FLAGELLIN SENSING Research Articles

Proteobacteria Contain Diverse flg22 Epitopes That Elicit Varying Immune Responses in Arabidopsis thaliana.

Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22-amino-acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum Proteobacteria, using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif (11[ST]xx[DN][DN]xAGxxI21) in the flg22 sequences, followed by sequence conservation analysis and protein structural modeling. These data led us to hypothesize that most flg22 epitopes of the γ- and β-Proteobacteria are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε-Proteobacteria are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in Arabidopsis thaliana. The flg22 peptides of γ- and β-Proteobacteria triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α-Proteobacteria triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum Proteobacteria. We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of A. thaliana FLS2 was driven by the flg22 epitopes of the γ- and β-Proteobacteria, a monophyletic group containing many common phytopathogens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

DYNAMIN-RELATED PROTEIN DRP1A functions with DRP2B in plant growth, flg22-immune responses, and endocytosis.

Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.

Lumi-Map, a Real-Time Luciferase Bioluminescence Screen of Mutants Combined with MutMap, Reveals Arabidopsis Genes Involved in PAMP-Triggered Immunity.

Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single-nucleotide polymorphisms (SNPs) for studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole-genome sequencing-based method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing the LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). Although five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2.

Plants mount defense responses by recognizing indicators of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin, from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), contains two MAMPs, flg22 and flgII-28, that are recognized by tomato (Solanum lycopersicum) receptors Flagellin sensing2 (Fls2) and Fls3, respectively, but to what degree each receptor contributes to immunity and whether they promote immune responses using the same molecular mechanisms are unknown. Here, we characterized CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants and found that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, we observed striking differences in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species and an increase in transcript abundance of 44 tomato genes, with two genes serving as specific reporters for the Fls3 pathway. Fls3 had greater in vitro kinase activity than Fls2 and could transphosphorylate a substrate. Using chimeric Fls2/Fls3 proteins, we found no evidence that a single receptor domain is responsible for the Fls3-sustained reactive oxygen species, suggesting involvement of multiple structural features or a nullified function of the chimeric construct. This work reveals differences in certain immunity outputs between Fls2 and Fls3, suggesting that they might use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.

Conserved exitrons of FLAGELLIN-SENSING 2 (FLS2) across dicot plants and their functions

The alternative splicing of pattern recognition receptor genes regulates immune signalling in mammals, but in plants its role is still unknown. Here, we detected alternatively spliced introns (exitrons) in the first annotated exons of FLAGELLIN-SENSING 2 (FLS2) genes in all the examined dicot plants across nine families. The 5′ splice site (SS) regions were conserved and with rare synonymous substitutions. Point mutations and gene swaps indicated that the position and efficiency of exitron splicing primarily depended on the nucleotide sequences of FLS2 genes. Single-nucleotide mutations in the invariable codon carrying 5′ SS dramatically altered the accumulation of poplar and tomato FLS2 transcripts, indicating the 5′-proximal exitrons of FLS2 function as stimulatory introns on gene expression. The 3′ SSs of exitrons are diverse and can be changed by 1–2 nucleotide mutations in Salicaceae FLS2. The alternative transcripts (ATs) of poplar and tobacco FLS2, which encode small secreted proteins, were specifically induced by flg22, and one such AT from tobacco FLS2 suppressed flg22-induced response. Our results indicated that the exitrons of FLS2 genes regulate the accumulation of transcripts by an intron mediated enhancement (IME) mechanism and some ATs have the potential to encode suppressors for FLS2 pathway.

STRESS INDUCED FACTOR 2 Regulates Arabidopsis Stomatal Immunity through Phosphorylation of the Anion Channel SLAC1.

Upon recognition of microbes, pattern recognition receptors (PRRs) activate pattern-triggered immunity. FLAGELLIN SENSING2 (FLS2) and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) form a typical PRR complex that senses bacteria. Here, we report that the kinase activity of the malectin-like receptor-like kinase STRESS INDUCED FACTOR 2 (SIF2) is critical for Arabidopsis (Arabidopsis thaliana) resistance to bacteria by regulating stomatal immunity. SIF2 physically associates with the FLS2-BAK1 PRR complex and interacts with and phosphorylates the guard cell SLOW ANION CHANNEL1 (SLAC1), which is necessary for abscisic acid (ABA)-mediated stomatal closure. SIF2 is also required for the activation of ABA-induced S-type anion currents in Arabidopsis protoplasts, and SIF2 is sufficient to activate SLAC1 anion channels in Xenopus oocytes. SIF2-mediated activation of SLAC1 depends on specific phosphorylation of Ser 65. This work reveals that SIF2 functions between the FLS2-BAK1 initial immunity receptor complex and the final actuator SLAC1 in stomatal immunity.

