Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1

Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.

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The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0904739107 ◽

2010 ◽

Vol 107 (5) ◽

pp. 2349-2354 ◽

Cited By ~ 92

Author(s):

Mike Wilton ◽

Rajagopal Subramaniam ◽

James Elmore ◽

Corinna Felsensteiner ◽

Gitta Coaker ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

Effector Proteins ◽

Type Iii ◽

Pathogen Effector ◽

Effector Triggered Immunity ◽

Type Iii Secretion Effector ◽

Virulence Target

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as “non-self” features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as “non-self” features or induce a “modified-self” state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2Pto also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2Pto were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2Pto interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2Pto. In support of this hypothesis, HopF2Pto interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2Pto did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2Pto and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.

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Differential N-end rule degradation of RIN4/NOI fragments generated by the AvrRpt2 effector protease

10.1101/583054 ◽

2019 ◽

Cited By ~ 1

Author(s):

Kevin Goslin ◽

Lennart Eschen-Lippold ◽

Christin Naumann ◽

Eric Linster ◽

Maud Sorel ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

Host Proteins ◽

Protein Fragments ◽

Effector Triggered Immunity ◽

Bona Fide ◽

Resistance Protein ◽

Domain Protein ◽

Physiological Substrates

AbstractThe protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both layers of plant immunity systems, the so-called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). RIN4 is targeted by several effectors, including thePseudomonas syringaeprotease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RPS2 (RESISTANT TO P. SYRINGAE2). Hence, identifying the determinants of RIN4 degradation is key to understanding RPS2-mediated ETI, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI-domain proteins by AvrRpt2 may contribute to PTI regulation, the (in)stability of these proteolytic fragments and the determinants that regulate their stability have not been examined. Notably, a common feature of RIN4 and of many NOI-domain protein fragments generated by AvrRpt2 cleavage is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed N-degron pathway). By contrast, several NOI-domain protein fragments arebona fidesubstrates of the N-degron pathway. The discovery of this novel set of substrates considerably expands the number of proteins targeted for degradation by this ubiquitin-dependent pathway, for which very few physiological substrates are known in plants. Our results also open new avenues of research to understand the role of AvrRpt2 in promoting bacterial virulence.One sentence summaryAnalysis of RIN4/NOI fragments released after cleavage by the bacterial effector protease AvrRpt2 reveals a novel role of the N-end rule in the degradation of NOI-domain proteins, but not of RIN4.

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The Arabidopsis homolog of human G3BP1 is a key regulator of stomatal and apoplastic immunity

Life Science Alliance ◽

10.26508/lsa.201800046 ◽

2018 ◽

Vol 1 (2) ◽

pp. e201800046 ◽

Cited By ~ 2

Author(s):

Aala A Abulfaraj ◽

Kiruthiga Mariappan ◽

Jean Bigeard ◽

Prabhu Manickam ◽

Ikram Blilou ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Plant Immunity ◽

Bacterial Pathogen ◽

Defense Responses ◽

Pathogen Resistance ◽

Negative Regulator ◽

Multifunctional Protein

Mammalian Ras-GTPase–activating protein SH3-domain–binding proteins (G3BPs) are a highly conserved family of RNA-binding proteins that link kinase receptor-mediated signaling to RNA metabolism. Mammalian G3BP1 is a multifunctional protein that functions in viral immunity. Here, we show that the Arabidopsis thaliana homolog of human G3BP1 negatively regulates plant immunity. Arabidopsis g3bp1 mutants showed enhanced resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated in Atg3bp1 mutants by altered stomatal and apoplastic immunity. Atg3bp1 mutants restricted pathogen entry into stomates showing insensitivity to bacterial coronatine–mediated stomatal reopening. AtG3BP1 was identified as a negative regulator of defense responses, which correlated with moderate up-regulation of salicylic acid biosynthesis and signaling without growth penalty.

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Convergent Evolution of Effector Protease Recognition by Arabidopsis and Barley

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-18-0202-fi ◽

2019 ◽

Vol 32 (5) ◽

pp. 550-565 ◽

Cited By ~ 15

Author(s):

Morgan E. Carter ◽

Matthew Helm ◽

Antony V. E. Chapman ◽

Emily Wan ◽

Ana Maria Restrepo Sierra ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Protease Activity ◽

Phylogenetic Analyses ◽

Defense Responses ◽

Host Protein ◽

Wheat Cultivars ◽

Wild Type ◽

Barley Cultivars ◽

Resistance Protein ◽

Effector Recognition

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We also show that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Response 1 (Pbr1). Transient coexpression of PBR1 with wild-type AvrPphB but not with a protease inactive mutant triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 coimmunoprecipitated with barley and Nicotiana benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain two putative Pbr1 orthologs. Phylogenetic analyses showed, however, that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently and imply that selection to guard PBS1-like proteins occurs across species. Also, these results suggest that PBS1-based decoys may be used to engineer protease effector recognition–based resistance in barley and wheat.

