Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

Plant cell wall-mediated disease resistance: Current understanding and future perspectives.

Beyond their function as structural barriers, plant cell walls are essential elements for the adaptation of plants to environmental conditions. Cell walls are dynamic structures whose composition and integrity can be altered in response to environmental challenges and developmental cues. These wall changes are perceived by plant sensors/receptors to trigger adaptative responses during development and upon stress perception. Plant cell wall damage caused by pathogen infection, wounding, or other stresses leads to the release of wall molecules, such as carbohydrates (glycans), that function as damage-associated molecular patterns (DAMPs). DAMPs are perceived by the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs) to activate pattern-triggered immunity (PTI) and disease resistance. Similarly, glycans released from the walls and extracellular layers of microorganisms interacting with plants are recognized as microbe-associated molecular patterns (MAMPs) by specific ECD-PRRs triggering PTI responses. The number of oligosaccharides DAMPs/MAMPs identified that are perceived by plants has increased in recent years. However, the structural mechanisms underlying glycan recognition by plant PRRs remain limited. Currently, this knowledge is mainly focused on receptors of the LysM-PRR family, which are involved in the perception of various molecules, such as chitooligosaccharides from fungi and lipo-chitooligosaccharides (i.e., Nod/MYC factors from bacteria and mycorrhiza, respectively) that trigger differential physiological responses. Nevertheless, additional families of plant PRRs have recently been implicated in oligosaccharide/polysaccharide recognition. These include receptor kinases (RKs) with leucine-rich repeat and Malectin domains in their ECDs (LRR-MAL RKs), Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE group (CrRLK1L) with Malectin-like domains in their ECDs, as well as wall-associated kinases, lectin-RKs, and LRR-extensins. The characterization of structural basis of glycans recognition by these new plant receptors will shed light on their similarities with those of mammalians involved in glycan perception. The gained knowledge holds the potential to facilitate the development of sustainable, glycan-based crop protection solutions.

A Glycosyl Hydrolase 5 Family Protein Is Essential for Virulence of Necrotrophic Fungi and Can Suppress Plant Immunity.

Phytopathogenic fungi normally secrete large amounts of CWDEs to enhance infection of plants. In this study, we identified and characterized a secreted glycosyl hydrolase 5 family member in Sclerotinia sclerotiorum (SsGH5, Sclerotinia sclerotiorum Glycosyl Hydrolase 5). SsGH5 was significantly upregulated during the early stages of infection. Knocking out SsGH5 did not affect the growth and acid production of S. sclerotiorum but resulted in decreased glucan utilization and significantly reduced virulence. In addition, Arabidopsis thaliana expressing SsGH5 became more susceptible to necrotrophic pathogens and basal immune responses were inhibited in these plants. Remarkably, the lost virulence of the ΔSsGH5 mutants was restored after inoculating onto SsGH5 transgenic Arabidopsis. In summary, these results highlight that S. sclerotiorum suppresses the immune responses of Arabidopsis through secreting SsGH5, and thus exerts full virulence for successful infection.

Cytoplasmic calcium influx mediated by plant MLKLs confers TNL-triggered immunity.

The plant homolog of vertebrate necroptosis inducer mixed-lineage kinase domain-like (MLKL) contributes to downstream steps in Toll-interleukin-1 receptor domain NLR (TNL)-receptor-triggered immunity. Here, we show that Arabidopsis MLKL1 (AtMLKL1) clusters into puncta at the plasma membrane upon TNL activation and that this sub-cellular reorganization is dependent on the TNL signal transducer, EDS1. We find that AtMLKLs confer TNL-triggered immunity in parallel with RPW8-type HeLo-domain-containing NLRs (RNLs) and that the AtMLKL N-terminal HeLo domain is indispensable for both immunity and clustering. We show that the AtMLKL HeLo domain mediates cytoplasmic Ca2+ ([Ca2+]cyt) influx in plant and human cells, and AtMLKLs are responsible for sustained [Ca2+]cyt influx during TNL-triggered, but not CNL-triggered, immunity. Our study reveals parallel immune signaling functions of plant MLKLs and RNLs as mediators of [Ca2+]cyt influx and a potentially common role of the HeLo domain fold in the Ca2+-signal relay of diverse organisms.

