Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

CPR5 positively regulates pattern-triggered immunity via a mediator protein.

Plant cells possess a two-layered immune system consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), mediated by cell surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), respectively. The CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) nuclear pore complex protein negatively regulates ETI, including ETI-associated hypersensitive response. Here, we show that CPR5 is essential for the activation of various PTI responses in Arabidopsis, such as resistance to the non-adapted bacterium Pseudomonas syringae pv. tomato DC3000 hrcC- . In a forward-genetic screen for suppressors of cpr5, we identified the mediator protein MED4. Mutation of MED4 in cpr5 greatly restored the defective PTI of cpr5. Our findings reveal that CPR5 plays opposite roles in regulating PTI and ETI, and genetically regulates PTI via MED4.

A pair of G-type lectin receptor-like kinases modulates nlp20-mediated immune responses by coupling to the RLP23 receptor complex.

Plant cells recognize microbial patterns with the plasma-membrane-localized pattern-recognition receptors consisting mainly of receptor kinases (RKs) and receptor-like proteins (RLPs). RKs, such as bacterial flagellin receptor FLS2, and their downstream signaling components have been studied extensively. However, newly discovered regulatory components of RLP-mediated immune signaling, such as the nlp20 receptor RLP23, await identification. Unlike RKs, RLPs lack a cytoplasmic kinase domain, instead recruiting the receptor-like kinases (RLKs) BAK1 and SOBIR1. SOBIR1 specifically works as an adapter for RLP-mediated immunity. To identify new regulators of RLP-mediated signaling, we looked for SOBIR1-binding proteins (SBPs) in Arabidopsis thaliana using protein immunoprecipitation and mass spectrometry, identifying two G-type lectin RLKs, SBP1 and SBP2, that physically interacted with SOBIR1. SBP1 and SBP2 showed high sequence similarity, were tandemly repeated on chromosome 4, and also interacted with both RLP23 and BAK1. sbp1 sbp2 double mutants obtained via CRISPR-Cas9 gene editing showed severely impaired nlp20-induced reactive oxygen species burst, mitogen-activated protein kinase (MAPK) activation, and defense gene expression, but normal flg22-induced immune responses. We showed that SBP1 regulated nlp20-induced immunity in a kinase activity-independent manner. Furthermore, the nlp20-induced the RLP23-BAK1 interaction, although not the flg22-induced FLS2-BAK1 interaction, was significantly reduced in sbp1 sbp2. This study identified SBPs as new regulatory components in RLP23 receptor complex that may specifically modulate RLP23-mediated immunity by positively regulating the interaction between the RLP23 receptor and the BAK1 co-receptor.

The cell-type specific role of Arabidopsis bZIP59 transcription factor in plant immunity.

Stomatal movement participates in plant immunity by directly affecting the invasion of bacteria, but the genes that regulate stomatal immunity have not been well identified. Here, we characterised the function of the bZIP59 transcription factor from Arabidopsis thaliana, which is constitutively expressed in guard cells. The bzip59 mutant is partially impaired in stomatal closure induced by Pseudomonas syringae pv. tomato strain (Pst) DC3000 and is more susceptible to Pst DC3000 infection. By contrast, the line overexpressing bZIP59 enhances resistance to Pst DC3000 infection. Furthermore, the bzip59 mutant is also partially impaired in stomatal closure induced by flagellin flg22 derived from Pst DC3000, and epistasis analysis revealed that bZIP59 acts upstream of reactive oxygen species (ROS) and nitric oxide (NO) and downstream of salicylic acid signalling in flg22-induced stomatal closure. In addition, the bzip59 mutant showed resistance and sensitivity to Sclerotinia sclerotiorum and Tobacco mosaic virus that do not invade through stomata, respectively. Collectively, our results demonstrate that bZIP59 plays an important role in the stomatal immunity and reveal that the same transcription factor can positively and negatively regulate disease resistance against different pathogens.

Arabidopsis PUB2 and PUB4 connect signaling components of pattern-triggered immunity.

