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The bacterial plant pathogen Pseudomonas syringae requires type III secreted effectors (T3SEs) for pathogenesis. However, a major facet of plant immunity entails the recognition of a subset of P. syringae's T3SEs by intracellular host receptors in a process called Effector-Triggered Immunity (ETI). Prior work has shown that ETI-eliciting T3SEs are pervasive in the P. syringae species complex raising the question of how P. syringae mitigates its ETI load to become a successful pathogen. While pathogens can evade ETI by T3SE mutation, recombination, or loss, there is increasing evidence that effector-effector (a.k.a., metaeffector) interactions can suppress ETI. To study the ETI-suppression potential of P. syringae T3SE repertoires, we compared the ETI-elicitation profiles of two genetically divergent strains: P. syringae pv. tomato DC3000 (PtoDC3000) and P. syringae pv. maculicola ES4326 (PmaES4326), which are both virulent on Arabidopsis thaliana but harbour largely distinct effector repertoires. Of the 529 T3SE alleles screened on A. thaliana Col-0 from the P. syringae T3SE compendium (PsyTEC), 69 alleles from 21 T3SE families elicited ETI in at least one of the two strain backgrounds, while 50 elicited ETI in both backgrounds, resulting in 19 differential ETI responses including two novel ETI-eliciting families: AvrPto1 and HopT1. Although most of these differences were quantitative, three ETI responses were completely absent in one of the pathogenic backgrounds. We performed ETI suppression screens to test if metaeffector interactions contributed to these ETI differences, and found that HopQ1a suppressed AvrPto1m-mediated ETI, while HopG1c and HopF1g suppressed HopT1b-mediated ETI. Overall, these results show that P. syringae strains leverage metaeffector interactions and ETI suppression to overcome the ETI load associated with their native T3SE repertoires., Competing Interests: The authors have declared that no competing interests exist.

The bacterial phytopathogen Pseudomonas syringae causes disease on a wide array of plants, including the model plant Arabidopsis thaliana and its agronomically important relatives in the Brassicaceae family. To cause disease, P. syringae delivers effector proteins into plant cells through a type III secretion system. In response, plant nucleotide-binding leucine-rich repeat proteins recognize specific effectors and mount effector-triggered immunity (ETI). While ETI is pervasive across A. thaliana, with at least 19 families of P. syringae effectors recognized in this model species, the ETI landscapes of crop species have yet to be systematically studied. Here, we investigated the conservation of the A. thaliana ETI landscape in two closely related oilseed crops, Brassica napus (canola) and Camelina sativa (false flax). We show that the level of immune conservation is inversely related to the degree of evolutionary divergence from A. thaliana, with the more closely related C. sativa losing ETI responses to only one of the 19 P. syringae effectors tested, while the more distantly related B. napus loses ETI responses to four effectors. In contrast to the qualitative conservation of immune response, the quantitative rank order is not as well-maintained across the three species and diverges increasingly with evolutionary distance from A. thaliana. Overall, our results indicate that the A. thaliana ETI profile is qualitatively conserved in oilseed crops, but quantitatively distinct., (© 2022. The Author(s).)

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system., Competing Interests: The authors declare no competing interest.

Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to the environment. However, a main limitation of traditional approaches to study protein interactions, such as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of interactions include many of the protein complexes that mediate the perception and response to cellular stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells, including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins (within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the study of proteomes in numerous organisms, its application to plant systems has only recently been realized. In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.

The phytopathogen Pseudomonas syringae delivers into host cells type III secreted effectors (T3SEs) that promote virulence. One virulence mechanism employed by T3SEs is to target hormone signaling pathways to perturb hormone homeostasis. The phytohormone abscisic acid (ABA) influences interactions between various phytopathogens and their plant hosts, and has been shown to be a target of P. syringae T3SEs. In order to provide insight into how T3SEs manipulate ABA responses, we generated an ABA-T3SE interactome network (ATIN) between P. syringae T3SEs and Arabidopsis proteins encoded by ABA-regulated genes. ATIN consists of 476 yeast-two-hybrid interactions between 97 Arabidopsis ABA-regulated proteins and 56 T3SEs from four pathovars of P. syringae. We demonstrate that T3SE interacting proteins are significantly enriched for proteins associated with transcription. In particular, the ETHYLENE RESPONSIVE FACTOR (ERF) family of transcription factors is highly represented. We show that ERF105 and ERF8 displayed a role in defense against P. syringae, supporting our overall observation that T3SEs of ATIN converge on proteins that influence plant immunity. In addition, we demonstrate that T3SEs that interact with a large number of ABA-regulated proteins can influence ABA responses. One of these T3SEs, HopF3 Pph6 , inhibits the function of ERF8, which influences both ABA-responses and plant immunity. These results provide a potential mechanism for how HopF3 Pph6 manipulates ABA-responses to promote P. syringae virulence, and also demonstrate the utility of ATIN as a resource to study the ABA-T3SE interface., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI., Competing Interests: The authors have declared that no competing interests exist.