Natural variation in the pattern-triggered immunity response in plants: Investigations, implications and applications.

The pattern-triggered immunity (PTI) response is triggered at the plant cell surface by the recognition of microbe-derived molecules known as microbe- or pathogen-associated molecular patterns or molecules derived from compromised host cells called damage-associated molecular patterns. Membrane-localized receptor proteins, known as pattern recognition receptors, are responsible for this recognition. Although much of the machinery of PTI is conserved, natural variation for the PTI response exists within and across species with respect to the components responsible for pattern recognition, activation of the response, and the strength of the response induced. This review describes what is known about this variation. We discuss how variation in the PTI response can be measured and how this knowledge might be utilized in the control of plant disease and in developing plant varieties with enhanced disease resistance.

Recent advances in understanding the role of two mitogen-activated protein kinase cascades in plant immunity.

The mitogen-activated protein kinase (MAPK/MPK) cascade is an important intercellular signaling module that regulates plant growth, development, reproduction, and responses to biotic and abiotic stresses. A MAPK cascade usually consists of a MAPK kinase kinase (MAPKKK/MEKK), a MAPK kinase (MAPKK/MKK/MEK), and a MAPK. The well-characterized MAPK cascades in plant immunity to date are the MEKK1-MKK1/2-MPK4 cascade and the MAPKKK3/4/5-MKK4/5-MPK3/6 cascade. Recently, major breakthroughs have been made in understanding the molecular mechanisms associated with the regulation of immune signaling by both of these MAPK cascades. In this review, we highlight the most recent advances in understanding the role of both MAPK cascades in activating plant defense and in suppressing or fine-tuning immune signaling. We also discuss the molecular mechanisms by which plants stabilize and maintain the activation of MAPK cascades during immune signaling. Based on this review, we reveal the complexity and importance of the MEKK1-MKK1/2-MPK4 cascade and the MAPKKK3/4/5-MKK4/5-MPK3/6 cascade, which are tightly controlled by their interacting partners or substrates, in plant immunity.

Calcium homeostasis and signaling in plant immunity.

Calcium (Ca2+) signaling consists of three steps: (1) initiation of a change in cellular Ca2+ concentration in response to a stimulus, (2) recognition of the change through direct binding of Ca2+ by its sensors, (3) transduction of the signal to elicit downstream responses. Recent studies have uncovered a central role for Ca2+ signaling in both layers of immune responses initiated by plasma membrane (PM) and intracellular receptors, respectively. These advances in our understanding are attributed to several lines of research, including invention of genetically-encoded Ca2+ reporters for the recording of intracellular Ca2+ signals, identification of Ca2+ channels and their gating mechanisms, and functional analysis of Ca2+ binding proteins (Ca2+ sensors). This review analyzes the recent literature that illustrates the importance of Ca2+ homeostasis and signaling in plant innate immunity, featuring intricate Ca2+dependent positive and negative regulations.

Temperature-dependent action of pepper mildew resistance locus O 1 in inducing pathogen immunity and thermotolerance.

Plant diseases tend to be more serious under conditions of high-temperature/high-humidity (HTHH) than under moderate conditions, and hence disease resistance under HTHH is an important determinant for plant survival. However, how plants cope with diseases under HTHH remains poorly understood. In this study, we used the pathosystem consisting of pepper (Capsicum annuum) and Ralstonia solanacearum (bacterial wilt) as a model to examine the functions of the protein mildew resistance locus O 1 (CaMLO1) and U-box domain-containing protein 21 (CaPUB21) under conditions of 80% humidity and either 28 °C or 37 °C. Expression profiling, loss- and gain-of-function assays involving virus-induced gene-silencing and overexpression in pepper plants, and protein-protein interaction assays were conducted, and the results showed that CaMLO1 acted negatively in pepper immunity against R. solanacearum at 28 °C but positively at 37 °C. In contrast, CaPUB21 acted positively in immunity at 28 °C but negatively at 37 °C. Importantly, CaPUB21 interacted with CaMLO1 under all of the tested conditions, but only the interaction in response to R. solanacearum at 37 °C or to exposure to 37 °C alone led to CaMLO1 degradation, thereby turning off defence responses against R. solanacearum at 37 °C and under high-temperature stress to conserve resources. Thus, we show that CaMLO1 and CaPUB21 interact with each other and function distinctly in pepper immunity against R. solanacearum in an environment-dependent manner.

