Plant-microbe interaction
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Activity Profiling of Vacuolar Processing Enzymes reveals a role for VPE during oomycete infection - Misas-Villamil - The Plant Journal - Wiley Online Library

Activity Profiling of Vacuolar Processing Enzymes reveals a role for VPE during oomycete infection - Misas-Villamil - The Plant Journal - Wiley Online Library | Plant-microbe interaction | Scoop.it

Summary
Vacuolar Processing Enzymes (VPEs) are important cysteine proteases implicated in maturation of e.g. seed storage proteins and involved in programmed cell death during plant-microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs and labeling is activity dependent, illustrated by sensitivity for inhibitors, pH and reducing agent. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in different tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role of VPE during compatible interactions that is presumably independent from programmed cell death. Our data indicate that as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.

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Plant-microbe interaction
Current research on plant immunity, effector proteins, the proteasome and autophagy
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The proteasome acts as a hub for plant immunity and is targeted by Pseudomonas type-III effectors

The proteasome acts as a hub for plant immunity and is targeted by Pseudomonas type-III effectors | Plant-microbe interaction | Scoop.it
Recent evidence suggests that the ubiquitin-proteasome system (UPS) is involved in several aspects of plant immunity and a range of plant pathogens subvert the UPS to enhance their virulence. Here we show that proteasome activity is strongly induced during basal defense in Arabidopsis. Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas syringae pv. maculicola ES4326. Both proteasome subunits are required for Pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic-acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR. In addition, we show that Pst inhibits proteasome activity in a type-III secretion dependent manner. A screen for type-III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1 and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass-spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome leading to its inhibition. Thus, the proteasome is an essential component of PTI and SAR, which is targeted by multiple bacterial effectors.
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MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity

MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity | Plant-microbe interaction | Scoop.it
Background

Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.
Results

Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.
Conclusions

By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

Via Christophe Jacquet
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Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types During Infection

Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types During Infection | Plant-microbe interaction | Scoop.it
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 utilized 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.
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Receptor oligomerization guides pathway choice between proteasomal and autophagic degradation : Nature Cell Biology : Nature Research

Receptor oligomerization guides pathway choice between proteasomal and autophagic degradation : Nature Cell Biology : Nature Research | Plant-microbe interaction | Scoop.it

Abnormal or aggregated proteins have a strong cytotoxic potential and are causative for human disorders such as Alzheimer’s, Parkinson’s, Huntington’s disease and amyotrophic lateral sclerosis. If not restored by molecular chaperones, abnormal proteins are typically degraded by proteasomes or eliminated by selective autophagy. The discovery that both pathways are initiated by substrate ubiquitylation but utilize different ubiquitin receptors incited a debate over how pathway choice is achieved. Here, we demonstrate in yeast that pathway choice is made after substrate ubiquitylation by competing ubiquitin receptors harbouring either proteasome- or autophagy-related protein 8 (Atg8/LC3)-binding modules. Proteasome pathway receptors bind ubiquitin moieties more efficiently, but autophagy receptors gain the upper hand following substrate aggregation and receptor bundling. Indeed, by using sets of modular artificial receptors harbouring identical ubiquitin-binding modules we found that proteasome/autophagy pathway choice is independent of the ubiquitin-binding properties of the receptors but largely determined by their oligomerization potentials. Our work thus suggests that proteasomal degradation and selective autophagy are two branches of an adaptive protein quality control pathway, which uses substrate ubiquitylation as a shared degradation signal.

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Frontiers | A Proteomic Approach Suggests Unbalanced Proteasome Functioning Induced by the Growth-Promoting Bacterium Kosakonia radicincitans in Arabidopsis | Plant Science

Frontiers | A Proteomic Approach Suggests Unbalanced Proteasome Functioning Induced by the Growth-Promoting Bacterium Kosakonia radicincitans in Arabidopsis | Plant Science | Plant-microbe interaction | Scoop.it
Endophytic plant growth-promoting bacteria have significant impact on the plant physiology and understanding this interaction at the molecular level is of particular interest to support crop productivity and sustainable production systems. We used a proteomics approach to investigate the molecular mechanisms underlying plant growth promotion in the interaction of Kosakonia radicincitans DSM 16656 with Arabidopsis thaliana. Four weeks after the inoculation, the proteome of roots from inoculated and control plants was compared using two-dimensional gel electrophoresis and differentially abundant protein spots were identified by liquid chromatography tandem mass spectrometry. Twelve protein spots were responsive to the inoculation, with the majority of them being related to cellular stress reactions. The protein expression of 20S proteasome alpha-3 subunit was increased by the presence of K. radicincitans. Determination of proteasome activity and immuno blotting analysis for ubiquitinated proteins revealed that endophytic colonisation interferes with ubiquitin-dependent protein degradation. Inoculation of rpn12a, defective in a 26S proteasome regulatory particle, enhanced the growth-promoting effect. This indicates that the plant proteasome, besides being a known target for plant pathogenic bacteria, is involved in the establishment of beneficial interactions of microorganisms with plants.
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Arabidopsis leucine-rich repeat receptor–like kinase NILR1 is required for induction of innate immunity to parasitic nematodes