Ligand-induced monoubiquitination of BIK1 regulates plant immunity.

Recognition of microbe-associated molecular patterns (MAMPs) via plasma membrane (PM)-resident pattern recognition receptors (PRRs) triggers the first line of inducible defense against invading pathogens1–3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms underlying PM-tethered RLCK activation remain elusive. We report here that, upon MAMP perception, RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent BIK1 release from the FLS2-BAK1 complex and immune signaling activation. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics distinct from those of PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and elucidates that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signaling.

The Arabidopsis exocyst subunits EXO70B1 and EXO70B2 regulate FLS2 homeostasis at the plasma membrane.

The plasma membrane (PM)-localized receptor kinase FLAGELLIN SENSING 2 (FLS2) recognizes bacterial flagellin or its immunogenic epitope flg22, and initiates microbe-associated molecular pattern-triggered immunity, which inhibits infection by bacterial pathogens. The localization, abundance and activity of FLS2 are under dynamic control. Here, we demonstrate that Arabidopsis thaliana EXO70B1, a subunit of the exocyst complex, plays a critical role in FLS2 signaling that is independent of the truncated Toll/interleukin-1 receptor-nucleotide binding sequence protein TIR-NBS2 (TN2). In the exo70B1-3 mutant, the abundance of FLS2 protein at the PM is diminished, consistent with the impaired flg22 response of this mutant. EXO70B1-GFP plants showed increased FLS2 accumulation at the PM and therefore enhanced FLS2 signaling. The EXO70B1-mediated trafficking of FLS2 to the PM is partially independent of the PENETRATION 1 (PEN1)-containing secretory pathway. In addition, EXO70B1 interacts with EXO70B2, a close homolog of EXO70B1, and both proteins associate with FLS2 and contribute to the accumulation of FLS2 at the PM. Taken together, our data suggest that the exocyst complex subunits EXO70B1 and EXO70B2 regulate the trafficking of FLS2 to the PM, which represents a new layer of regulation of FLS2 function in plant immunity.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

Perception of Agrobacterium tumefaciens flagellin by FLS2XL confers resistance to crown gall disease.

Bacterial flagella are perceived by the innate immune systems of plants1 and animals2 alike, triggering resistance. Common to higher plants is the immunoreceptor FLAGELLIN-SENSING 2 (FLS2)3, which detects flagellin via its most conserved epitope, flg22. Agrobacterium tumefaciens, which causes crown gall disease in many crop plants, has a highly diverged flg22 epitope and evades immunodetection by plants so far studied. We asked whether, as a next step in this game of 'hide and seek', there are plant species that have evolved immunoreceptors with specificity for the camouflaged flg22Atum of A. tumefaciens. In the wild grape species Vitis riparia, we discovered FLS2XL, a previously unknown form of FLS2, that provides exquisite sensitivity to typical flg22 and to flg22Atum. As exemplified by ectopic expression in tobacco, FLS2XL can limit crown gall disease caused by A. tumefaciens.

Silencing GmFLS2 enhances the susceptibility of soybean to bacterial pathogen through attenuating the activation of GmMAPK signaling pathway

The plasma membrane (PM)-localized receptor-like kinases (RLKs) play important roles in pathogen defense. One of the first cloned RLKs is the Arabidopsis receptor kinase FLAGELLIN SENSING 2 (FLS2), which specifically recognizes a conserved 22 amino acid N-terminal sequence of Pseudomonas syringae pv．tomato DC3000 (Pst) flagellin protein (flg22). Although extensively studied in Arabidopsis, the functions of RLKs in crop plants remain largely uninvestigated. To understand the roles of RLKs in soybean (Glycine max), GmFLS2 was silenced via virus induced gene silencing (VIGS) mediated by Bean pod mottle virus (BPMV). No significant morphological differences were observed between GmFLS2-silenced plants and the vector control plants. However, silencing GmFLS2 significantly enhanced the susceptibility of the soybean plants to Pseudomonas syringae pv．glycinea (Psg). Kinase activity assay showed that silencing GmFLS2 significantly reduced the phosphorylation level of GmMPK6 in response to flg22 treatment. However, reduced phosphorylation level of both GmMPK3 and GmMPK6 in response to Psg infection was observed in GmFLS2-silenced plants, implying that defense response is likely transduced through activation of the downstream GmMAPK signaling pathway upon recognition of bacterial pathogen by GmFLS2. The core peptides of flg22 from Pst and Psg were highly conserved and only 4 amino acid differences were seen at their N-termini. Interestingly, it appeared that the Psg-flg22 was more effective in activating soybean MAPKs than activating Arabidopsis MAPKs, and conversely, Pst-flg22 was more effective in activating Arabidopsis MAPKs than activating soybean MAPKs, suggesting that the cognate recognition is more potent than heterologous recognition in activating downstream signaling. Taken together, our results suggest that the function of FLS2 is conserved in immunity against bacteria pathogens across different plant species.

