Flagellin-sensitive 2 Research Articles

Single-molecule analysis reveals the phosphorylation of FLS2 governs its spatiotemporal dynamics and immunity

The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following Förster resonance energy transfer-fluorescence lifetime imaging microscopy and protein proximity indexes assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2S938D but not FLS2S938A with AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2S938 affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functional AtRem1.3-associated nanodomains, which fills the gap between the FLS2S938 phosphorylation and FLS2-mediated immunity.

Single-molecule analysis reveals the phosphorylation of FLS2 governs its spatiotemporal dynamics and immunity.

The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following Förster resonance energy transfer-fluorescence lifetime imaging microscopy and protein proximity indexes assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2S938D but not FLS2S938A with AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2S938 affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functional AtRem1.3-associated nanodomains, which fills the gap between the FLS2S938 phosphorylation and FLS2-mediated immunity.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

LESION SIMULATING DISEASE 3 regulates disease resistance via fine-tuning histone acetylation in cassava.

Bacterial blight seriously affects the growth and production of cassava (Manihot esculenta Crantz), but disease resistance genes and the underlying molecular mechanism remain unknown. In this study, we found that LESION SIMULATING DISEASE 3 (MeLSD3) is essential for disease resistance in cassava. MeLSD3 physically interacts with SIRTUIN 1 (MeSRT1), inhibiting MeSRT1-mediated deacetylation modification at the acetylation of histone 3 at K9 (H3K9Ac). This leads to increased H3K9Ac levels and transcriptional activation of SUPPRESSOR OF BIR1 (SOBIR1) and FLAGELLIN-SENSITIVE2 (FLS2) in pattern-triggered immunity, resulting in immune responses in cassava. When MeLSD3 was silenced, the release of MeSRT1 directly decreased H3K9Ac levels and inhibited the transcription of SOBIR1 and FLS2, leading to decreased disease resistance. Notably, DELLA protein GIBBERELLIC ACID INSENSITIVE 1 (MeGAI1) also interacted with MeLSD3, which enhanced the interaction between MeLSD3 and MeSRT1 and further strengthened the inhibition of MeSRT1-mediated deacetylation modification at H3K9Ac of defense genes. In summary, this study illustrates the mechanism by which MeLSD3 interacts with MeSRT1 and MeGAI1, thereby mediating the level of H3K9Ac and the transcription of defense genes and immune responses in cassava.

FLS2-RBOHD module regulates changes in the metabolome of Arabidopsis in response to abiotic stress.

Through crosstalk, FLAGELLIN SENSITIVE 2 (FLS2) and RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) are involved in regulating the homeostasis of cellular reactive oxygen species (ROS) and are linked to the metabolic response of plants toward both biotic and abiotic stress. In the present study, we examined the metabolome of Arabidopsis seedlings under drought and salt conditions to better understand the potential role of FLS2 and RBOHD-dependent signaling in the regulation of abiotic stress response. We identified common metabolites and genes that are regulated by FLS2 and RBOHD, and are involved in the response to drought and salt stress. Under drought conditions, D-aspartic acid and the expression of associated genes, such as ASPARAGINE SYNTHASE 2 (ASN2), increased in both fls2 and robed/f double mutants. The accumulation of amino acids, carbohydrates, and hormones, such as L-proline, D-ribose, and indoleacetaldehyde increased in both fls2 and rbohd/f double mutants under salt conditions, as did the expression of related genes, such as PROLINE IMINOPEPTIDASE, PHOSPHORIBOSYL PYROPHOSPHATE SYNTHASE 5, and NITRILASE 3. Collectively, these results indicate that the FLS2-RBOHD module regulates plant response to drought and salt stress through ROS signaling by adjusting the accumulation of metabolites and expression of genes related to metabolite synthesis.