Arabidopsis leucine-rich repeat receptor–like kinase NILR1 is required for induction of innate immunity to parasitic nematodes | Plant-microbe interaction | Scoop.it
Plant-parasitic nematodes are destructive pests causing losses of billions of dollars annually. An effective plant defence against pathogens relies on the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localised receptors leading to the activation of PAMP-triggered immunity (PTI). Extensive studies have been conducted to characterise the role of PTI in various models of plant-pathogen interactions. However, far less is known about the role of PTI in roots in general and in plant-nematode interactions in particular. Here we show that nematode-derived proteinaceous elicitor/s is/are capable of inducing PTI in Arabidopsis in a manner dependent on the common immune co-receptor BAK1. Consistent with the role played by BAK1, we identified a leucine-rich repeat receptor-like kinase, termed NILR1 that is specifically regulated upon infection by nematodes. We show that NILR1 is essential for PTI responses initiated by nematodes and nilr1 loss-of-function mutants are hypersusceptible to a broad category of nematodes. To our knowledge, NILR1 is the first example of an immune receptor that is involved in induction of basal immunity (PTI) in plants or in animals in response to nematodes. Manipulation of NILR1 will provide new options for nematode control in crop plants in future.
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SINAT E3 Ligases Control the Light-Mediated Stability of the Brassinosteroid-Activated Transcription Factor BES1 in Arabidopsis

SINAT E3 Ligases Control the Light-Mediated Stability of the Brassinosteroid-Activated Transcription Factor BES1 in Arabidopsis | Plant-microbe interaction | Scoop.it
The plant hormones brassinosteroids (BRs) participate in light-mediated regulation of plant growth, although the underlying mechanisms are far from being fully understood. In addition, the function of the core transcription factor in the BR signaling pathway, BRI1-EMS-SUPPRESSOR 1 (BES1), largely depends on its phosphorylation status and its protein stability, but the regulation of BES1 is not well understood. Here, we report that SINA of Arabidopsis thaliana (SINATs) specifically interact with dephosphorylated BES1 and mediate its ubiquitination and degradation. Our genetic data demonstrated that SINATs inhibit BR signaling in a BES1-dependent manner. Interestingly, we found that the protein levels of SINATs were decreased in the dark and increased in the light, which changed BES1 protein levels accordingly. Thus, our study not only uncovered a new mechanism of BES1 degradation but also provides significant insights into how light conditionally regulates plant growth through controlling accumulation of different forms of BES1.
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Ten simple rules for considering preprints

So why make your work available as preprints? There are perceived positives and negatives to disclosing scientific work in the form of a preprint, explored here in the form of 10 Simple Rules. These rules, if they pass review, will appear as part of the PLOS Computational Biology Ten Simple Rules Collection. The rules cover such issues as reward, incentives, speed of dissemination, quality, scooping, and record of priority. You cannot have an article describing preprints, without itself being a preprint!!
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Bodyguards: Pathogen-Derived Decoys That Protect Virulence Factors

Bodyguards: Pathogen-Derived Decoys That Protect Virulence Factors | Plant-microbe interaction | Scoop.it
Recent studies on plant-pathogen interactions have exposed a new strategy used by plant pathogens: decoy effectors that protect virulence factors. Examples of these “bodyguards” include the recently discovered PsXLP1 from Phytophthora sojae and truncated TALEs from Xanthomonas oryzae. These examples suggest important roles for seemingly non-functional effector proteins in distracting the host.
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CALCIUM-DEPENDENT PROTEIN KINASE5 associates with the truncated NLR protein TIR-NBS2 to contribute to exo70B1-mediated immunity