Background: ETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses., Results: In this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively., Conclusions: Together these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.

Proximity dependent biotin identification (BioID) has emerged as a powerful tool for studies of proteome architecture, including insoluble or membrane-associated proteins. The technique has been well established in mammalian cells but has yet to be applied to whole plant systems. Here we demonstrate the application of BioID on leaf tissues of the model plant Arabidopsis thaliana, thereby expanding the versatility of this important technique and providing a powerful proteomics tool for plant biologists.

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSI LRR ) that consists of 567 interactions. To demonstrate the power of CSI LRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSI LRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

Nucleotide-binding domain and leucine-rich repeat domain-containing (NLR) proteins are sentinels of plant immunity that monitor host proteins for perturbations induced by pathogenic effector proteins. Here we show that the Arabidopsis ZAR1 NLR protein requires the ZRK3 kinase to recognize the Pseudomonas syringae type III effector (T3E) HopF2a. These results support the hypothesis that ZAR1 associates with an expanded ZRK protein family to broaden its effector recognition spectrum.

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Effector-triggered immunity (ETI) is conferred by dominant plant resistance (R) genes, which encode predominantly nucleotide-binding and leucine-rich repeat domain proteins (NLRs), against cognate microbial avirulence (Avr) genes, which include bacterial type III secreted effectors (T3Es). The 'guard model' describes the mechanism of T3E perception by plants, whereby NLRs monitor host proteins ('sensors') for T3E-induced perturbations. This model has provided a molecular framework to understand T3E perception and has rationalized how plants can use a limited number of NLRs (∼160 in Arabidopsis) to contend with a potentially limitless number of evolving effectors. In this review we provide a characteristic overview of plant T3E sensors and discuss how these sensors convey the presence of T3Es to NLR proteins to activate ETI., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The sesquiterpenoid abscisic acid (ABA) mediates an assortment of responses across a variety of kingdoms including both higher plants and animals. In plants, where most is known, a linear core ABA signaling pathway has been identified. However, the complexity of ABA-dependent gene expression suggests that ABA functions through an intricate network. Here, using systems biology approaches that focused on genes transcriptionally regulated by ABA, we defined an ABA signaling network of over 500 interactions among 138 proteins. This map greatly expanded ABA core signaling but was still manageable for systematic analysis. For example, functional analysis was used to identify an ABA module centered on two sucrose nonfermenting (SNF)-like kinases. We also used coexpression analysis of interacting partners within the network to uncover dynamic subnetwork structures in response to different abiotic stresses. This comprehensive ABA resource allows for application of approaches to understanding ABA functions in higher plants., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Arabidopsis thaliana GOLDEN2-LIKE (GLK1 and 2) transcription factors regulate chloroplast development in a redundant manner. Overexpression of AtGLK1 (35S:AtGLK1) in Arabidopsis also confers resistance to the cereal pathogen Fusarium graminearum. To further elucidate the role of GLK transcription factors in plant defence, the Arabidopsis glk1 glk2 double-mutant and 35S:AtGLK1 plants were challenged with the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2. Compared with Col-0, glk1 glk2 plants were highly resistant to Hpa Noco2, whereas 35S:AtGLK1 plants showed enhanced susceptibility to this pathogen. Genetic studies suggested that AtGLK-mediated plant defence to Hpa Noco2 was partially dependent on salicylic acid (SA) accumulation, but independent of the SA signalling protein NONEXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1). Pretreatment with jasmonic acid (JA) dramatically reversed Hpa Noco2 resistance in the glk1 glk2 double mutant, but only marginally affected the 35S:AtGLK1 plants. In addition, overexpression of AtGLK1 in the JA signalling mutant coi1-16 did not increase susceptibility to Hpa Noco2. Together, our GLK gain-of-function and loss-of-function experiments suggest that GLK acts upstream of JA signalling in disease susceptibility to Hpa Noco2. In contrast, glk1 glk2 plants were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, whereas 35S:AtGLK1 plants exhibited heightened resistance which could be maintained in the absence of JA signalling. Together, the data reveal that AtGLK1 is involved in JA-dependent susceptibility to the biotrophic pathogen Hpa Noco2 and in JA-independent resistance to the necrotrophic pathogen B. cinerea., (© 2013 HER MAJESTY THE QUEEN IN RIGHT OF CANADA. MOLECULAR PLANT PATHOLOGY © 2013 BSPP. REPRODUCED WITH THE PERMISSION OF THE MINISTER OF AGRICULTURE AND AGRI-FOOD CANADA.)

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 bacterial pathogens of both animals and plants. These effectors can either suppress host immunity or elicit defense responses depending on the host genotype. In a recent report, we identified an Arabidopsis thaliana pseudokinase ZED1 that is required for the recognition of the Pseudomonas syringae HopZ1a effector. Here we discuss the role of ZED1 in HopZ1a recognition, and present models of effector recognition in plants. We draw parallels between HopZ1a and YopJ effector proteins, and between ZED1 and other immunity-related kinases that can be targeted by pathogen effectors.

Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1-resistance complex, resulting in ETI activation.

Background: Identification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system., Results: Here we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data make it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence., Conclusions: We demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.

Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the approximately 170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T-DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-activated resistance 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS-LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1-mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a., Competing Interests: The authors have declared that no competing interests exist.

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 HopF2(Pto) also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2(Pto) were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2(Pto) interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2(Pto). In support of this hypothesis, HopF2 (Pto) interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2(Pto) 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 HopF2(Pto) and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.

Type 2C protein phosphatases (PP2Cs) are vitally involved in abscisic acid (ABA) signaling. Here, we show that a synthetic growth inhibitor called pyrabactin functions as a selective ABA agonist. Pyrabactin acts through PYRABACTIN RESISTANCE 1 (PYR1), the founding member of a family of START proteins called PYR/PYLs, which are necessary for both pyrabactin and ABA signaling in vivo. We show that ABA binds to PYR1, which in turn binds to and inhibits PP2Cs. We conclude that PYR/PYLs are ABA receptors functioning at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting PP2Cs. Our results illustrate the power of the chemical genetic approach for sidestepping genetic redundancy.

The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.

Plant pathogenic Pseudomonas syringae deliver type III effector proteins into the host cell, where they function to manipulate host defense and metabolism to benefit the extracellular bacterial colony. The activity of these virulence factors can be monitored by plant disease resistance proteins deployed to "guard" the targeted host proteins. The Arabidopsis RIN4 protein is targeted by three different type III effectors. Specific manipulation of RIN4 by each of them leads to activation of either the RPM1 or RPS2 disease resistance proteins. The type III effector AvrRpt2 is a cysteine protease that is autoprocessed inside the host cell where it activates RPS2 by causing RIN4 disappearance. RIN4 contains two sites related to the AvrRpt2 cleavage site (RCS1 and RCS2). We demonstrate that AvrRpt2-dependent cleavage of RIN4 at RCS2 is functionally critical in vivo. This event leads to proteasome-mediated elimination of all but a membrane-embedded approximately 6.4-kDa C-terminal fragment of RIN4. One or more of three consecutive cysteines in this C-terminal fragment are required for RIN4 localization; these are likely to be palmitoylation and/or prenylation sites. AvrRpt2-dependent cleavage at RCS2, and release of the remainder of RIN4 from the membrane, consequently prevents RPM1 activation by AvrRpm1 or AvrB. RCS2 is contained within the smallest tested fragment of RIN4 that binds AvrB in vitro. Thus, at least two bacterial virulence factors target the same domain of RIN4, a approximately 30-aa plant-specific signature sequence found in a small Arabidopsis protein family that may be additional targets for these bacterial virulence factors.

Transcriptional reprogramming is critical for plant disease resistance responses; its global control is not well understood. Salicylic acid (SA) can induce plant defense gene expression and a long-lasting disease resistance state called systemic acquired resistance (SAR). Plant-specific "Whirly" DNA binding proteins were previously implicated in defense gene regulation. We demonstrate that the potato StWhy1 protein is a transcriptional activator of genes containing the PBF2 binding PB promoter element. DNA binding activity of AtWhy1, the Arabidopsis StWhy1 ortholog, is induced by SA and is required for both SA-dependent disease resistance and SA-induced expression of an SAR response gene. AtWhy1 is required for both full basal and specific disease resistance responses. The transcription factor-associated protein NPR1 is also required for SAR. Surprisingly, AtWhy1 activation by SA is NPR1 independent, suggesting that AtWhy1 works in conjunction with NPR1 to transduce the SA signal. Our analysis of AtWhy1 adds a critical component to the SA-dependent plant disease resistance response.

The Arabidopsis NPR1 protein is essential for regulating salicylic acid-dependent gene expression during systemic acquired resistance. NPR1 interacts differentially with members of the TGA class of basic domain/Leu zipper transcription factors and regulates their DNA binding activity. Here, we report that although TGA1 does not interact with NPR1 in yeast two-hybrid assays, treatment with salicylic acid induces the interaction between these proteins in Arabidopsis leaves. This phenomenon is correlated with a reduction of TGA1 Cys residues. Furthermore, site-directed mutagenesis of TGA1 Cys-260 and Cys-266 enables the interaction with NPR1 in yeast and Arabidopsis. Together, these results indicate that TGA1 relies on the oxidation state of Cys residues to mediate the interaction with NPR1. An intramolecular disulfide bridge in TGA1 precludes interaction with NPR1, and NPR1 can only stimulate the DNA binding activity of the reduced form of TGA1. Unlike its animal and yeast counterparts, the DNA binding activity of TGA1 is not redox regulated; however, this property is conferred by interaction with the NPR1 cofactor.