Involvement of a receptor-like kinase complex of FvFLS2 and FvBAK1 in brassinosteroids-induced immunity in postharvest strawberry fruit

Conserved pathogen-derived features of microbial invaders are sensed by plants via pattern-recognition receptors (PRRs) and induce both local and systemic immune responses. The plant PRR flagellin sensitive 2 (FLS2), a leucine-rich repeat-receptor kinase, recognizes pathogen-derived molecules and initiates pattern-triggered immunity (PTI) responses by associating with other potential kinases to propagate downstream signalling cascades. Here, we identified a plasma membrane-localized receptor-like kinase BAK1 from strawberry (FvBAK1) that is rapidly induced upon exposure to the plant-specific hormone brassinosteroid (BR), which provides strawberry fruit with immunity to the necrotrophic fungus Botrytis cinerea. FvBAK1 reciprocally interacts with FvFLS2 and the receptor-like cytoplasmic kinase FvBIK1 in yeast cells in vivo. Subsequently, FvBIK1 can directly interact with respiratory burst oxidase homologue D (FvRBOHD), contributing to FvRBOHD-dependent ROS accumulation to amplify the SA signalling pathway in the BR-stimulated immunity of postharvest strawberry fruit to B. cinerea infection. Importantly, overexpression of bak1 promotes ROS accumulation and BR-induced defence responses, whereas bak1 loss-of-function mutants have compromised BR-induced SA signalling and immunity to necrotrophic fungal pathogens. In such a situation, FvBAK1 is an essential component of BR-mediated PTI signalling, which links the pathogen-derived receptor complex to downstream intracellular signalling pathways. Taken together, the findings of the current study highlight the importance of FvBAK1 as an upstream regulator of FvBIK1 and as a coreceptor of FvFLS2 in establishing BR-induced PTI signalling in postharvest strawberry fruit.

Super-Resolution Imaging of Plant Receptor-Like Kinases Uncovers Their Colocalization and Coordination with Nanometer Resolution.

Plant cell signaling often relies on the cellular organization of receptor-like kinases (RLKs) within membrane nanodomains to enhance signaling specificity and efficiency. Thus, nanometer-scale quantitative analysis of spatial organizations of RLKs could provide new understanding of mechanisms underlying plant responses to environmental stress. Here, we used stochastic optical reconstruction fluorescence microscopy (STORM) to quantify the colocalization of the flagellin-sensitive-2 (FLS2) receptor and the nanodomain marker, remorin, within Arabidopsis thaliana root hair cells. We found that recovery of FLS2 and remorin in the plasma membrane, following ligand-induced internalization by bacterial-flagellin-peptide (flg22), reached ~85% of their original membrane density after ~90 min. The pairs colocalized at the membrane at greater frequencies, compared with simulated randomly distributed pairs, except for directly after recovery, suggesting initial uncoordinated recovery followed by remorin and FLS2 pairing in the membrane. The purinergic receptor, P2K1, colocalized with remorin at similar frequencies as FLS2, while FLS2 and P2K1 colocalization occurred at significantly lower frequencies, suggesting that these RLKs mostly occupy distinct nanodomains. The chitin elicitor receptor, CERK1, colocalized with FLS2 and remorin at much lower frequencies, suggesting little coordination between CERK1 and FLS2. These findings emphasize STORM's capacity to observe distinct nanodomains and degrees of coordination between plant cell receptors, and their respective immune pathways.

The host exocyst complex is targeted by a conserved bacterial type-III effector that promotes virulence.

For most Gram-negative bacteria, pathogenicity largely depends on the type-III secretion system that delivers virulence effectors into eukaryotic host cells. The subcellular targets for the majority of these effectors remain unknown. Xanthomonas campestris, the causal agent of black rot disease of crucifers such as Brassica spp., radish, and turnip, delivers XopP, a highly conserved core-effector protein produced by X. campestris, which is essential for virulence. Here, we show that XopP inhibits the function of the host-plant exocyst complex by direct targeting of Exo70B, a subunit of the exocyst complex, which plays a significant role in plant immunity. XopP interferes with exocyst-dependent exocytosis and can do this without activating a plant NOD-like receptor that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host's pathogen-associated molecular pattern (PAMP)-triggered immunity by blocking exocytosis of pathogenesis-related protein-1A, callose deposition, and localization of the FLAGELLIN SENSITIVE2 (FLS2) immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defenses represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.

