A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component.

Beyond its role in cellular homeostasis, autophagy plays anti- and promicrobial roles in host-microbe interactions, both in animals and plants. One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense-related autophagy in plant-bacteria interactions.

Arabidopsis UGT76B1 glycosylates N-hydroxy-pipecolic acid and inactivates systemic acquired resistance in tomato.

Systemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in this process are currently unclear. Here, we report that Arabidopsis thaliana UGT76B1 generated glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants. During infection, Arabidopsis ugt76b1 mutants did not accumulate NHP-Glc and accumulated less glycosylated salicylic acid (SA-Glc) than wild-type plants. The metabolic changes in ugt76b1 plants were accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of the SAR molecules NHP and salicylic acid by UGT76B1 plays an important role in modulating defense responses. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato (Solanum lycopersicum) increased NHP-Glc production and reduced NHP accumulation in local tissue and abolished the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling.

Arabidopsis bZIP11 Is a Susceptibility Factor During Pseudomonas syringae Infection.

The induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens (e.g., Xanthomonas spp.) use transcription activator-like (TAL) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pv. tomato strain DC3000 lacks TAL effectors yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Because bacteria require other nutrients in addition to sugars for efficient reproduction, we hypothesized that Pseudomonas spp. may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11-regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tomato Atypical Receptor Kinase1 Is Involved in the Regulation of Preinvasion Defense.

Tomato Atypical Receptor Kinase 1 (TARK1) is a pseudokinase required for postinvasion immunity. TARK1 was originally identified as a target of the Xanthomonas euvesicatoria effector protein Xanthomonas outer protein N (XopN), a suppressor of early defense signaling. How TARK1 participates in immune signal transduction is not well understood. To gain insight into TARK1's role in tomato (Solanum lycopersicum) immunity, we used a proteomics approach to isolate and identify TARK1-associated immune complexes formed during infection. We found that TARK1 interacts with proteins predicted to be associated with stomatal movement. TARK1 CRISPR mutants and overexpression (OE) lines did not display differences in light-induced stomatal opening or abscisic acid-induced stomatal closure; however, they did show altered stomatal movement responses to bacteria and biotic elicitors. Notably, we found that TARK1 CRISPR plants were resistant to Pseudomonas syringae pathovar tomato strain DC3000-induced stomatal reopening, and TARK1 OE plants were insensitive to P syringae pathovar tomato strain DC3118 (coronatine deficit)-induced stomatal closure. We also found that TARK1 OE in leaves resulted in increased susceptibility to bacterial invasion. Collectively, our results indicate that TARK1 functions in stomatal movement only in response to biotic elicitors and support a model in which TARK1 regulates stomatal opening postelicitation.

N-hydroxy-pipecolic acid is a mobile metabolite that induces systemic disease resistance in Arabidopsis.

Systemic acquired resistance (SAR) is a global response in plants induced at the site of infection that leads to long-lasting and broad-spectrum disease resistance at distal, uninfected tissues. Despite the importance of this priming mechanism, the identity and complexity of defense signals that are required to initiate SAR signaling is not well understood. In this paper, we describe a metabolite, N-hydroxy-pipecolic acid (N-OH-Pip) and provide evidence that this mobile molecule plays a role in initiating SAR signal transduction in Arabidopsis thaliana We demonstrate that FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of SAR-associated defense priming, can synthesize N-OH-Pip from pipecolic acid in planta, and exogenously applied N-OH-Pip moves systemically in Arabidopsis and can rescue the SAR-deficiency of fmo1 mutants. We also demonstrate that N-OH-Pip treatment causes systemic changes in the expression of pathogenesis-related genes and metabolic pathways throughout the plant and enhances resistance to a bacterial pathogen. This work provides insight into the chemical nature of a signal for SAR and also suggests that the N-OH-Pip pathway is a promising target for metabolic engineering to enhance disease resistance.

Tomato bHLH132 Transcription Factor Controls Growth and Defense and Is Activated by Xanthomonas euvesicatoria Effector XopD During Pathogenesis.

