Antje Heese

The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants

In pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), plant cell surface receptors sense potential microbial pathogens by recognizing elicitors called PAMPs. Although diverse PAMPs trigger PTI through distinct receptors, the resulting intracellular responses overlap extensively. Despite this, a common component(s) linking signal perception with transduction remains unknown. In this study, we identify SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)3/brassinosteroid-associated kinase (BAK)1, a receptor-like kinase previously implicated in hormone signaling, as a component of plant PTI. In Arabidopsis thaliana, AtSERK3/BAK1 rapidly enters an elicitor-dependent complex with FLAGELLIN SENSING 2 (FLS2), the receptor for the bacterial PAMP flagellin and its peptide derivative flg22. In the absence of AtSERK3/BAK1, early flg22-dependent responses are greatly reduced in both A. thaliana and Nicotiana benthamiana. Furthermore, N. benthamiana Serk3/Bak1 is required for full responses to unrelated PAMPs and, importantly, for restriction of bacterial and oomycete infections. Thus, SERK3/BAK1 appears to integrate diverse perception events into downstream PAMP responses, leading to immunity against a range of invading microbes.

Bacterial Effectors Target the Common Signaling Partner BAK1 to Disrupt Multiple MAMP Receptor-Signaling Complexes and Impede Plant Immunity

Successful pathogens have evolved strategies to interfere with host immune systems. For example, the ubiquitous plant pathogen Pseudomonas syringae injects two sequence-distinct effectors, AvrPto and AvrPtoB, to intercept convergent innate immune responses stimulated by multiple microbe-associated molecular patterns (MAMPs). However, the direct host targets and precise molecular mechanisms of bacterial effectors remain largely obscure. We show that AvrPto and AvrPtoB bind the Arabidopsis receptor-like kinase BAK1, a shared signaling partner of both the flagellin receptor FLS2 and the brassinosteroid receptor BRI1. This targeting interferes with ligand-dependent association of FLS2 with BAK1 during infection. It also impedes BAK1-dependent host immune responses to diverse other MAMPs and brassinosteroid signaling. Significantly, the structural basis of AvrPto-BAK1 interaction appears to be distinct from AvrPto-Pto association required for effector-triggered immunity. These findings uncover a unique strategy of bacterial pathogenesis where virulence effectors block signal transmission through a key common component of multiple MAMP-receptor complexes.

Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity.

Targeted degradation of bacterial sensing proteins keeps plant defenses from running amok. Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLAGELLIN-SENSING 2 (FLS2) senses bacterial flagellin and initiates immune signaling through association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases, PUB12 and PUB13, to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12 and PUB13 and is required for FLS2-PUB12/13 association. PUB12 and PUB13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.

Sensitivity to Flg22 Is Modulated by Ligand-Induced Degradation and de Novo Synthesis of the Endogenous Flagellin-Receptor FLAGELLIN-SENSING2

Ligand-induced degradation of endogenous flagellin receptor desensitizes cells to its stimulus, likely to remove ligand-bound receptors from the site of perception, and subsequent de novo receptor synthesis resensitizes cells for a new round of stimulus perception. FLAGELLIN-SENSING2 (FLS2) is the plant cell surface receptor that perceives bacterial flagellin or flg22 peptide, initiates flg22-signaling responses, and contributes to bacterial growth restriction. Flg22 elicitation also leads to ligand-induced endocytosis and degradation of FLS2 within 1 h. Why plant cells remove this receptor precisely at the time during which its function is required remains mainly unknown. Here, we assessed in planta flg22-signaling competency in the context of ligand-induced degradation of endogenous FLS2 and chemical interference known to impede flg22-dependent internalization of FLS2 into endocytic vesicles. Within 1 h after an initial flg22 treatment, Arabidopsis (Arabidopsis thaliana) leaf tissue was unable to reelicit flg22 signaling in a ligand-, time-, and dose-dependent manner. These results indicate that flg22-induced degradation of endogenous FLS2 may serve to desensitize cells to the same stimulus (homologous desensitization), likely to prevent continuous signal output upon repetitive flg22 stimulation. In addition to impeding ligand-induced FLS2 degradation, pretreatment with the vesicular trafficking inhibitors Wortmannin or Tyrphostin A23 impaired flg22-elicited reactive oxygen species production that was partially independent of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1. Interestingly, these inhibitors did not affect flg22-induced mitogen-activated protein kinase phosphorylation, indicating the ability to utilize vesicular trafficking inhibitors to target different flg22-signaling responses. For Tyrphostin A23, reduced flg22-induced reactive oxygen species could be separated from the defect in FLS2 degradation. At later times (>2 h) after the initial flg22 elicitation, recovery of FLS2 protein levels positively correlated with resensitization to flg22, indicating that flg22-induced new synthesis of FLS2 may prepare cells for a new round of monitoring the environment for flg22.