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Two Arabidopsis Homologs of Human Lysine-Specific Demethylase Function in Epigenetic Regulation of Plant Defense Responses

Frontiers in Plant Science ◽

10.3389/fpls.2021.688003 ◽

2021 ◽

Vol 12 ◽

Author(s):

Seong Woo Noh ◽

Ri-Ra Seo ◽

Hee Jin Park ◽

Ho Won Jung

Keyword(s):

Pseudomonas Syringae ◽

Epigenetic Regulation ◽

Plant Immunity ◽

Systemic Disease ◽

Defense Responses ◽

Plant Defense Responses ◽

Promoter Regions ◽

Lysine Specific Demethylase ◽

Genome Wide ◽

Histone H3k4

Epigenetic marks such as covalent histone modification and DNA methylation are crucial for mitotically and meiotically inherited cellular memory-based plant immunity. However, the roles of individual players in the epigenetic regulation of plant immunity are not fully understood. Here we reveal the functions of two Arabidopsis thaliana homologs of human lysine-specific demethylase1-like1, LDL1 and LDL2, in the maintenance of methyl groups at lysine 4 of histone H3 and in plant immunity to Pseudomonas syringae infection. The growth of virulent P. syringae strains was reduced in ldl1 and ldl2 single mutants compared to wild-type plants. Local and systemic disease resistance responses, which coincided with the rapid, robust transcription of defense-related genes, were more stably expressed in ldl1 ldl2 double mutants than in the single mutants. At the nucleosome level, mono-methylated histone H3K4 accumulated in ldl1 ldl2 plants genome-wide and in the mainly promoter regions of the defense-related genes examined in this study. Furthermore, in silico comparative analysis of RNA-sequencing and chromatin immunoprecipitation data suggested that several WRKY transcription factors, e.g., WRKY22/40/70, might be partly responsible for the enhanced immunity of ldl1 ldl2. These findings suggest that LDL1 and LDL2 control the transcriptional sensitivity of a group of defense-related genes to establish a primed defense response in Arabidopsis.

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molecular link from PAMP perception to a MAPK cascade associated with tomato disease resistance

10.32747/2012.7597918.bard ◽

2012 ◽

Author(s):

Guido Sessa ◽

Gregory B. Martin

Keyword(s):