Calcium-dependent protein kinases (CPKs) function as calcium sensors and play important roles in plant immunity. Loss of function of the exocyst complex subunit EXO70B1 leads to autoimmunity caused by activation of TN2, a truncated Toll/interleukin-1 receptor-nucleotide-binding sequence protein (TIR-NBS). Here we show, based on a screen for suppressors of exo70B1, that exo70B1-activated autoimmune responses require CPK5. However, the CPK5 homologs CPK4, CPK6, and CPK11, which were previously reported to function redundantly with CPK5 in effector-triggered immunity, did not contribute to exo70B1-associated phenotypes, indicating that CPK5 plays a unique role in plant immunity. Overexpressing CPK5 results in TN2-dependent autoimmunity and enhanced disease resistance, reminiscent of the exo70B1 phenotypes. Ectopic expression of CPK5 in the exo70B1 mutant led to constitutive CPK5 protein kinase activity, which was not detectable in tn2 mutants. Furthermore, TN2 interacts with the CPK5 N-terminal variable and kinase domains, stabilizing CPK5 kinase activity in vitro. This work uncovers a direct functional link between an atypical immune receptor and a crucial component of early immune signaling: increased immunity in exo70B1 depends on TN2 and CPK5 and, in a positive feedback loop, TN2 keeps CPK5 enzymatically active beyond the initiating stimulus.
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A Phytophthora Effector Manipulates Host Histone Acetylation and Reprograms Defense Gene Expression to Promote Infection

A Phytophthora Effector Manipulates Host Histone Acetylation and Reprograms Defense Gene Expression to Promote Infection | Plant-microbe interaction | Scoop.it
Immune response during pathogen infection requires extensive transcription reprogramming. A fundamental mechanism of transcriptional regulation is histone acetylation. However, how pathogens interfere with this process to promote disease remains largely unknown. Here we demonstrate that the cytoplasmic effector PsAvh23 produced by the soybean pathogen Phytophthora sojae acts as a modulator of histone acetyltransferase (HAT) in plants. PsAvh23 binds to the ADA2 subunit of the HAT complex SAGA and disrupts its assembly by interfering with the association of ADA2 with the catalytic subunit GCN5. As such, PsAvh23 suppresses H3K9 acetylation mediated by the ADA2/GCN5 module and increases plant susceptibility. Expression of PsAvh23 or silencing of GmADA2/GmGCN5 resulted in misregulation of defense-related genes, most likely due to decreased H3K9 acetylation levels at the corresponding loci. This study highlights an effective counter-defense mechanism by which a pathogen effector suppresses the activation of defense genes by interfering with the function of the HAT complex during infection.
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Zeng Xuan's curator insight, March 29, 12:48 AM
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Rescooped by Suayib Üstün from Publications from The Sainsbury Laboratory
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eLife: Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains (2017)

eLife: Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains (2017) | Plant-microbe interaction | Scoop.it
Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

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The Sainsbury Lab's curator insight, March 6, 11:55 AM
Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.
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Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles | Plant-microbe interaction | Scoop.it
Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana. We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis.

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PNAS: NLR network mediates immunity to diverse plant pathogens (2017)

PNAS: NLR network mediates immunity to diverse plant pathogens (2017) | Plant-microbe interaction | Scoop.it

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that “sensor” NLR proteins are paired with “helper” NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.


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A bacterial acetyltransferase triggers immunity in Arabidopsis thaliana independent of hypersensitive response

A bacterial acetyltransferase triggers immunity in Arabidopsis thaliana independent of hypersensitive response | Plant-microbe interaction | Scoop.it

Type-III secreted effectors (T3Es) play critical roles during bacterial pathogenesis in plants. Plant recognition of certain T3Es can trigger defence, often accompanied by macroscopic cell death, termed the hypersensitive response (HR). Economically important species of kiwifruit are susceptible to Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker. Although Psa is non-pathogenic in Arabidopsis thaliana, we observed that a T3E, HopZ5 that is unique to a global outbreak clade of Psa, triggers HR and defence in Arabidopsis accession Ct-1. Ws-2 and Col-0 accessions are unable to produce an HR in response to Pseudomonas-delivered HopZ5. While Ws-2 is susceptible to virulent bacterial strain Pseudomonas syringae pv. tomato DC3000 carrying HopZ5, Col-0 is resistant despite the lack of an HR. We show that HopZ5, like other members of the YopJ superfamily of acetyltransferases that it belongs to, autoacetylates lysine residues. Through comparisons to other family members, we identified an acetyltransferase catalytic activity and demonstrate its requirement for triggering defence in Arabidopsis and Nicotiana species. Collectively, data herein indicate that HopZ5 is a plasma membrane-localized acetyltransferase with autoacetylation activity required for avirulence.