The phagocytosis oxidase/Bem1p domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity.

Perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation. To understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD with mass spectrometry analysis and identified PHAGOCYTOSIS OXIDASE/BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP competes with BIK1 for binding to RBOHD in vitro. Furthermore, PAMP treatment enhances the PB1CP-RBOHD interaction, thereby leading to the dissociation of phosphorylated BIK1 from RBOHD in vivo. PB1CP localizes at the cell periphery and PAMP treatment induces relocalization of PB1CP and RBOHD to the same small endomembrane compartments. Additionally, overexpression of PB1CP in Arabidopsis leads to a reduction in the abundance of RBOHD protein, suggesting the possible involvement of PB1CP in RBOHD endocytosis. We found PB1CP, a novel negative regulator of RBOHD, and revealed its possible regulatory mechanisms involving the removal of phosphorylated BIK1 from RBOHD and the promotion of RBOHD endocytosis.

BTL2 phospho-switch surveils plant immunity.

BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) is a co-receptor involved in the recognition of pattern-associated molecular patterns (PAMPs) via plasma membrane-localized pattern recognition receptors (PRRs). Absence of BAK1/SERK4 leads to the activation of autoimmunity in plants. Yu et al. recently showed that BAK-TO-LIFE 2 (BTL2) is required for the surveillance of BAK1/SERK4 integrity to maintain immune homeostasis.

BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling.

Pathogen-associated molecular patterns (PAMPs) trigger plant innate immunity that acts as the first line of inducible defense against pathogen infection. A receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) functions as a signaling hub immediately downstream of multiple pattern recognition receptors (PRRs). It is known that PLANT U-BOX PROTEIN 25 (PUB25) and PUB26 ubiquitinate BIK1 and mediate BIK1 degradation. However, how BIK1 homeostasis is maintained is not fully understood. Here, we show that two closely related ubiquitin ligases, RING DOMAIN LIGASE 1 (RGLG1) and RGLG2, preferentially associate with the hypo-phosphorylated BIK1 and promote the association of BIK1 with the co-receptor for several PRRs, BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). PUB25 interacts with RGLG2 and mediates its degradation. In turn, RGLG2 represses the ubiquitin ligase activity of PUB25. RGLG1/2 suppress PUB25-mediated BIK1 degradation, promote BIK1 protein accumulation, and positively regulate immune signaling in a ubiquitin ligase activity-dependent manner. Our work reveals how BIK1 homeostasis is maintained by the interplay of different ubiquitin ligases.

Autophagy promotes jasmonate-mediated defense against nematodes.

Autophagy, as an intracellular degradation system, plays a critical role in plant immunity. However, the involvement of autophagy in the plant immune system and its function in plant nematode resistance are largely unknown. Here, we show that root-knot nematode (RKN; Meloidogyne incognita) infection induces autophagy in tomato (Solanum lycopersicum) and different atg mutants exhibit high sensitivity to RKNs. The jasmonate (JA) signaling negative regulators JASMONATE-ASSOCIATED MYC2-LIKE 1 (JAM1), JAM2 and JAM3 interact with ATG8s via an ATG8-interacting motif (AIM), and JAM1 is degraded by autophagy during RKN infection. JAM1 impairs the formation of a transcriptional activation complex between ETHYLENE RESPONSE FACTOR 1 (ERF1) and MEDIATOR 25 (MED25) and interferes with transcriptional regulation of JA-mediated defense-related genes by ERF1. Furthermore, ERF1 acts in a positive feedback loop and regulates autophagy activity by transcriptionally activating ATG expression in response to RKN infection. Therefore, autophagy promotes JA-mediated defense against RKNs via forming a positive feedback circuit in the degradation of JAMs and transcriptional activation by ERF1.

Utilising natural diversity of kinases to rationally engineer interactions with the angiosperm immune receptor ZAR1.