Cell Death ◽

Disease Resistance ◽

Immune Responses ◽

Map Kinase ◽

Pseudomonas Syringae ◽

Molecular Mechanisms ◽

Plant Immunity ◽

Defense Responses ◽

Research Activities ◽

Mapk Cascades

The research problem: The detection of pathogen-associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is a key mechanism by which plants activate an effective immune response against pathogen attack. MAPK cascades are important signaling components downstream of PRRs that transduce the PAMP signal to activate various defense responses. Preliminary experiments suggested that the receptor-like cytoplasmickinase (RLCK) Mai5 plays a positive role in pattern-triggered immunity (PTI) and interacts with the MAPKKK M3Kε. We thus hypothesized that Mai5, as other RLCKs, functions as a component PRR complexes and acts as a molecular link between PAMP perception and activation of MAPK cascades. Original goals: The central goal of this research was to investigate the molecular mechanisms by which Mai5 and M3Kε regulate plant immunity. Specific objectives were to: 1. Determine the spectrum of PAMPs whose perception is transmitted by M3Kε; 2. Identify plant proteins that act downstream of M3Kε to mediate PTI; 3. Investigate how and where Mai5 interacts with M3Kε in the plant cell; 4. Examine the mechanism by which Mai5 contributes to PTI. Changes in research directions: We did not find convincing evidence for the involvement of M3Kε in PTI signaling and substituted objectives 1 and 3 with research activities aimed at the analysis of transcriptomic profiles of tomato plants during the onset of plant immunity, isolation of the novel tomato PRR FLS3, and investigation of the involvement of the RLCKBSKs in PTI. Main achievements during this research program are in the following major areas: 1. Functional characterization of Mai5. The function of Mai5 in PTI signaling was demonstrated by testing the effect of silencing the Mai5 gene by virus-induced gene silencing (VIGS) experiments and in cell death assays. Domains of Mai5 that interact with MAPKKKs and subcellular localization of Mai5 were analyzed in detail. 2. Analysis of transcriptional profiles during the tomato immune responses to Pseudomonas syringae (Pombo et al., 2014). We identified tomato genes whose expression is induced specifically in PTI or in effector-triggered immunity (ETI). Thirty ETI-specific genes were examined by VIGS for their involvement in immunity and the MAPKKK EPK1, was found to be required for ETI. 3. Dissection of MAP kinase cascades downstream of M3Kε (Oh et al., 2013; Teper et al., 2015). We identified genes that encode positive (SGT and EDS1) and negative (WRKY1 and WRKY2) regulators of the ETI-associated cell death mediated by M3Kε. In addition, the MKK2 MAPKK, which acts downstream of M3Kε, was found to interact with the MPK3 MAPK and specific MPK3 amino acids involved interaction were identified and found to be required for induction of cell death. We also identified 5 type III effectors of the bacterial pathogen Xanthomonaseuvesicatoria that inhibited cell death induced by components of ETI-associated MAP kinase cascades. 4. Isolation of the tomato PRR FLS3 (Hind et al., submitted). FLS3, a novel PRR of the LRR-RLK family that specifically recognizes the flagellinepitope flgII-28 was isolated. FLS3 was shown to bind flgII-28, to require kinase activity for function, to act in concert with BAK1, and to enhance disease resistance to Pseudomonas syringae. 5. Functional analysis of RLCKs of the brassinosteroid signaling kinase (BSK) family.Arabidopsis and tomato BSKs were found to interact with PRRs. In addition, certain ArabidospsisBSK mutants were found to be impaired in PAMP-induced resistance to Pseudomonas syringae. Scientific and agricultural significance: Our research activities discovered and characterized new molecular components of signaling pathways mediating recognition of invading pathogens and activation of immune responses against them. Increased understanding of molecular mechanisms of immunity will allow them to be manipulated by both molecular breeding and genetic engineering to produce plants with enhanced natural defense against disease.

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A High-Throughput, Seedling Screen for Plant Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-19-0295-ta ◽

2020 ◽

Vol 33 (3) ◽

pp. 394-401

Author(s):

Alexandre Martel ◽

Timothy Lo ◽

Darrell Desveaux ◽

David S. Guttman

Keyword(s):

Pseudomonas Syringae ◽

High Throughput ◽

Biological Diversity ◽

Association Studies ◽

Plant Immunity ◽

Genome Wide Association Studies ◽

Pathogen Effector ◽

Immunity Genes ◽

Microbe Interactions

An understanding of how biological diversity affects plant–microbe interactions is becoming increasingly important, particularly with respect to components of the pathogen effector arsenal and the plant immune system. Although technological improvements have greatly advanced our ability to examine molecular sequences and interactions, relatively few advances have been made that facilitate high-throughput, in vivo pathology screens. Here, we present a high-throughput, microplate-based, nondestructive seedling pathology assay, and apply it to identify Arabidopsis thaliana effector-triggered immunity (ETI) responses against Pseudomonas syringae type III secreted effectors. The assay was carried out in a 48-well microplate format with spray inoculation, and disease symptoms were quantitatively recorded in a semiautomated manner, thereby greatly reducing both time and costs. The assay requires only slight modifications of common labware and uses no proprietary software. We validated the assay by recapitulating known ETI responses induced by P. syringae in Arabidopsis. We also demonstrated that we can quantitatively differentiate responses from a diversity of plant genotypes grown in the same microplate. Finally, we showed that the results obtained from our assay can be used to perform genome-wide association studies to identify host immunity genes, recapitulating results that have been independently obtained with mature plants.

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The Majority of the Type III Effector Inventory of Pseudomonas syringae pv. tomato DC3000 Can Suppress Plant Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-9-1069 ◽

2009 ◽

Vol 22 (9) ◽

pp. 1069-1080 ◽

Cited By ~ 147

Author(s):