The C4 Protein from Tomato Yellow Leaf Curl Virus Can Broadly Interact with Plant Receptor-Like Kinases.

Plant receptor-like kinases (RLKs) exert an essential function in the transduction of signals from the cell exterior to the cell interior, acting as important regulators of plant development and responses to environmental conditions. A growing body of evidence suggests that RLKs may play relevant roles in plant-virus interactions, although the details and diversity of effects and underlying mechanisms remain elusive. The C4 protein from different geminiviruses has been found to interact with RLKs in the CLAVATA 1 (CLV1) clade. However, whether C4 can interact with RLKs in other subfamilies and, if so, what the biological impact of such interactions might be, is currently unknown. In this work, we explore the interaction landscape of C4 from the geminivirus Tomato yellow leaf curl virus within the Arabidopsis RLK family. Our results show that C4 can interact with RLKs from different subfamilies including, but not restricted to, members of the CLV1 clade. Functional analyses of the interaction of C4 with two well-characterized RLKs, FLAGELLIN SENSING 2 (FLS2) and BRASSINOSTEROID INSENSITIVE 1 (BRI1), indicate that C4 might affect some, but not all, RLK-derived outputs. The results presented here offer novel insight on the interface between RLK signaling and the infection by geminiviruses, and point at C4 as a potential broad manipulator of RLK-mediated signaling.

PAMP-induced peptide 1 cooperates with salicylic acid to regulate stomatal immunity in Arabidopsis thaliana

ABSTRACTVarious pathogenic species are capable of penetrating plant leaves through stomata on the leaf surface for propagation by absorbing nutrients in plant interiors. Plants have evolved abilities to close stomata to restrict pathogen infections. The model plant Arabidopsis (Arabidopsis thaliana) closes stomata when FLAGELLIN SENSING2 (FLS2), a receptor protein localized in the plasma membrane (PM) of stomatal guard cells, detects flagellin, a pathogen-associated molecular pattern (PAMP) derived from the bacterial pathogen Pseudomonas syringae. It currently remains largely unknown how flagellin-FLS2 signaling initiates stomatal closure. Our previous studies showed that PAMP-INDUCED PEPTIDE1 (PIP1), an Arabidopsis endogenous peptide, activates immune responses through a PM-localized receptor, RECEPTOR-LIKE KINASE7 (RLK7). Here, we demonstrate that PIP1-RLK7 act downstream of FLS2 to activate stomatal immunity against the bacterial strain Pseudomonas syringe pv. tomato (Pst) DC3118. PIP1 promotes the expression of genes involved in salicylic acid (SA) biosynthesis. SA contributes to the expression of PIP1 preligand prePIP1 and the PIP1-induced stomatal closure. In contrast, methl jasmonate (MJ) and a pathogen-derived jasmonate mimic coronatine (COR) performs an opposite function of SA. SA also promotes the PIP1-induced production of reactive oxygen species (ROS) which is required for PIP1-induced stomatal closure. Overall, PIP1 and SA may form a positive feedback loop to regulate ROS-mediated stomatal immunity in Arabidopsis.

Temporary heat stress suppresses PAMP-triggered immunity and resistance to bacteria in Arabidopsis thaliana.

SummaryRecognition of pathogen‐associated molecular patterns (PAMPs) is crucial for plant defence against pathogen attack. The best characterized PAMP is flg22, a 22 amino acid conserved peptide from flagellin protein. In Arabidopsis thaliana, flg22 is recognized by the flagellin sensing 2 (FLS2) receptor. In this study, we focused on biotic stress responses triggered by flg22 after exposure to temporary heat stress (HS). It is important to study the reactions of plants to multiple stress conditions because plants are often exposed simultaneously to a combination of both abiotic and biotic stresses. Transient early production of reactive oxygen species (ROS) is a well‐characterized response to PAMP recognition. We demonstrate the strong reduction of flg22‐induced ROS production in A. thaliana after HS treatment. In addition, a decrease in FLS2 transcription and a decrease of the FLS2 presence at the plasma membrane are shown after HS. In summary, our data show the strong inhibitory effect of HS on flg22‐triggered events in A. thaliana. Subsequently, temporary HS strongly decreases the resistance of A. thaliana to Pseudomonas syringae. We propose that short exposure to high temperature is a crucial abiotic stress factor that suppresses PAMP‐triggered immunity, which subsequently leads to the higher susceptibility of plants to pathogens.

