Predictors of Vascular Wilt in Tomato.

PubMed

Jelinski, Nicolas A; Broz, Karen; Jonkers, Wilfried; Ma, Li-Jun; Kistler, H Corby

2017-07-01

Seventy-four Fusarium oxysporum soil isolates were assayed for known effector genes present in an F. oxysporum f. sp. lycopersici race 3 tomato wilt strain (FOL MN-25) obtained from the same fields in Manatee County, Florida. Based on the presence or absence of these genes, four haplotypes were defined, two of which represented 96% of the surveyed isolates. These two most common effector haplotypes contained either all or none of the assayed race 3 effector genes. We hypothesized that soil isolates with all surveyed effector genes, similar to FOL MN-25, would be pathogenic toward tomato, whereas isolates lacking all effectors would be nonpathogenic. However, inoculation experiments revealed that presence of the effector genes alone was not sufficient to ensure pathogenicity on tomato. Interestingly, a nonpathogenic isolate containing the full suite of unmutated effector genes (FOS 4-4) appears to have undergone a chromosomal rearrangement yet remains vegetatively compatible with FOL MN-25. These observations confirm the highly dynamic nature of the F. oxysporum genome and support the conclusion that pathogenesis among free-living populations of F. oxysporum is a complex process. Therefore, the presence of effector genes alone may not be an accurate predictor of pathogenicity among soil isolates of F. oxysporum.

Putative terminator and/or effector functions of Arf GAPs in the trafficking of clathrin-coated vesicles.

PubMed

Kon, Shunsuke; Funaki, Tomo; Satake, Masanobu

2011-05-01

The role of ArfGAP1 as a terminator or effector in COPi-vesicle formation has been the subject of ongoing discussions. Here, the discussion on the putative terminator/effector functions has been enlarged to include Arf GAP members involved in the formation of clathrin-coated vesicles. ACAP1, whose role has been studied extensively, enhances the recycling of endocytosed proteins to the plasma membrane. Importantly, this positive role appears to be an overall reflection of both the terminator and effector activities attributed to ACAP1. Other Arf GAP subtypes have also been suggested to possess both terminator and effector activities. Interestingly, while most Arf GAP proteins regulate membrane trafficking by acting as facilitators, a few Arf GAP subtypes act as inhibitors.

Fast Evolution and Lineage-Specific Gene Family Expansions of Aphid Salivary Effectors Driven by Interactions with Host-Plants.

PubMed

Boulain, Hélène; Legeai, Fabrice; Guy, Endrick; Morlière, Stéphanie; Douglas, Nadine E; Oh, Jonghee; Murugan, Marimuthu; Smith, Michael; Jaquiéry, Julie; Peccoud, Jean; White, Frank F; Carolan, James C; Simon, Jean-Christophe; Sugio, Akiko

2018-05-18

Effector proteins play crucial roles in plant-parasite interactions by suppressing plant defenses and hijacking plant physiological responses to facilitate parasite invasion and propagation. Although effector proteins have been characterized in many microbial plant pathogens, their nature and role in adaptation to host plants are largely unknown in insect herbivores. Aphids rely on salivary effector proteins injected into the host plants to promote phloem sap uptake. Therefore, gaining insight into the repertoire and evolution of aphid effectors is key to unveiling the mechanisms responsible for aphid virulence and host plant specialization. With this aim in mind, we assembled catalogues of putative effectors in the legume specialist aphid, Acyrthosiphon pisum, using transcriptomics and proteomics approaches. We identified 3603 candidate effector genes predicted to be expressed in A. pisum salivary glands (SGs), and 740 of which displayed up-regulated expression in SGs in comparison to the alimentary tract. A search for orthologs in 17 arthropod genomes revealed that SG-up-regulated effector candidates of A. pisum are enriched in aphid-specific genes and tend to evolve faster compared to the whole gene set. We also found that a large fraction of proteins detected in the A. pisum saliva belonged to three gene families, of which certain members show evidence consistent with positive selection. Overall, this comprehensive analysis suggests that the large repertoire of effector candidates in A. pisum constitutes a source of novelties promoting plant adaptation to legumes.