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Autophagy as an emerging arena for plant–pathogen interactions

Autophagy as an emerging arena for plant–pathogen interactions | Plant-microbe interaction | Scoop.it

Highlights

• Autophagy is an integral part of plant–pathogen interactions.
• A large variety of microbial pathogens target or are targeted by plant autophagy.
• Autophagy in eukaryotic microbial pathogens is essential for pathogenesis.
• Plant autophagy participates in defense responses against invading microbes.
• Successful pathogens have evolved strategies to manipulate plant autophagy.



Autophagy is a highly conserved degradation and recycling process that controls cellular homeostasis, stress adaptation, and programmed cell death in eukaryotes. Emerging evidence indicates that autophagy is a key regulator of plant innate immunity and contributes with both pro-death and pro-survival functions to antimicrobial defences, depending on the pathogenic lifestyle. In turn, several pathogens have co-opted and evolved strategies to manipulate host autophagy pathways to the benefit of infection, while some eukaryotic microbes require their own autophagy machinery for successful pathogenesis. In this review, we present and discuss recent advances that exemplify the important role of pro- and antimicrobial autophagy in plant–pathogen interactions.


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An Oomycete Effector Protein Induces Shade Avoidance In Arabidopsis And Attenuates Salicylate Signaling By Binding To Host Proteins Of The RADICAL-INDUCED CELL DEATH1 Family

An Oomycete Effector Protein Induces Shade Avoidance In Arabidopsis And Attenuates Salicylate Signaling By Binding To Host Proteins Of The RADICAL-INDUCED CELL DEATH1 Family | Plant-microbe interaction | Scoop.it
The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. During infection, Hpa like other biotrophic pathogens, suppresses activation of plant innate immunity by translocating effector proteins into host cells. Some of these effectors localize to the host cell nucleus where they may manipulate transcriptional reprogramming of plant defense genes. Here we report that the nuclear-localized Hpa effector HaRxL106, when expressed in Arabidopsis, induces shade avoidance and attenuates the transcriptional response to the defense signaling molecule salicylic acid. HaRxL106 interacts with RADICAL-INDUCED CELL DEATH1 (RCD1) and loss of RCD1 function renders Arabidopsis resilient against HaRxL106-mediated suppression of immunity. To further characterize the molecular functions of RCD1 we solved a crystal structure of the RCD1 Poly-(ADP-ribose)-Polymerase (PARP) domain and, based on non-conservation of amino acids constituting the active site of canonical PARPs, conclude that RCD1 has no PARP activity. We report that RCD1-type proteins are phosphorylated and identified histone-modifying Mut9-like kinases (MLKs) as RCD1-interacting proteins. A mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared to wild-type plants. Our data suggest that HaRxL106 suppresses Arabidopsis innate immunity by manipulating the function(s) of RCD1 in the host cell nucleus and point towards a role of RCD1 as a transcriptional co-regulator that integrates signals from light and pathogen sensors.
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Translatome analysis of an NB-LRR immune response identifies important contributors to plant immunity in Arabidopsis | Journal of Experimental Botany | Oxford Academic

Translatome analysis of an NB-LRR immune response identifies important contributors to plant immunity in Arabidopsis | Journal of Experimental Botany | Oxford Academic | Plant-microbe interaction | Scoop.it
An important branch of plant immunity involves the recognition of pathogens by nucleotide-binding, leucine-rich repeat (NB-LRR) proteins. However, signaling events downstream of NB-LRR activation are poorly understood. We have analysed the Arabidopsis translatome using ribosome affinity purification and RNA sequencing. Our results show that the translational status of hundreds of transcripts is differentially affected upon activation of the NB-LRR protein RPM1, showing an overall pattern of a switch away from growth-related activities to defense. Among these is the central translational regulator and growth promoter, Target of Rapamycin (TOR) kinase. Suppression of TOR expression leads to increased resistance to pathogens while overexpression of TOR results in increased susceptibility, indicating an important role for translational control in the switch from growth to defense. Furthermore, we show that several additional genes whose mRNAs are translationally regulated, including BIG, CCT2, and CIPK5, are required for both NB-LRR-mediated and basal plant innate immunity, identifying novel actors in plant defense.