Plants use pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity (PTI). Precise regulation of information from PRRs to downstream signaling components is vital to mounting an appropriate immune response and requires dynamic interactions of these PTI components. We used transcriptome profiling, phenotypic analysis, molecular genetics, and protein-protein interaction analysis to understand the roles of the Arabidopsis plant U-box (PUB) proteins PUB2 and PUB4 in disease resistance and PTI signaling. Loss of function of both PUB2 and PUB4 diminishes the PAMP-triggered oxidative bursts and dampens mitogen-activated protein kinase signaling, resulting in a severe compromise in resistance to not only pathogenic but also nonpathogenic strains of Pseudomonas syringae. Within PUB4, the E3 ligase activity is dispensable, but the armadillo repeat region is essential and sufficient for its function in immunity. PUB2 and PUB4 interact with PTI signaling components, including FLS2, BIK1, PBL27, and RbohD, and enhance FLS2-BIK1 and BIK1-RbohD interactions. Our study reveals that PUB2 and PUB4 are critical components of plant immunity and connect PTI components to positively regulate defense responses.

Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalk between SA-dependent immunity and phosphate-starvation responses.

KEY MESSAGE: Selective Arabidopsis thaliana inositol phosphate kinase functions modulate response amplitudes in innate immunity by balancing signalling adjustments with phosphate homeostasis networks. Pyrophosphorylation of InsP6 generates InsP7 and/or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. As bona fide co-factors for several phytohormone networks, InsP7/InsP8 modulate key developmental processes. With requirements in transducing jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 exemplifies a versatile metabolite for crosstalks between different cellular pathways during diverse stress exposures. Here we show that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2) implicated in InsP8 biosynthesis, suppress salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, constitutive activation of defenses lead to enhanced resistance against the Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that upregulated SA-signaling sectors potentiate increased expression of several phosphate-starvation inducible (PSI)-genes, previously known in these mutants. In reciprocation, upregulated PSI-genes moderate expression amplitudes of defense-associated markers. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators in defense-phosphate homeostasis and in reprogramming stress-appropriate response intensities.

Melatonin functions in priming of stomatal immunity in Panax notoginseng and Arabidopsis thaliana.

Melatonin (MT) plays important roles in plant disease response, but the mechanisms are largely unknown. Here, we show that MT functions in stomatal immunity in Panax notoginseng and Arabidopsis thaliana. Biochemical analyses showed that MT-induced stomatal closure plays a prominent role in preventing invasion of bacteria Pseudomonas syringe pv. tomato (Pst) DC3000 via activation of mitogen-activated protein kinase (MAPK) and NADPH oxidase-mediated reactive oxygen species production in P. notoginseng. The first putative phytomelatonin receptor 1 (PMTR1) is a plasma membrane protein required for perceiving MT signaling in stomatal closure and activation of MAPK. Biochemical and genetic tests found PMTR1 is essential for flg22- and MT-induced MAPK activation in a heterotrimeric GTP-binding protein Gα subunit GPA1-independent manner. GPA1 functions in the same genetic pathways of FLS2/BAK1 (Flagellin Sensing 2/Brassinosteroid Insensitive 1-associated kinase 1)- as well as PMTR1-mediated flg22 and MT signaling in stomatal closure. The stomata in pmtr1 are insensitive to MT and flg22, but the application of MT induces stomatal closure and reduces the bacterial growth in fls2 and bak1 plants, indicating that PMTR1 might be a downstream signaling component in FLS2- and BAK1-mediated stomatal immunity. In summary, our results (i) demonstrate that phytomelatonin functions in the priming of stomatal immunity and (ii) provide insights into the phytomelatonin signaling transduction pathway.

Atypical molecular features of RNA silencing against the phloem-restricted polerovirus TuYV.

In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection.

Synaptotagmin 5 Controls SYP132-VAMP721/722 Interaction for Arabidopsis Immunity to Pseudomonas syringae pv tomato DC3000.

Vesicle-associated membrane proteins 721 and 722 (VAMP721/722) are secretory vesicle-localized arginine-conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) to drive exocytosis in plants. They are involved in diverse physiological processes in plants by interacting with distinct plasma membrane (PM) syntaxins. Here, we show that synaptotagmin 5 (SYT5) is involved in plant defense against Pseudomonas syringae pv tomato (Pst) DC3000 by regulating SYP132-VAMP721/722 interactions. Calcium-dependent stimulation of in vitro SYP132-VAMP722 interaction by SYT5 and reduced in vivo SYP132-VAMP721/722 interaction in syt5 plants suggest that SYT5 regulates the interaction between SYP132 and VAMP721/722. We interestingly found that disease resistance to Pst DC3000 bacterium but not to Erysiphe pisi fungus is compromised in syt5 plants. Since SYP132 plays an immune function to bacteria, elevated growth of surface-inoculated Pst DC3000 in VAMP721/722-deficient plants suggests that SYT5 contributes to plant immunity to Pst DC3000 by promoting the SYP132-VAMP721/722 immune secretory pathway.