Ming Guo ◽

Fang Tian ◽

Yashitola Wamboldt ◽

James R. Alfano

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

In Planta ◽

Tomato Dc3000 ◽

Type Iii Effector ◽

Type Iii Effectors ◽

Type Iii ◽

Molecular Pattern ◽

Effector Triggered Immunity ◽

Type Iii Protein Secretion

The Pseudomonas syringae type III protein secretion system (T3SS) and the type III effectors it injects into plant cells are required for plant pathogenicity and the ability to elicit a hypersensitive response (HR). The HR is a programmed cell death that is associated with effector-triggered immunity (ETI). A primary function of P. syringae type III effectors appears to be the suppression of ETI and pathogen-associated molecular pattern–triggered immunity (PTI), which is induced by conserved molecules on microorganisms. We reported that seven type III effectors from P. syringae pv. tomato DC3000 were capable of suppressing an HR induced by P. fluorescens(pHIR11) and have now tested 35 DC3000 type III effectors in this assay, finding that the majority of them can suppress the HR induced by HopA1. One newly identified type III effector with particularly strong HR suppression activity was HopS2. We used the pHIR11 derivative pLN1965, which lacks hopA1, in related assays and found that a subset of the type III effectors that suppressed HopA1-induced ETI also suppressed an ETI response induced by AvrRpm1 in Arabidopsis thaliana. A. thaliana plants expressing either HopAO1 or HopF2, two type III effectors that suppressed the HopA1-induced HR, were reduced in the flagellin-induced PTI response as well as PTI induced by other PAMPs and allowed enhanced in planta growth of P. syringae. Collectively, our results suggest that the majority of DC3000 type III effectors can suppress plant immunity. Additionally, the construct pLN1965 will likely be a useful tool in determining whether other type III effectors or effectors from other types of pathogens can suppress either ETI, PTI, or both.

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Jasmonic Acid at the Crossroads of Plant Immunity and Pseudomonas syringae Virulence

International Journal of Molecular Sciences ◽

10.3390/ijms21207482 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7482

Author(s):

Aarti Gupta ◽

Mamta Bhardwaj ◽

Lam-Son Phan Tran

Keyword(s):

Jasmonic Acid ◽

Plant Defense ◽

Pseudomonas Syringae ◽

Regulatory Networks ◽

Genetic Manipulation ◽

Plant Immunity ◽

Stomatal Closure ◽

Defense Responses ◽

Plant Defense Responses ◽

Callose Deposition

Sensing of pathogen infection by plants elicits early signals that are transduced to affect defense mechanisms, such as effective blockage of pathogen entry by regulation of stomatal closure, cuticle, or callose deposition, change in water potential, and resource acquisition among many others. Pathogens, on the other hand, interfere with plant physiology and protein functioning to counteract plant defense responses. In plants, hormonal homeostasis and signaling are tightly regulated; thus, the phytohormones are qualified as a major group of signaling molecules controlling the most widely tinkered regulatory networks of defense and counter-defense strategies. Notably, the phytohormone jasmonic acid mediates plant defense responses to a wide array of pathogens. In this review, we present the synopsis on the jasmonic acid metabolism and signaling, and the regulatory roles of this hormone in plant defense against the hemibiotrophic bacterial pathogen Pseudomonas syringae. We also elaborate on how this pathogen releases virulence factors and effectors to gain control over plant jasmonic acid signaling to effectively cause disease. The findings discussed in this review may lead to ideas for the development of crop cultivars with enhanced disease resistance by genetic manipulation.

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Analysis of Resistance Gene-Mediated Defense Responses in Arabidopsis thaliana Plants Carrying a Mutation in CPR5

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.12.1196 ◽

1998 ◽

Vol 11 (12) ◽

pp. 1196-1206 ◽

Cited By ~ 51

Author(s):

Jens Boch ◽

Michelle L. Verbsky ◽

Tara L. Robertson ◽

John C. Larkin ◽

Barbara N. Kunkel

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Resistance Gene ◽

Pseudomonas Syringae ◽

Bacterial Pathogen ◽

Defense Responses ◽

Negative Regulator ◽

Suppressor Mutations ◽

Defense Related Gene ◽

Rapid Activation

In resistant plants, pathogen attack often leads to rapid activation of defense responses that limit multiplication and spread of the pathogen. To investigate the signaling mechanisms underlying this process, we carried out a screen for mutants in the signaling pathway governing resistance in Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae. This involved screening for suppressor mutations that restored resistance to a susceptible line carrying a mutation in the RPS2 resistance gene. A mutant that conferred resistance by activating defense responses in the absence of pathogens was isolated. This mutant, which carries a mutation at the CPR5 locus and was thus designated cpr5-2, exhibited resistance to P. syringae, spontaneous development of necrotic lesions, elevated PR gene expression in the absence of pathogens, and abnormal trichomes. Resistance gene-mediated defenses, including the hypersensitive response, restriction of pathogen growth, and induction of defense-related gene expression, were functional in cpr5-2 mutant plants. Additionally, in cpr5-2 plants RPS2-mediated induction of PR-1 expression was enhanced, whereas RPM1-mediated induction of ELI3 was not. These findings suggest that CPR5 encodes a negative regulator of the RPS2 signal transduc-tion pathway.

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