RGI‐GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity

Plant immune responses must be tightly controlled for proper allocation of resources for growth and development. In plants, endogenous signaling peptides regulate developmental and growth‐related processes. Recent research indicates that some of these peptides also have regulatory functions in the control of plant immune responses. This classifies these peptides as phytocytokines as they show analogies with metazoan cytokines. However, the mechanistic basis for phytocytokine‐mediated regulation of plant immunity remains largely elusive. Here, we identify GOLVEN2 (GLV2) peptides as phytocytokines in Arabidopsis thaliana. GLV2 signaling enhances sensitivity of plants to elicitation with immunogenic bacterial elicitors and contributes to resistance against virulent bacterial pathogens. GLV2 is perceived by ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors. RGI mutants show reduced elicitor sensitivity and enhanced susceptibility to bacterial infection. RGI3 forms ligand‐induced complexes with the pattern recognition receptor (PRR) FLAGELLIN SENSITIVE 2 (FLS2), suggesting that RGIs are part of PRR signaling platforms. GLV2‐RGI signaling promotes PRR abundance independent of transcriptional regulation and controls plant immunity via a previously undescribed mechanism of phytocytokine activity.

FLS2-RBOHD-PIF4 Module Regulates Plant Response to Drought and Salt Stress.

As sessile organisms, plants are constantly challenged by several environmental stresses. Different kinds of stress often occur simultaneously, leading to the accumulation of reactive oxygen species (ROS) produced by respiratory burst oxidase homolog (RBOHD) and calcium fluctuation in cells. Extensive studies have revealed that flagellin sensitive 2 (FLS2) can sense the infection by pathogenic microorganisms and activate cellular immune response by regulating intracellular ROS and calcium signals, which can also be activated during plant response to abiotic stress. However, little is known about the roles of FLS2 and RBOHD in regulating abiotic stress. In this study, we found that although the fls2 mutant showed tolerance, the double mutant rbohd rbohf displayed hypersensitivity to abiotic stress, similar to its performance in response to immune stress. An analysis of the transcriptome of the fls2 mutant and rbohd rbohf double mutant revealed that phytochrome interacting factor 4 (PIF4) acted downstream of FLS2 and RBOHD to respond to the abiotic stress. Further analysis showed that both FLS2 and RBOHD regulated the response of plants to drought and salt stress by regulating the expression of PIF4. These findings revealed an FLS2–RBOHD–PIF4 module in regulating plant response to biotic and abiotic stresses.

Phosphorylation of BIK1 is critical for interaction with downstream signaling components.

Botrytis-induced Kinase 1 (BIK1) is a receptor-like cytoplasmic kinase (RLCK) involved in the defense, growth, and development of higher plants. It interacts with various receptor-like kinases (RLKs) such as Brassinosteroid Insensitive 1 (BRI1), Flagellin Sensitive 2 (FLS2), and Perception of the Arabidopsis Danger Signal Peptide 1 (PEPR1), but little is known about signaling downstream of BIK1. In this study, we aimed to identify Arabidopsis thaliana BIK1 (AtBIK1) and Brassica rapa BIK1 (BrBIK1) interacting proteins, which is downstream signaling components in Arabidopsis. In addition, the effect of BIK1 phosphorylation on their interaction were examined. For yeast two hybrid (Y2H) screening, a B. rapa cDNA activation domain (AD) library and an A. thaliana cDNA library were used. Reverse reaction (LR) recombinations of appropriate open reading frames (AtBIK1, BrBIK1, AtRGP2, AtPATL2, AtPP7) in either pDONR207 or pDONR/zeo were performed with the split-YFP destination vectors pDEST-GWVYNE and pDEST-GWVYCE to generate N- or C-terminal fusions with the N- and C-terminal yellow fluorescent protein (YFP) moieties, respectively. Recombined vectors were transformed into Agrobacterium strain GV3101. The described GST-AtBIK1, Flag-AtBIK1, and Flag-BrBIK1 constructs were used as templates for site-directed mutagenesis with a QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). In results, A. thaliana BIK1 (AtBIK1) displays strong autophosphorylation kinase activity on tyrosine and threonine residues, whereas B. rapa BIK1 (BrBIK1) does not exhibit autophosphorylation kinase activity in vitro. Herein, we demonstrated that four proteins (RGP2, PATL2, PP7, and SULTR4.1) interact with BrBIK1 but not AtBIK1 in a Y2H system. To confirm interactions between BIK1 and protein candidates in Nicotiana benthamiana, BiFC analysis was performed and it was found that only BrBIK1 bound the three proteins tested. Three phosphosites, T90, T362, and T368, based on amino acid sequence alignment between AtBIK1 and BrBIK1, and performed site-directed mutagenesis (SDM) on AtBIK1 and BrBIK. S90T, P362T, and A369T mutations in BrBIK1 restored autophosphorylation kinase activity on threonine residues comparable to AtBIK1. However, T90A, T362P, and T368A mutations in AtBIK1 did not alter autophosphorylation kinase activity on threonine residues compared with wild-type AtBIK1. BiFC results showed that BIK1 mutations restored kinase activity led to the loss of the binding activity to RGP2, PATL2, or PP7 proteins. Phospho-BIK1 might be involved in plant innate immunity, while non-phospho BIK1 may regulate plant growth and development through interactions with RGP2, PATL2, and PP7.