Novel functions of STOMATAL CYTOKINESIS-DEFECTIVE 1 (SCD1) in innate immune responses against bacteria

Eukaryotes employ complex immune mechanisms for protection against microbial pathogens. Here, we identified SCD1 (Stomatal Cytokinesis-Defective 1), previously implicated in growth and development through its role in cytokinesis and polarized cell expansion (Falbel, T. G., Koch, L. M., Nadeau, J. A., Segui-Simarro, J. M., Sack, F. D., and Bednarek, S. Y. (2003) Development 130, 4011–4024) as a novel component of innate immunity. In Arabidopsis, SCD1 is a unique gene encoding for the only protein containing a complete DENN (Differentially Expressed in Normal and Neoplastic cells) domain. The DENN domain is a largely uncharacterized tripartite protein motif conserved among eukaryotic proteins. We show that conditional scd1-1 plants containing a point mutation in a conserved DENN residue affected a subset of signaling responses to some bacterial pathogen-associated molecular patterns (PAMPs). Consistent with increased transcript accumulation of Pathogen-related (PR) genes, scd1-1 plants were more resistant to Pseudomonas syringae pathovar tomato (Pst) DC3000 infection implicating SCD1 as a negative regulator of basal resistance against bacteria. scd1-1 plants were different from known mutants exhibiting constitutive expressor of PR (cpr)-like phenotypes, in that growth impairment of scd1-1 plants was genetically independent of constitutive immune response activation. For scd1-1, shift to elevated temperature or introduction of a mutant allele in Salicylic acid Induction-Deficient 2 (SID2) suppressed constitutive defense response activation. sid2-2 also repressed the resistance phenotype of scd1-1. Temperature shift and sid2-2, however, did not rescue conditional growth and sterility defects of scd1-1. These results implicate SCD1 in multiple cellular pathways, possibly by affecting different proteins. Overall, our studies identified a novel role for eukaryotic DENN proteins in immunity against bacteria.

Loss of Arabidopsis thaliana Dynamin-Related Protein 2B Reveals Separation of Innate Immune Signaling Pathways

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC −, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC−. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.

Rapid Phosphorylation of a Syntaxin during the Avr9/Cf-9-Race-Specific Signaling Pathway

The tomato (Lycopersicon esculentum) resistance (R) gene Cf-9 is required for resistance to races of the fungal pathogen Cladosporium fulvum expressing the elicitor Avr9 and also confers responsiveness to Avr9 in Cf-9-containing transgenic tobacco (Nicotiana tabacum; Cf9 tobacco). Although protein phosphorylation is required for many early Avr9/Cf-9-signaling events, so far the only phosphorylation targets known in this race-specific signaling pathway are three kinases: the two mitogen-activated protein kinases, wound-induced protein kinase and salicylic acid-induced protein kinase, and the calcium-dependent protein kinase NtCDPK2. Here, we provide evidence that a tobacco syntaxin is rapidly and transiently phosphorylated after Avr9 elicitation. The syntaxin was detected with an antibody against NtSyp121, a plasma membrane-localized syntaxin implicated in abscisic acid responses and secretion. Consistent with the gene-for-gene hypothesis, syntaxin phosphorylation required the presence of both Avr9 and Cf-9. This phosphorylation event occurred either upstream of the pathway leading to reactive oxygen species production or in a parallel pathway. Interestingly, rapid syntaxin phosphorylation was triggered by the race-specific elicitor Avr9 but not by flg22P.aer, a general elicitor capable of inducing other defense-related signaling events in Cf9 tobacco such as reactive oxygen species production, mitogen-activated protein kinase activation, and PR5 transcript up-regulation. Furthermore, NtSyp121 transcript levels were increased at 24 h after elicitation with Avr9 but not with flg22P.aer. Because most other previously described Avr9- and flg22P.aer-elicited responses are similar, syntaxin phosphorylation and NtSyp121 transcript up-regulation may serve as novel early biochemical and late molecular markers, respectively, to elucidate further differences in the signaling responses between these two elicitors.