Effector-dependent manipulation of host transcription is a key virulence mechanism used by Xanthomonas species causing bacterial spot disease in tomato and pepper. Transcription activator-like (TAL) effectors employ novel DNA-binding domains to directly activate host transcription, whereas the non-TAL effector XopD uses a small ubiquitin-like modifier (SUMO) protease activity to represses host transcription. The targets of TAL and non-TAL effectors provide insight to the genes governing susceptibility and resistance during Xanthomonas infection. In this study, we investigated the extent to which the X. euvesicatoria non-TAL effector strain Xe85-10 activates tomato transcription to gain new insight to the transcriptional circuits and virulence mechanisms associated with Xanthomonas euvesicatoria pathogenesis. Using transcriptional profiling, we identified a putative basic helix-loop-helix (bHLH) transcription factor, bHLH132, as a pathogen-responsive gene that is moderately induced by microbe-associated molecular patterns and defense hormones and is highly induced by XopD during X. euvesicatoria infection. We also found that activation of bHLH132 transcription requires the XopD SUMO protease activity. Silencing bHLH132 mRNA expression results in stunted tomato plants with enhanced susceptibility to X. euvesicatoria infection. Our work suggests that bHLH132 is required for normal vegetative growth and development as well as resistance to X. euvesicatoria. It also suggests new transcription-based models describing XopD virulence and recognition in tomato.

Aphid effector Me10 interacts with tomato TFT7, a 14-3-3 isoform involved in aphid resistance.

We demonstrated previously that expression of Macrosiphum euphorbiae salivary protein Me10 enhanced aphid reproduction on its host tomato (Solanum lycopersicum). However, the mechanism of action of Me10 remained elusive. To confirm the secretion of Me10 by the aphid into plant tissues, we produced Me10 polyclonal antibodies. To identify the plant targets of Me10, we developed a tomato immune induced complementary DNA yeast two-hybrid library and screened it with Me10 as bait. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays were performed to validate one of the interactions in planta, and virus-induced gene silencing was used for functional characterization in tomato. We demonstrated that Me10 is secreted into the plant tissues and interacts with tomato 14-3-3 isoform 7 (TFT7) in yeast. Immunoprecipitation assays confirmed that Me10 and its homologue in Aphis gossypii, Ag10k, interact with TFT7 in planta. Further, BiFC revealed that Me10 interaction with TFT7 occurs in the plant cell cytoplasm. While silencing of TFT7 in tomato leaves did not affect tomato susceptibility to M. euphorbiae, it enhanced longevity and fecundity of A. gossypii, the non-host aphid. Our results suggest the model whereby TFT7 plays a role in aphid resistance in tomato and effectors of the Me10/Ag10k family interfere with TFT7 function during aphid infestation.

Tomato 14-3-3 Proteins Are Required for Xv3 Disease Resistance and Interact with a Subset of Xanthomonas euvesicatoria Effectors.

The 14-3-3 phospho-binding proteins with scaffolding activity play central roles in the regulation of enzymes and signaling complexes in eukaryotes. In plants, 14-3-3 isoforms are required for disease resistance and key targets of pathogen effectors. Here, we examined the requirement of the tomato (Solanum lycopersicum) 14-3-3 isoform (TFT) protein family for Xv3 disease resistance in response to the bacterial pathogen Xanthomonas euvesicatoria. In addition, we determined whether TFT proteins interact with the repertoire of X. euvesicatoria type III secretion effector proteins, including AvrXv3, the elicitor of Xv3 resistance. We show that multiple TFT contribute to Xv3 resistance. We also show that one or more TFT proteins physically interact with multiple effectors (AvrXv3, XopE1, XopE2, XopN, XopO, XopQ, and XopAU). Genetic analyses indicate that none of the identified effectors interfere with AvrXv3-elicited resistance into Xv3 tomato leaves; however, XopE1, XopE2, and XopO are required to suppress symptom development in susceptible tomato leaves. Phospho-peptide mapping revealed that XopE2 is phosphorylated at multiple residues in planta and residues T66, T131, and S334 are required for maximal binding to TFT10. Together, our data support the hypothesis that multiple TFT proteins are involved in immune signaling during X. euvesicatoria infection.