The receptor-like cytoplasmic kinase CDG1 negatively regulates Arabidopsis pattern-triggered immunity and is involved in AvrRpm1-induced RIN4 phosphorylation.

Arabidopsis CDG1 negatively regulates flg22- and chitin-triggered immunity by promoting FLS2 and CERK1 degradation and is partially required for bacterial effector AvrRpm1-induced RIN4 phosphorylation. Negative regulators play indispensable roles in pattern-triggered immunity in plants by preventing sustained immunity impeding growth. Here, we report Arabidopsis thaliana CONSTITUTIVE DIFFERENTIAL GROWTH1 (CDG1), a receptor-like cytoplasmic kinase VII member, as a negative regulator of bacterial flagellin/flg22- and fungal chitin-triggered immunity. CDG1 can interact with the flg22 receptor FLAGELLIN SENSITIVE2 (FLS2) and chitin co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (CERK1). CDG1 overexpression impairs flg22 and chitin responses by promoting the degradation of FLS2 and CERK1. This process requires the kinase activity of MEK KINASE1 (MEKK1), but not the Plant U-Box (PUB) ubiquitin E3 ligases PUB12 and PUB13. Interestingly, the Pseudomonas syringae effector AvrRpm1 can induce CDG1 to interact with its host target RPM1-INTERACTING PROTEIN4 (RIN4), which depends on the ADP-ribosyl transferase activity of AvrRpm1. CDG1 is capable of phosphorylating RIN4 in vitro at multiple sites including Thr166 and the AvrRpm1-induced Thr166 phosphorylation of RIN4 is diminished in cdg1 null plants. Accordingly, CDG1 knockout attenuates AvrRpm1-induced hypersensitive response and increases the growth of AvrRpm1-secreting bacteria in plants. Unexpectedly, AvrRpm1 can also induce FLS2 depletion, which is fully dependent on RIN4 and partially dependent on CDG1, but does not require the kinase activity of MEKK1. Collectively, this study reveals previously unknown functions of CDG1 in both pattern-triggered immunity and effector-triggered susceptibility in plants.

Multi-Omics Revealed Molecular Mechanisms Underlying Guard Cell Systemic Acquired Resistance.

Systemic Acquired Resistance (SAR) improves immunity of plant systemic tissue after local exposure to a pathogen. Guard cells that form stomatal pores on leaf surfaces recognize bacterial pathogens via pattern recognition receptors, such as Flagellin Sensitive 2 (FLS2). However, how SAR affects stomatal immunity is not known. In this study, we aim to reveal molecular mechanisms underlying the guard cell response to SAR using multi-omics of proteins, metabolites and lipids. Arabidopsis plants previously exposed to pathogenic bacteria Pseudomonas syringae pv. tomato DC3000 (Pst) exhibit an altered stomatal response compared to control plants when they are later exposed to the bacteria. Reduced stomatal apertures of SAR primed plants lead to decreased number of bacteria in leaves. Multi-omics has revealed molecular components of SAR response specific to guard cells functions, including potential roles of reactive oxygen species (ROS) and fatty acid signaling. Our results show an increase in palmitic acid and its derivative in the primed guard cells. Palmitic acid may play a role as an activator of FLS2, which initiates stomatal immune response. Improved understanding of how SAR signals affect stomatal immunity can aid biotechnology and marker-based breeding of crops for enhanced disease resistance.