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Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins

Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins | Plant-microbe interaction | Scoop.it
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.
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Selective Autophagy of BES1 Mediated by DSK2 Balances Plant Growth and Survival

Selective Autophagy of BES1 Mediated by DSK2 Balances Plant Growth and Survival | Plant-microbe interaction | Scoop.it
Plants encounter a variety of stresses and must fine-tune their growth and stress-response programs to best suit their environment. BES1 functions as a master regulator in the brassinosteroid (BR) pathway that promotes plant growth. Here, we show that BES1 interacts with the ubiquitin receptor protein DSK2 and is targeted to the autophagy pathway during stress via the interaction of DSK2 with ATG8, a ubiquitin-like protein directing autophagosome formation and cargo recruitment. Additionally, DSK2 is phosphorylated by the GSK3-like kinase BIN2, a negative regulator in the BR pathway. BIN2 phosphorylation of DSK2 flanking its ATG8 interacting motifs (AIMs) promotes DSK2-ATG8 interaction, thereby targeting BES1 for degradation. Accordingly, loss-of-function dsk2 mutants accumulate BES1, have altered global gene expression profiles, and have compromised stress responses. Our results thus reveal that plants coordinate growth and stress responses by integrating BR and autophagy pathways and identify the molecular basis of this crosstalk.
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Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination | Plant-microbe interaction | Scoop.it
p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar what is seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT-expressing cells increases ubiquitinated inclusion formation and p62's association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62’s UBA domain at lysine 420 may regulate p62’s function and be disrupted in p62-associated disease.
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TRAF-Family Proteins Regulate Autophagy Dynamics by Modulating AUTOPHAGY PROTEIN6 Stability in Arabidopsis

TRAF-Family Proteins Regulate Autophagy Dynamics by Modulating AUTOPHAGY PROTEIN6 Stability in Arabidopsis | Plant-microbe interaction | Scoop.it
Eukaryotic cells use autophagy to recycle cellular components. During autophagy, autophagosomes deliver cytoplasmic contents to the vacuole or lysosome for breakdown. Mammalian cells regulate the dynamics of autophagy via ubiquitin-mediated proteolysis of autophagy proteins. Here, we show that the Arabidopsis thaliana Tumor necrosis factor Receptor-Associated Factor (TRAF)-family proteins, TRAF1a and TRAF1b (previously named MUSE14 and MUSE13, respectively) help regulate autophagy via ubiquitination. Upon starvation, cytoplasmic TRAF1a and TRAF1b translocated to autophagosomes. Knockout traf1a/b lines showed reduced tolerance to nutrient deficiency, increased salicylic acid and reactive oxygen species levels, and constitutive cell death in rosettes, resembling the phenotypes of autophagy-defective mutants. Starvation-activated autophagosome accumulation decreased in traf1a/b root cells, indicating that TRAF1a and TRAF1b function redundantly in regulating autophagosome formation. TRAF1a and TRAF1b interacted in planta with ATG6 and the RING finger E3 ligases SINAT1, SINAT2, and SINAT6 (with a truncated RING-finger domain). SINAT1 and SINAT2 require the presence of TRAF1a and TRAF1b to ubiquitinate and destabilize AUTOPHAGY PROTEIN6 (ATG6) in vivo. Conversely, starvation-induced SINAT6 reduced SINAT1- and SINAT2-mediated ubiquitination and degradation of ATG6. Consistently, SINAT1/SINAT2 and SINAT6 knockout mutants exhibited increased tolerance and sensitivity, respectively, to nutrient starvation. Therefore, TRAF1a and TRAF1b function as molecular adaptors that help regulate autophagy by modulating ATG6 stability in Arabidopsis.
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Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation | Plant-microbe interaction | Scoop.it
Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3‐interacting region (LIR) at the C‐terminus of the protein and a novel motif at the N‐terminus. Although both sites are important for Atg4–Atg8 interaction in vivo, only the new N‐terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE‐bound Atg8.
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Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response | Plant-microbe interaction | Scoop.it
Crosstalk between post-translational modifications such as ubiquitination and phosphorylation play key roles in controlling the duration and intensity of signalling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signalling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and thus autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo.
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Tweet from @PlantoPhagy: Three recent papers demonstrate antiviral role of autophagy in plants (2017)

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles.
Hafrén A, Macia JL, Love AJ, Milner JJ, Drucker M, Hofius D.
http://www.pnas.org/content/early/2017/02/17/1610687114.abstract

 

A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana.
Li F, Zhao N, Li Z, Xu X, Wang Y, Yang X, Liu SS, Wang A, Zhou X.
http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006213#ppat-1006213-g003

 

Autophagy functions as an antiviral mechanism against geminiviruses in plants.
Haxim Y, Ismayil A, Jia Q, Wang Y, Zheng X, Chen T, Qian L, Liu N, Wang Y, Shaojie H, Cheng J, Yijun Q, Hong Y, Liu Y.
https://elifesciences.org/content/6/e23897


Via Kamoun Lab @ TSL
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A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana

A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana | Plant-microbe interaction | Scoop.it
A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway.
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