The bHLH transcription factor HBI1 mediates the trade-off between growth and pathogen-associated molecular pattern-triggered immunity in Arabidopsis.

The trade-off between growth and immunity is crucial for survival in plants. However, the mechanism underlying growth-immunity balance has remained elusive. The PRE-IBH1-HBI1 tripartite helix-loop-helix/basic helix-loop-helix module is part of a central transcription network that mediates growth regulation by several hormonal and environmental signals. Here, genome-wide analyses of HBI1 target genes show that HBI1 regulates both overlapping and unique targets compared with other DNA binding components of the network in Arabidopsis thaliana, supporting a role in specifying network outputs and fine-tuning feedback regulation. Furthermore, HBI1 negatively regulates a subset of genes involved in immunity, and pathogen-associated molecular pattern (PAMP) signals repress HBI1 transcription. Constitutive overexpression and loss-of-function experiments show that HBI1 inhibits PAMP-induced growth arrest, defense gene expression, reactive oxygen species production, and resistance to pathogen. These results show that HBI1, as a component of the central growth regulation circuit, functions as a major node of crosstalk that mediates a trade-off between growth and immunity in plants.

AvrBsT acetylates Arabidopsis ACIP1, a protein that associates with microtubules and is required for immunity.

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1's defense function, which is linked to the activation of ETI.

Sugar transporters for intercellular exchange and nutrition of pathogens.

Sugar efflux transporters are essential for the maintenance of animal blood glucose levels, plant nectar production, and plant seed and pollen development. Despite broad biological importance, the identity of sugar efflux transporters has remained elusive. Using optical glucose sensors, we identified a new class of sugar transporters, named SWEETs, and show that at least six out of seventeen Arabidopsis, two out of over twenty rice and two out of seven homologues in Caenorhabditis elegans, and the single copy human protein, mediate glucose transport. Arabidopsis SWEET8 is essential for pollen viability, and the rice homologues SWEET11 and SWEET14 are specifically exploited by bacterial pathogens for virulence by means of direct binding of a bacterial effector to the SWEET promoter. Bacterial symbionts and fungal and bacterial pathogens induce the expression of different SWEET genes, indicating that the sugar efflux function of SWEET transporters is probably targeted by pathogens and symbionts for nutritional gain. The metazoan homologues may be involved in sugar efflux from intestinal, liver, epididymis and mammary cells.

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

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

Functional Analysis of Plant Defense Suppression and Activation by the Xanthomonas Core Type III Effector XopX.

Many phytopathogenic type III secretion effector proteins (T3Es) have been shown to target and suppress plant immune signaling but perturbation of the plant immune system by T3Es can also elicit a plant response. XopX is a "core" Xanthomonas T3E that contributes to growth and symptom development during Xanthomonas euvesicatoria infection of tomato but its functional role is undefined. We tested the effect of XopX on several aspects of plant immune signaling. XopX promoted ethylene production and plant cell death (PCD) during X. euvesicatoria infection of susceptible tomato and in transient expression assays in Nicotiana benthamiana, which is consistent with its requirement for the development of X. euvesicatoria-induced disease symptoms. Additionally, although XopX suppressed flagellin-induced reactive oxygen species, it promoted the accumulation of pattern-triggered immunity (PTI) gene transcripts. Surprisingly, XopX coexpression with other PCD elicitors resulted in delayed PCD, suggesting antagonism between XopX-dependent PCD and other PCD pathways. However, we found no evidence that XopX contributed to the suppression of effector-triggered immunity during X. euvesicatoria-tomato interactions, suggesting that XopX's primary virulence role is to modulate PTI. These results highlight the dual role of a core Xanthomonas T3E in simultaneously suppressing and activating plant defense responses.

A comprehensive analysis of microProteins reveals their potentially widespread mechanism of transcriptional regulation.