Twisting mode of supercoil leucine-rich domain mediates peptide sensing in FLS2–flg22–BAK1 complex**Project supported by the National Natural Science Foundation of China (Grant No. 11704140), self-determined research funds of CCNU from the Colleges’ Basic Research and Operation of MOE (Grant No. CCNU20TS004) (Y. Z.), and the China Scholarship Council Fund (Grant No. 201708420039) (L. P.).

Plants and animals recognize microbial invaders by detecting pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs). This recognition plays a crucial role in plant immunity. The newly discovered protein in plants that responds to bacterial flagellin, i.e., flagellin-sensitive 2 (FLS2), is ubiquitously expressed and present in many plants. The association of FLS2 and BAK1, facilitated by a highly conserved epitope flg22 of flagellin, triggers such downstream immune responses as activated MAPK pathway and elevated reactive oxygen species (ROS) for bacterial defense and plant immunity. Here we study the intrinsic dynamics and conformational change of FLS2 upon the formation of the FLS2–flg22–BAK1 complex. The top intrinsic normal modes and principal structural fluctuation components are very similar, showing two bending modes and one twisting mode. The twisting mode alone, however, accounts for most of the conformational change of FLS2 induced by binding with flg22 and BAK1. This study indicates that flg22 binding suppresses FLS2 conformational fluctuation, especially on the twisting motion, thus facilitating FLS2–BAK1 interaction. A detailed analysis of this sensing mechanism may aid better design on both PRR and peptide mimetics for plant immunity.

SUMO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity

Detection of conserved microbial patterns by host cell surface pattern recognition receptors (PRRs) activates innate immunity. The FLAGELLIN-SENSITIVE 2 (FLS2) receptor perceives bacterial flagellin and recruits another PRR, BAK1 and the cytoplasmic-kinase BIK1 to form an active co-receptor complex that initiates antibacterial immunity in Arabidopsis. Molecular mechanisms that transmit flagellin perception from the plasma-membrane FLS2-associated receptor complex to intracellular events are less well understood. Here, we show that flagellin induces the conjugation of the SMALL UBIQUITIN-LIKE MODIFIER (SUMO) protein to FLS2 to trigger release of BIK1. Disruption of FLS2 SUMOylation can abolish immune responses, resulting in susceptibility to bacterial pathogens in Arabidopsis. We also identify the molecular machinery that regulates FLS2 SUMOylation and demonstrate a role for the deSUMOylating enzyme, Desi3a in innate immunity. Flagellin induces the degradation of Desi3a and enhances FLS2 SUMOylation to promote BIK1 dissociation and trigger intracellular immune signalling.

Variable Effects of C-Terminal Fusions on FLS2 Function: Not All Epitope Tags Are Created Equal.

Receptor-like kinases (RLKs) are the largest family of proteins in plants and are responsible for perceiving the vast majority of extracellular stimuli. Thus, RLKs function in diverse processes, including sensing pathogen attacks, regulating symbiotic interactions, transducing hormone and peptide signals, and monitoring cell wall status. However, despite their fundamental role in plant biology, very few antibodies are available against RLKs, which necessitates the use of epitope tags and fluorescent protein fusions in biochemical analyses such as immunoblot analysis and intracellular visualization. Epitope tags are widely used and are typically assumed to be benign, with no influence on protein function. FLAGELLIN SENSITIVE2 (FLS2) is the receptor for bacterial flagellin and often is used as a model for RLK function. Previous work implies that carboxyl-terminal epitope fusions to FLS2 maintain protein function. Here, a detailed complementation analysis of Arabidopsis (Arabidopsis thaliana) fls2 mutant plants expressing various FLS2 C-terminal epitope fusions revealed highly variable and unpredictable FLS2-mediated signaling outputs. In addition, only one out of four FLS2 epitope fusions maintained the ability to inhibit plant growth in response to flg22 treatment comparable to that in the wild type or control untagged transgenic lines. These results raise concerns over the widespread use of RLK epitope tag fusions for functional studies. Many of the subtleties of FLS2 function, and by extension those of other RLKs, may have been overlooked or inappropriately interpreted through the use of RLK epitope tag fusions.