The highly conserved angiosperm immune receptor HOPZ-ACTIVATED RESISTANCE1 (ZAR1) recognises the activity of diverse pathogen effector proteins by monitoring the ZED1-related kinase (ZRK) family. Understanding how ZAR1 achieves interaction specificity for ZRKs may allow for the expansion of the ZAR1-kinase recognition repertoire to achieve novel pathogen recognition outside of model species. We took advantage of the natural diversity of Arabidopsis thaliana kinases to probe the ZAR1-kinase interaction interface and found that A. thaliana ZAR1 (AtZAR1) can interact with most ZRKs, except ZRK7. We found evidence of alternative splicing of ZRK7, resulting in a protein that can interact with AtZAR1. Despite high sequence conservation of ZAR1, interspecific ZAR1-ZRK pairings resulted in the autoactivation of cell death. We showed that ZAR1 interacts with a greater diversity of kinases than previously thought, while still possessing the capacity for specificity in kinase interactions. Finally, using AtZAR1-ZRK interaction data, we rationally increased ZRK10 interaction strength with AtZAR1, demonstrating the feasibility of the rational design of a ZAR1-interacting kinase. Overall, our findings advance our understanding of the rules governing ZAR1 interaction specificity, with promising future directions for expanding ZAR1 immunodiversity.

A novel protein elicitor (PeSy1) from Saccharothrix yanglingensis induces plant resistance and interacts with a receptor-like cytoplasmic kinase in Nicotiana benthamiana.

Previously, we reported a rare actinomycete Saccharothrix yanglingensis Hhs.015 with strong biocontrol ability, which can colonize plant tissues and induce resistance, but the key elicitor and immune mechanisms were unclear. In this study, a novel protein elicitor screened from the genome of Hhs.015, PeSy1 (protein elicitor of S. yanglingensis 1), could induce a strong hypersensitive response (HR) and resistance in plants. The PeSy1 gene encodes an 11 kDa protein with 109 amino acids that is conserved in Saccharothrix species. PeSy1-His recombinant protein induced early defence events such as a cellular reactive oxygen species burst, callose deposition, and the activation of defence hormone signalling pathways, which enhanced Nicotiana benthamiana resistance to Sclerotinia sclerotiorum and Phytophthora capsici, and Solanum lycopersicum resistance to Pseudomonas syringae pv. tomato DC3000. Through pull-down and mass spectrometry, candidate proteins that interacted with PeSy1 were obtained from N. benthamiana. We confirmed the interaction between receptor-like cytoplasmic kinase RSy1 (Response to PeSy1) and PeSy1 using co-immunoprecipitation, bimolecular fluorescence complementation, and microscale thermophoresis. PeSy1 treatment promoted up-regulation of marker genes in pattern-triggered immunity. The cell death it elicited was dependent on the co-receptors NbBAK1 and NbSOBIR1, suggesting that PeSy1 acts as a microbe-associated molecular pattern from Hhs.015. Additionally, RSy1 positively regulated PeSy1-induced plants resistant to S. sclerotiorum. In conclusion, our results demonstrated a novel receptor-like cytoplasmic kinase in the plant perception of microbe-associated molecular patterns, and the potential of PeSy1 in induced resistance provided a new strategy for biological control of actinomycetes in agricultural diseases.

Plant latent defense response against compatibility.

Managing the association with microbes is crucial for plants. Evidence is emerging for the plant latent defense response, which is conditionally elicited by certain microbial nonpathogenic factors and thereby guards against potential risks from beneficial or commensal microbes. Latent defense response is an exciting new research area with a number of key issues immediately awaiting exploration. A detailed understanding of latent defense response will underpin the applications of beneficial microbes.

SHOU4/4L link cell wall cellulose synthesis to pattern-triggered immunity.