RIN4 homologs from important crop species differentially regulate the Arabidopsis NB-LRR immune receptor, RPS2.

KEY MESSAGE: RIN4 homologs from important crop species differ in their ability to prevent ectopic activity of the nucleotide binding-leucine rich repeat resistance protein, RPS2. Pathogens deploy virulence effectors to perturb host processes. Plants utilize intracellular resistance (R) proteins to recognize pathogen effectors either by direct interaction or indirectly via effector-mediated perturbations of host components. RPM1-INTERACTING PROTEIN4 (RIN4) is a plant immune regulator that mediates the indirect activation of multiple, independently evolved R-proteins by multiple, unrelated effector proteins. One of these, RPS2 (RESISTANT TO P. SYRINGAE2), is activated upon cleavage of Arabidopsis (At)RIN4 by the Pseudomonas syringae effector AvrRpt2. To gain insight into the AvrRpt2-RIN4-RPS2 defense-activation module, we compared the function of AtRIN4 with RIN4 homologs present in a diverse range of plant species. We selected seven homologs containing conserved features of AtRIN4, including two NOI (Nitrate induced) domains, each containing a predicted cleavage site for AvrRpt2, and a C-terminal palmitoylation site predicted to mediate membrane tethering of the proteins. Palmitoylation-mediated tethering of AtRIN4 to the plasma membrane and cleavage by AvrRpt2 are required for suppression and activation of RPS2, respectively. While all seven homologs are localized at the plasma membrane, only four suppress RPS2 when transiently expressed in Nicotiana benthamiana. All seven homologs are cleaved by AvrRpt2 and, for those homologs that are able to suppress RPS2, cleavage relieves suppression of RPS2. Further, we demonstrate that the membrane-tethered, C-terminal AvrRpt2-generated cleavage fragment is sufficient for the suppression of RPS2. Lastly, we show that the membrane localization of RPS2 is unaffected by its suppression or activation status.

The RING-type protein BOI negatively regulates the protein level of a CC-NBS-LRR in Arabidopsis.

Nucleotide-binding site and leucine-rich repeat receptors (NLRs) play pivotal roles in plant immunity. The regulation of NLR stability is essential to ensure effective immunity, whereas the exact mechanism is largely unclear. The Arabidopsis CC-NBS-LRR protein L5 (At1g12290) can induce cell death in Nicotiana benthamiana, but not in Arabidopsis thaliana. We screened the interactors of L5 by yeast two-hybrid, and found that the BOI can interact with the CC domain of L5. Transiently expressed BOI reduced the protein level of L5, and suppressed the auoactivity of L5 in N. benthamiana. BOI can interact and ubiquitinate L5 in vitro, and mediate the proteasomal degradation of L5 in N. benthamiana and Arabidopsis. The Lys425 in the NBS domain of L5 is the critical unbiquitin site for the degradation. In conclusion, our results reveal a mechanism for the control of the stability of L5 protein and for the suppressed of L5-triggered cell death by a RING-type E3 ligase through the ubiquitin proteasome system.

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms.

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

The Arabidopsis Iron-Sulfur (Fe-S) Cluster Gene MFDX1 Plays a Role in Host and Nonhost Disease Resistance by Accumulation of Defense-Related Metabolites.

Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.

A karyopherin constrains nuclear activity of the NLR protein SNC1 and is essential to prevent autoimmunity in Arabidopsis.