A Plant Immune Receptor Degraded by Selective Autophagy

Plants recycle non-activated immune receptors to maintain a functional immune system. The Arabidopsis immune receptor kinase FLAGELLIN-SENSING 2 (FLS2) recognizes bacterial flagellin. However, the molecular mechanisms by which non-activated FLS2 and other non-activated plant PRRs are recycled remain not well understood. Here, we provide evidence showing that Arabidopsis orosomucoid (ORM) proteins, which have been known to be negative regulators of sphingolipid biosynthesis, act as selective autophagy receptors to mediate the degradation of FLS2. Arabidopsis plants overexpressing ORM1 or ORM2 have undetectable or greatly diminished FLS2 accumulation, nearly lack FLS2 signaling, and are more susceptible to the bacterial pathogen Pseudomonas syringae. On the other hand, ORM1/2 RNAi plants and orm1 or orm2 mutants generated by the CRISPR/Cas9-mediated gene editing have increased FLS2 accumulation and enhanced FLS2 signaling, and are more resistant to P. syringae. ORM proteins interact with FLS2 and the autophagy-related protein ATG8. Interestingly, overexpression of ORM1 or ORM2 in autophagy-defective mutants showed FLS2 abundance that is comparable to that in wild-type plants. Moreover, FLS2 levels were not decreased in Arabidopsis plants overexpressing ORM1/2 derivatives that do not interact with ATG8. Taken together, these results suggest that selective autophagy functions in maintaining the homeostasis of a plant immune receptor and that beyond sphingolipid metabolic regulation ORM proteins can also act as selective autophagy receptors.

RECEPTOR-LIKE KINASE 902 Associates with and Phosphorylates BRASSINOSTEROID-SIGNALING KINASE1 to Regulate Plant Immunity

Plants employ receptor-like kinases (RLKs) and receptor-like proteins for rapid recognition of invading pathogens, and RLKs then transmit signals to receptor-like cytoplasmic kinases (RLCKs) to activate immune responses. RLKs are under fine regulation mediated by subcellular trafficking, which contributes to proper activation of plant immunity. In this study, we show that Arabidopsis thaliana RECEPTOR-LIKE KINASE 902 (RLK902) plays important roles in resistance to the bacterial pathogen Pseudomonas syringae, but not to the fungal powdery mildew pathogen Golovinomyces cichoracearum. RLK902 localizes at the plasma membrane and associates with ENHANCED DISEASE RESISTANCE 4 (EDR4), a protein involved in clathrin-mediated trafficking pathways. EDR4 and CLATHRIN HEAVY CHAIN 2 (CHC2) regulate the subcellular trafficking and accumulation of RLK902 protein. Furthermore, we found that RLK902 directly associates with the RLCK BRASSINOSTEROID-SIGNALING KINASE1 (BSK1), a key component of plant immunity, but not with other members of the FLAGELLIN SENSING 2 immune complex. RLK902 phosphorylates BSK1, and its Ser-230 is a key phosphorylation site critical for RLK902-mediated defense signaling. Taken together, our data indicate that EDR4 regulates plant immunity by modulating the subcellular trafficking and protein accumulation of RLK902, and that RLK902 transmits immune signals by phosphorylating BSK1.

Transcriptional Regulation of the Immune Receptor FLS2 Controls the Ontogeny of Plant Innate Immunity.

Innate immunity plays a vital role in protecting plants and animals from pathogen infections. Immunity varies with age in both animals and plants. However, little is known about the ontogeny of plant innate immunity during seedling development. We report here that the Arabidopsis (Arabidopsis thaliana) microRNA miR172b regulates the transcription of the immune receptor gene FLAGELLIN-SENSING2 (FLS2) through TARGET OF EAT1 (TOE1) and TOE2, which directly bind to the FLS2 promoter and inhibit its activity. The level of miR172b is very low in the early stage of seedling development but increases over time, which results in decreased TOE1/2 protein accumulation and, consequently, increased FLS2 transcription and the ontogeny of FLS2-mediated immunity during seedling development. Our study reveals a role for the miR172b-TOE1/2 module in regulating plant innate immunity and elucidates a regulatory mechanism underlying the ontogeny of plant innate immunity.plantcell;30/11/2779/FX1F1fx1.