Pattern recognition receptors (PRRs) are plasma membrane-localised proteins that sense molecular patterns to initiate pattern-triggered immunity (PTI). Receptor-like cytoplasmic kinases (RLCKs) function downstream of PRRs to propagate signal transduction via the phosphorylation of substrate proteins. The identification and characterisation of RLCK-regulated substrate proteins are critical for our understanding of plant immunity. We showed that SHOU4 and SHOU4L are rapidly phosphorylated upon various patterns elicitation and are indispensable for plant resistance to bacterial and fungal pathogens. Protein-protein interaction and phosphoproteomic analysis revealed that BOTRYTIS-INDUCED KINASE 1, a prominent protein kinase of RLCK subfamily VII (RLCK-VII), interacted with SHOU4/4L and phosphorylated multiple serine residues on SHOU4L N-terminus upon pattern flg22 treatment. Neither phospho-dead nor phospho-mimic SHOU4L variants complemented pathogen resistance and plant development defect of the loss-of-function mutant, suggesting that reversible phosphorylation of SHOU4L is critical to plant immunity and plant development. Co-immunoprecipitation data revealed that flg22 induced SHOU4L dissociation from cellulose synthase 1 (CESA1) and that a phospho-mimic SHOU4L variant inhibited the interaction between SHOU4L and CESA1, indicating the link between SHOU4L-mediated cellulose synthesis and plant immunity. This study thus identified SHOU4/4L as new components of PTI and preliminarily revealed the mechanism governing SHOU4L regulation by RLCKs.

Amino Acid Availability Determines Plant Immune Homeostasis in the Rhizosphere Microbiome.

Microbes possess conserved microbe-associated molecular patterns (MAMPs) that are recognized by plant receptors to induce pattern-triggered immunity (PTI). Despite containing the same MAMPs as pathogens, commensals thrive in the plant rhizosphere microbiome, indicating they must suppress or evade host immunity. Previous work found that bacterial-secreted gluconic acid is sufficient to suppress PTI. Here, we show that gluconic acid biosynthesis is not necessary for immunity suppression by the beneficial bacterial strain Pseudomonas simiae WCS417. We performed a forward genetic screen with EMS-mutagenized P. simiae WCS417 and a flagellin-inducible CYP71A12pro:GUS reporter as a PTI readout. We identified a loss of function mutant in ornithine carbamoyltransferase argF, which is required for ornithine conversion to arginine, that cannot suppress PTI or acidify the rhizosphere. Fungal pathogens use alkalization through production of ammonia and glutamate, and arginine biosynthetic precursors, to promote their own growth and virulence. While a ΔargF mutant has a growth defect in the rhizosphere, we found that restoring growth with exogenous arginine resulted in rhizosphere alkalization in a mutant that cannot make gluconic acid, indicating that arginine biosynthesis is required for both growth and acidification. Furthermore, blocking bacterial arginine, glutamine, or proline biosynthesis through genetic mutations or feedback inhibition by adding corresponding amino acids, resulted in rhizosphere alkalization. Untargeted metabolomics determined that ornithine, an alkaline molecule, accumulates under conditions associated with rhizosphere alkalization. Our findings show that bacterial amino acid biosynthesis contributes to acidification by preventing accumulation of ornithine and the resulting alkalization. IMPORTANCE Understanding how microbiota evade and suppress host immunity is critical to our knowledge of how beneficial microbes persist in association with a host. Prior work has shown that secretion of organic acids by beneficial microbes is sufficient to suppress plant immunity. This work shows that microbial amino acid metabolism is not only critical for growth in the plant rhizosphere microbiome, but also for regulation of plant rhizosphere pH, and, consequentially, regulation of plant immunity. We found that, in the absence of microbial glutamate and arginine metabolism, rhizosphere alkalization and microbial overgrowth occurs. Collectively, our findings suggest that, by regulating nutrient availability, plants have the potential to regulate their immune homeostasis in the rhizosphere microbiome.

Arabidopsis clathrin adaptor EPSIN1 but not MODIFIED TRANSPORT TO THE VACOULE1 contributes to effective plant immunity against pathogenic Pseudomonas bacteria.

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.

The Phytophthora capsici RxLR effector CRISIS2 triggers cell death via suppressing plasma membrane H+-ATPase in the host plant.

Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we screened the Phytophthora capsici RxLR cell death-inducer suppressing immune system (CRISIS). We found that the cell death induced by the CRISIS2 effector in Nicotiana benthamiana was inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene-silencing analyses revealed that CRISIS2 physically and functionally associated with PMAs and induced host cell death independent of immune receptors. CRISIS2 induced apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici, whereas host-induced gene-silencing of CRISIS2 compromised the disease symptoms and the biomass of the pathogen. Thus, our study has identified a novel RxLR effector that plays multiple roles in the suppression of plant defense and in the induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.