The nucleotide-binding and leucine-rich repeat (NLR) proteins comprise a major class of intracellular immune receptors that are capable of detecting pathogen-derived molecules and activating immunity and cell death in plants. The activity of some NLRs, particularly the Toll-like/interleukin-1 receptor (TIR) type, is highly correlated with their nucleocytoplasmic distribution. However, whether and how the nucleocytoplasmic homeostasis of NLRs is coordinated through a bidirectional nuclear shuttling mechanism remains unclear. Here, we identified a nuclear transport receptor, KA120, which is capable of affecting the nucleocytoplasmic distribution of an NLR protein and is essential in preventing its autoactivation. We showed that the ka120 mutant displays an autoimmune phenotype and NLR-induced transcriptome features. Through a targeted genetic screen using an artificial NLR microRNA library, we identified the TIR-NLR gene SNC1 as a genetic interactor of KA120. Loss-of-function snc1 mutations as well as compromising SNC1 protein activities all substantially suppressed ka120-induced autoimmune activation, and the enhanced SNC1 activity upon loss of KA120 functionappeared to occur at the protein level. Overexpression of KA120 efficiently repressed SNC1 activity and led to a nearly complete suppression of the autoimmune phenotype caused by the gain-of-function snc1-1 mutation or SNC1 overexpression in transgenic plants. Further florescence imaging analysis indicated that SNC1 undergoes altered nucleocytoplasmic distribution with significantly reduced nuclear signal when KA120 is constitutively expressed, supporting a role of KA120 in coordinating SNC1 nuclear abundance and activity. Consistently, compromising the SNC1 nuclear level by disrupting the nuclear pore complex could also partially rescue ka120-induced autoimmunity. Collectively, our study demonstrates that KA120 is essential to avoid autoimmune activation in the absence of pathogens and is required to constrain the nuclear activity of SNC1, possibly through coordinating SNC1 nucleocytoplasmic homeostasis as a potential mechanism.

S-acylation of P2K1 mediates extracellular ATP-induced immune signaling in Arabidopsis.

S-acylation is a reversible protein post-translational modification mediated by protein S-acyltransferases (PATs). How S-acylation regulates plant innate immunity is our main concern. Here, we show that the plant immune receptor P2K1 (DORN1, LecRK-I.9; extracellular ATP receptor) directly interacts with and phosphorylates Arabidopsis PAT5 and PAT9 to stimulate their S-acyltransferase activity. This leads, in a time-dependent manner, to greater S-acylation of P2K1, which dampens the immune response. pat5 and pat9 mutants have an elevated extracellular ATP-induced immune response, limited bacterial invasion, increased phosphorylation and decreased degradation of P2K1 during immune signaling. Mutation of S-acylated cysteine residues in P2K1 results in a similar phenotype. Our study reveals that S-acylation effects the temporal dynamics of P2K1 receptor activity, through autophosphorylation and protein degradation, suggesting an important role for this modification in regulating the ability of plants in respond to external stimuli.

Coordination of microbe-host homeostasis by crosstalk with plant innate immunity.

Plants grown in natural soil are colonized by phylogenetically structured communities of microbes known as the microbiota. Individual microbes can activate microbe-associated molecular pattern (MAMP)-triggered immunity (MTI), which limits pathogen proliferation but curtails plant growth, a phenomenon known as the growth-defence trade-off. Here, we report that, in monoassociations, 41% (62 out of 151) of taxonomically diverse root bacterial commensals suppress Arabidopsis thaliana root growth inhibition (RGI) triggered by immune-stimulating MAMPs or damage-associated molecular patterns. Amplicon sequencing of bacterial 16S rRNA genes reveals that immune activation alters the profile of synthetic communities (SynComs) comprising RGI-non-suppressive strains, whereas the presence of RGI-suppressive strains attenuates this effect. Root colonization by SynComs with different complexities and RGI-suppressive activities alters the expression of 174 core host genes, with functions related to root development and nutrient transport. Furthermore, RGI-suppressive SynComs specifically downregulate a subset of immune-related genes. Precolonization of plants with RGI-suppressive SynComs, or mutation of one commensal-downregulated transcription factor, MYB15, renders the plants more susceptible to opportunistic Pseudomonas pathogens. Our results suggest that RGI-non-suppressive and RGI-suppressive root commensals modulate host susceptibility to pathogens by either eliciting or dampening MTI responses, respectively. This interplay buffers the plant immune system against pathogen perturbation and defence-associated growth inhibition, ultimately leading to commensal-host homeostasis.

The novel pathogen-responsive glycosyltransferase UGT73C7 mediates the redirection of phenylpropanoid metabolism and promotes SNC1-dependent Arabidopsis immunity.