The Arabidopsis Dynamin-Related Protein2 Family Is Essential for Gametophyte Development

Both the DRP2 family of classical dynamins and the plant-specific DRP1s are thought to be required for clathrin-mediated trafficking. This study shows that the Arabidopsis DRP2 and DRP1 families have distinct developmental roles. DRP2 function was found to be necessary for cell cycle progression in the early stages of both the male and the female gametophyte development. Clathrin-mediated membrane trafficking is critical for multiple stages of plant growth and development. One key component of clathrin-mediated trafficking in animals is dynamin, a polymerizing GTPase that plays both regulatory and mechanical roles. Other eukaryotes use various dynamin-related proteins (DRP) in clathrin-mediated trafficking. Plants are unique in the apparent involvement of both a family of classical dynamins (DRP2) and a family of dynamin-related proteins (DRP1) in clathrin-mediated membrane trafficking. Our analysis of drp2 insertional mutants demonstrates that, similar to the DRP1 family, the DRP2 family is essential for Arabidopsis thaliana development. Gametophytes lacking both DRP2A and DRP2B were inviable, arresting prior to the first mitotic division in both male and female gametogenesis. Mutant pollen displayed a variety of defects, including branched or irregular cell plates, altered Golgi morphology and ectopic callose deposition. Ectopic callose deposition was also visible in the pollen-lethal drp1c-1 mutant and appears to be a specific feature of pollen-defective mutants with impaired membrane trafficking. However, drp2ab pollen arrested at earlier stages in development than drp1c-1 pollen and did not accumulate excess plasma membrane or display other gross defects in plasma membrane morphology. Therefore, the DRP2 family, but not DRP1C, is necessary for cell cycle progression during early gametophyte development. This suggests a possible role for DRP2-dependent clathrin-mediated trafficking in the transduction of developmental signals in the gametophyte.

Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae

BackgroundArabidopsis thaliana and Pseudomonas syringae pathovar tomato (Pto) provide an excellent plant-bacteria model system to study innate immunity. During pattern-triggered immunity (PTI), cognate host receptors perceive pathogen-associated molecular patterns (PAMPs) as non-self molecules. Pto harbors many PAMPs; thus for experimental ease, many studies utilize single synthesized PAMPs such as flg22, a short protein peptide derived from Pseudomonas flagellin. Flg22 recognition by Arabidopsis Flagellin Sensing 2 (FLS2) initiates a plethora of signaling responses including rapid production of apoplastic reactive oxygen species (ROS). Assessing flg22-ROS has been instrumental in identifying novel PAMP-signaling components; but comparably little is known whether in Arabidopsis, ROS is produced in response to intact live Pto and whether this response can be used to dissect genetic requirements of the plant host and live bacterial pathogens in planta.ResultsHere, we report of a fast and robust bioassay to quantitatively assess early ROS in Arabidopsis leaves, a tissue commonly used for pathogen infection assays, in response to living bacterial Pto strains. We establish that live Pto elicits a transient and dose-dependent ROS that differed in timing of initiation, amplitude and duration compared to flg22-induced ROS. Our control experiments confirmed that the detected ROS was dependent on the presence of the bacterial cells. Utilizing Arabidopsis mutants previously shown to be defective in flg22-induced ROS, we demonstrate that ROS elicited by live Pto was fully or in part dependent on RbohD and BAK1, respectively. Because fls2 mutants did not produce any ROS, flagellin perception by FLS2 is the predominant recognition event in live Pto-elicited ROS in Arabidopsis leaves. Furthermore using different Pto strains, our in planta results indicate that early ROS production appeared to be independent of the Type III Secretion System.ConclusionsWe provide evidence and necessary control experiments demonstrating that in planta, this ROS bioassay can be utilized to rapidly screen different Arabidopsis mutant lines and ecotypes in combination with different bacterial strains to investigate the genetic requirements of a plant host and its pathogen. For future experiments, this robust bioassay can be easily extended beyond Arabidopsis-Pto to diverse plant-pathosystems including crop species and their respective microbial pathogens.

The Major Specificity-Determining Amino Acids of the Tomato Cf-9 Disease Resistance Protein Are at Hypervariable Solvent-Exposed Positions in the Central Leucine-Rich Repeats

The interaction between tomato and the leaf mold pathogen Cladosporium fulvum is controlled in a gene-for-gene manner by plant Cf genes that encode membrane-anchored extracytoplasmic leucine-rich repeat (LRR) glycoproteins, which confer recognition of their cognate fungal avirulence (Avr) proteins. Cf-9 and Cf-4 are two such proteins that are 91% identical yet recognize the sequence-unrelated fungal avirulence determinants Avr9 and Avr4, respectively. As shown previously, Cf-4 specificity is determined by three putative solvent-exposed residues in the central LRR and a deletion of two LRR relative to Cf-9. In this study, we focused on identifying the specificity determinants of Cf-9. We generated chimeras between Cf-9 and its close homologue Cf-9B and identified five amino acid residues that constitute major specificity determinants of Cf-9. Introduction of these residues into Cf-9B allowed recognition of Avr9. Consistent with a role in recognition specificity, the identified residues are putatively solvent exposed in the central LRR and occupy hypervariable positions in the global Cf alignment. One of the specificity residues is not found in any other known Cf protein, suggesting the importance of diversifying selection rather than sequence exchange between homologues. Interestingly, there is an overlap between the Cf-4 and Cf-9 specificity-determining residues, precluding a protein with dual specificity.