BRASSINOSTEROID-SIGNALING KINASE1 Phosphorylates MAPKKK5 to Regulate Immunity in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) immune receptor FLAGELLIN SENSING2 (FLS2) rapidly forms a complex to activate pathogen-associated molecular pattern-triggered immunity (PTI) upon perception of the bacterial protein flagellin. The receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALINGKINASE1 (BSK1) interacts with FLS2 and is critical for the activation of PTI. However, it is unknown how BSK1 transduces signals to activate downstream immune responses. We identified MEK Kinase5 (MAPKKK5) as a potential substrate of BSK1 by whole-genome phosphorylation analysis. In addition, we demonstrated that BSK1 interacts with and phosphorylates MAPKKK5. In the bsk1-1 mutant, the Ser-289 residue of MAPKKK5 was not phosphorylated as it was in the wild type. Similar to the bsk1 mutant, the mapkkk5 mutant displayed enhanced susceptibility to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000, and to the fungal powdery mildew pathogen Golovinomyces cichoracearum Phosphorylation of the Ser-289 residue is not involved in MAPKKK5-triggered cell death but is critical for MAPKKK5-mediated resistance to both bacterial and fungal pathogens. Furthermore, MAPKKK5 interacts with multiple MAPK kinases, including MKK1, MKK2, MKK4, MKK5, and MKK6. Overall, these results indicate that BSK1 regulates plant immunity by phosphorylating MAPKKK5 and suggest a direct regulatory mode of signaling from the immune complex to the MAPK cascade.

Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis.

The plant transmembrane receptor kinase FLAGELLIN SENSING 2 (FLS2) is crucial for innate immunity. Although previous studies have reported FLS2-mediated signal transduction and endocytosis via the clathrin-mediated pathway, whether additional endocytic pathways affect FLS2-mediated defense responses remains unclear. Here, we show that the Arabidopsis thaliana sterol-deficient mutant steroid methyltransferase 1 displays defects in immune responses induced by the flagellin-derived peptide flg22. Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) coupled with single-particle tracking showed that the spatiotemporal dynamics of FLS2-GFP changed on a millisecond time scale and that the FLS2-GFP dwell time at the plasma membrane increased in cells treated with a sterol-extracting reagent when compared with untreated counterparts. We further demonstrate that flg22-induced FLS2 clustering and endocytosis involves the sterol-associated endocytic pathway, which is distinct from the clathrin-mediated pathway. Moreover, flg22 enhanced the colocalization of FLS2-GFP with the membrane microdomain marker Flot 1-mCherry and FLS2 endocytosis via the sterol-associated pathway. This indicates that plants may respond to pathogen attacks by regulating two different endocytic pathways. Taken together, our results suggest the key role of sterol homeostasis in flg22-induced plant defense responses.

Nonredundant functions of Arabidopsis LecRK-V.2 and LecRK-VII.1 in controlling stomatal immunity and jasmonate-mediated stomatal closure.

Stomatal immunity restricts bacterial entry to leaves through the recognition of microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors (PRRs) and downstream abscisic acid and salicylic acid signaling. Through a reverse genetics approach, we characterized the function of the L-type lectin receptor kinase-V.2 (LecRK-V.2) and -VII.1 (LecRK-VII.1). Analyses of interactions with the PRR FLAGELLIN SENSING2 (FLS2) were performed by co-immunoprecipitation and bimolecular fluorescence complementation and whole-cell patch-clamp analyses were used to evaluate guard cell Ca2+ -permeable cation channels. The Arabidopsis thaliana LecRK-V.2 and LecRK-VII.1 and notably their kinase activities were required for full activation of stomatal immunity. Knockout lecrk-V.2 and lecrk-VII.1 mutants were hyper-susceptible to Pseudomonas syringae infection and showed defective stomatal closure in response to bacteria or to the MAMPs flagellin and EF-Tu. By contrast, Arabidopsis over-expressing LecRK-V.2 or LecRK-VII.1 demonstrated a potentiated stomatal immunity. LecRK-V.2 and LecRK-VII.1 are shown to be part of the FLS2 PRR complex. In addition, LecRK-V.2 and LecRK-VII.1 were critical for methyl jasmonate (MeJA)-mediated stomatal closure, notably for MeJA-induced activation of guard cell Ca2+ -permeable cation channels. This study highlights the role of LecRK-V.2 and LecRK-VII.1 in stomatal immunity at the FLS2 PRR complex and in MeJA-mediated stomatal closure.