A Technical Framework for Studying the Signaling Nexus of Brassinosteroids and Immunity.

Pathway cross-communication cannot be simply tackled by studying isolated signaling systems. Yet understanding how signal transduction pathways attenuate or reinforce each other in vivo is a challenging task. In plants, biosynthesis and signaling of brassinosteroids (BRs) finely regulate growth and defense programs through a complex array of mechanistic and physiological interactions. Conversely, induction of defenses also impacts on the BR biosynthesis at the transcriptional level. In this chapter, we present an experimental framework to study the physiological connection between BR-controlled growth and defenses. We focus on the signaling pathways regulated by the two archetypal cell surface receptors, BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2 (FLS2), to illustrate the signaling nexus of BRs and plant immunity. In Arabidopsis thaliana, these pathways provide one of the very few systems in which the tools and mechanistic details exist to study cross talk at the molecular level.

Regulation of sugar transporter activity for antibacterial defense in Arabidopsis.

Microbial pathogens strategically acquire metabolites from their hosts during infection. Here we show that the host can intervene to prevent such metabolite loss to pathogens. Phosphorylation-dependent regulation of sugar transport protein 13 (STP13) is required for antibacterial defense in the plant Arabidopsis thaliana STP13 physically associates with the flagellin receptor flagellin-sensitive 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1). BAK1 phosphorylates STP13 at threonine 485, which enhances its monosaccharide uptake activity to compete with bacteria for extracellular sugars. Limiting the availability of extracellular sugar deprives bacteria of an energy source and restricts virulence factor delivery. Our results reveal that control of sugar uptake, managed by regulation of a host sugar transporter, is a defense strategy deployed against microbial infection. Competition for sugar thus shapes host-pathogen interactions.

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase.

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.

Rice OsFLS2-Mediated Perception of Bacterial Flagellins Is Evaded by Xanthomonas oryzae pvs. oryzae and oryzicola

Bacterial flagellins are often recognized by the receptor kinase FLAGELLIN SENSITIVE2 (FLS2) and activate MAMP-triggered immunity in dicotyledonous plants. However, the capacity of monocotyledonous rice to recognize flagellins of key rice pathogens and its biological relevance remain poorly understood. We demonstrate that ectopically expressed OsFLS2 in Arabidopsis senses the eliciting flg22 peptide and in vitro purified Acidovorax avenae (Aa) flagellin in an expression level-dependent manner, but does not recognize purified flagellins or derivative flg22Xo peptides of Xanthomonas oryzae pvs. oryzae (Xoo) and oryzicola (Xoc). Consistently, the flg22 peptide and purified Aa flagellin, but not Xoo/Xoc flagellins, induce various immune responses such as defense gene induction and MAPK activation in rice. Perception of flagellin by rice does induce strong resistance to Xoo infection, as shown after pre-treatment of rice leaves with Aa flagellin. OsFLS2 was found to differ from AtFLS2 in its perception specificities or sensitivities to different flg22 sequences. In addition, post-translational modification of Xoc flagellin was altered by deletion of glycosyltransferase-encoding rbfC, but this had little effect on Xoc motility and rpfC mutation did not detectably reduce Xoc virulence on rice. Deletion of flagellin-encoding fliC from Xoo/Xoc blocked swimming motility but also did not significantly alter Xoo/Xoc virulence. These results suggest that Xoo/Xoc carry flg22-region amino acid changes that allow motility while evading the ancient flagellin detection system in rice, which retains recognition capacity for other bacterial pathogens.