Truncated transcription factor-like proteins called microProteins (miPs) can modulate transcription factor activities, thereby increasing transcriptional regulatory complexity. To understand their prevalence, evolution, and function, we predicted over 400 genes that encode putative miPs from Arabidopsis (Arabidopsis thaliana) using a bioinformatics pipeline and validated two novel miPs involved in flowering time and response to abiotic and biotic stress. We provide an evolutionary perspective for a class of miPs targeting homeodomain transcription factors in plants and metazoans. We identify domain loss as one mechanism of miP evolution and suggest the possible roles of miPs on the evolution of their target transcription factors. Overall, we reveal a prominent layer of transcriptional regulation by miPs, show pervasiveness of such proteins both within and across genomes, and provide a framework for studying their function and evolution.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence.

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344-733) is sufficient to bind TFT1. Removal of amino acids 605-733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.

Dynamic changes of the Prf/Pto tomato resistance complex following effector recognition.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen recognition and activation of innate immunity. In plants, NLRs recognise pathogen-derived effector proteins and initiate effector-triggered immunity (ETI). However, the molecular mechanisms that link NLR-mediated effector recognition and downstream signalling are not fully understood. By exploiting the well-characterised tomato Prf/Pto NLR resistance complex, we identified the 14-3-3 proteins TFT1 and TFT3 as interacting partners of both the NLR complex and the protein kinase MAPKKKα. Moreover, we identified the helper NRC proteins (NLR-required for cell death) as integral components of the Prf /Pto NLR recognition complex. Notably our studies revealed that TFTs and NRCs interact with distinct modules of the NLR complex and, following effector recognition, dissociate facilitating downstream signalling. Thus, our data provide a mechanistic link between activation of immune receptors and initiation of downstream signalling cascades.

SOBER1 phospholipase activity suppresses phosphatidic acid accumulation and plant immunity in response to bacterial effector AvrBsT.

Arabidopsis thaliana ecotype Pi-0 is resistant to Pseudomonas syringae pathovar tomato (Pst) strain DC3000 expressing the T3S effector protein AvrBsT. Resistance is due to a loss of function mutation (sober1-1) in a conserved alpha/beta hydrolase, SOBER1 (Suppressor of AvrBsT Elicited Resistance1). Members of this superfamily possess phospholipase and carboxylesterase activity with diverse substrate specificity. The nature of SOBER1 enzymatic activity and substrate specificity was not known. SOBER1-dependent suppression of the hypersensitive response (HR) in Pi-0 suggested that it might hydrolyze a plant lipid or precursor required for HR induction. Here, we show that Pi-0 leaves infected with Pst DC3000 expressing AvrBsT accumulated higher levels of phosphatidic acid (PA) compared to leaves infected with Pst DC3000. Phospholipase D (PLD) activity was required for high PA levels and AvrBsT-dependent HR in Pi-0. Overexpression of SOBER1 in Pi-0 reduced PA levels and inhibited HR. These data implicated PA, phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) as potential SOBER1 substrates. Recombinant His(6)-SOBER1 hydrolyzed PC but not PA or LysoPC in vitro indicating that the enzyme has phospholipase A(2) (PLA(2)) activity. Chemical inhibition of PLA(2) activity in leaves expressing SOBER1 resulted in HR in response to Pst DC3000 AvrBsT. These data are consistent with the model that SOBER1 PLA(2) activity suppresses PLD-dependent production of PA in response to AvrBsT elicitation. This work highlights an important role for SOBER1 in the regulation of PA levels generated in plants in response to biotic stress.

Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases.

Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development and immunity in plants, but the mechanisms that link LRR-RKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLAGELLIN SENSING 2 (FLS2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We surprisingly found that the loss of the BSU1 family phosphatases activates effector-triggered immunity and impairs flagellin-triggered MAP kinase activation and immunity. The flagellin-activated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine 251. Mutation of serine 251 reduces BSU1's ability to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress effector-triggered immunity and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine 764 upon brassinosteroid signalling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid-BRI1 and flagellin-FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signalling specificities for diverse plant receptor kinases.