Recent studies have shown that global metabolic reprogramming is a common event in plant innate immunity; however, the relevant molecular mechanisms remain largely unknown. Here, we identified a pathogen-induced glycosyltransferase, UGT73C7, that plays a critical role in Arabidopsis disease resistance through mediating redirection of the phenylpropanoid pathway. Loss of UGT73C7 function resulted in significantly decreased resistance to Pseudomonas syringae pv. tomato DC3000, whereas constitutive overexpression of UGT73C7 led to an enhanced defense response. UGT73C7-activated immunity was demonstrated to be dependent on the upregulated expression of SNC1, a Toll/interleukin 1 receptor-type NLR gene. Furthermore, in vitro and in vivo assays indicated that UGT73C7 could glycosylate p-coumaric acid and ferulic acid, the upstream metabolites in the phenylpropanoid pathway. Mutations that lead to the loss of UGT73C7 enzyme activities resulted in the failure to induce SNC1 expression. Moreover, glycosylation activity of UGT73C7 resulted in the redirection of phenylpropanoid metabolic flux to biosynthesis of hydroxycinnamic acids and coumarins. The disruption of the phenylpropanoid pathway suppressed UGT73C7-promoted SNC1 expression and the immune response. This study not only identified UGT73C7 as an important regulator that adjusts phenylpropanoid metabolism upon pathogen challenge, but also provided a link between phenylpropanoid metabolism and an NLR gene.

Bacterial effector targeting of a plant iron sensor facilitates iron acquisition and pathogen colonization.

Acquisition of nutrients from different species is necessary for pathogen colonization. Iron is an essential mineral nutrient for nearly all organisms, but little is known about how pathogens manipulate plant hosts to acquire iron. Here, we report that AvrRps4, an effector protein delivered by Pseudomonas syringae bacteria to plants, interacts with and targets the plant iron sensor protein BRUTUS (BTS) to facilitate iron uptake and pathogen proliferation in Arabidopsis thaliana. Infection of rps4 and eds1 by P. syringae pv. tomato (Pst) DC3000 expressing AvrRps4 resulted in iron accumulation, especially in the plant apoplast. AvrRps4 alleviates BTS-mediated degradation of bHLH115 and ILR3(IAA-Leucine resistant 3), two iron regulatory proteins. In addition, BTS is important for accumulating immune proteins Enhanced Disease Susceptibility1 (EDS1) at both the transcriptional and protein levels upon Pst (avrRps4) infections. Our findings suggest that AvrRps4 targets BTS to facilitate iron accumulation and BTS contributes to RPS4/EDS1-mediated immune responses.

SUMO enables substrate selectivity by mitogen-activated protein kinases to regulate immunity in plants.

The versatility of mitogen-activated protein kinases (MAPKs) in translating exogenous and endogenous stimuli into appropriate cellular responses depends on its substrate specificity. In animals, several mechanisms have been proposed about how MAPKs maintain specificity to regulate distinct functional pathways. However, little is known of mechanisms that enable substrate selectivity in plant MAPKs. Small ubiquitin-like modifier (SUMO), a posttranslational modification system, plays an important role in plant development and defense by rapid reprogramming of cellular events. In this study we identified a functional SUMO interaction motif (SIM) in Arabidopsis MPK3 and MPK6 that reveals a mechanism for selective interaction of MPK3/6 with SUMO-conjugated WRKY33, during defense. We show that WRKY33 is rapidly SUMOylated in response to Botrytis cinerea infection and flg22 elicitor treatment. SUMOylation mediates WRKY33 phosphorylation by MPKs and consequent transcription factor activity. Disruption of either WRKY33 SUMO or MPK3/6 SIM sites attenuates their interaction and inactivates WRKY33-mediated defense. However, MPK3/6 SIM mutants show normal interaction with a non-SUMOylated form of another transcription factor, SPEECHLESS, unraveling a role for SUMOylation in differential substrate selectivity by MPKs. We reveal that the SUMO proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2 control WRKY33 SUMOylation and demonstrate a role for these SUMO proteases in defense. Our data reveal a mechanism by which MPK3/6 prioritize molecular pathways by differentially selecting substrates using the SUMO-SIM module during defense responses.