Libo Shan

Differential innate immune signalling via Ca2+ sensor protein kinases

Innate immunity represents the first line of inducible defence against microbial infection in plants and animals. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively), such as flagellin, initiates convergent signalling pathways involving mitogen-activated protein kinase (MAPK) cascades and global transcriptional changes to boost immunity. Although Ca2+ has long been recognized as an essential and conserved primary mediator in plant defence responses, how Ca2+ signals are sensed and relayed into early MAMP signalling is unknown. Using a functional genomic screen and genome-wide gene expression profiling, here we show that four calcium-dependent protein kinases (CDPKs) are Ca2+-sensor protein kinases critical for transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in the synthesis of defence peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by the 22-amino-acid peptide flg22, as well as compromised pathogen defence. In contrast to negative roles of calmodulin and a calmodulin-activated transcription factor in plant defence, the present study reveals Ca2+ signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.

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.

A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity

Plants and animals rely on innate immunity to prevent infections by detection of microbe-associated molecular patterns (MAMPs) through pattern-recognition receptors (PRRs). The plant PRR FLS2, a leucine-rich repeat-receptor kinase, recognizes bacterial flagellin and initiates immune signaling by association with another leucine-rich repeat-receptor-like kinase, BAK1. It remains unknown how the FLS2/BAK1 receptor complex activates intracellular signaling cascades. Here we identified the receptor-like cytoplasmic kinase BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1. BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro. The flagellin phosphorylation site Thr237 of BIK1 is required for its phosphorylation on BAK1 and FLS2, suggesting that BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/BAK1 to propagate flagellin signaling. Importantly, bik1 mutants are compromised in diverse flagellin-mediated responses and immunity to the nonpathogenic bacterial infection. Thus, BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling.

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.

Genomewide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000.

The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and/or translocated into Arabidopsis in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in P. syringae revealed an export-associated pattern of equivalent solvent-exposed amino acids in the N-terminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADP-ribosyltransferases, a homolog of SrfC (a candidate effector in Salmonella enterica), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.

Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis thaliana auxin physiology

The Pseudomonas syringae type III effector AvrRpt2 promotes bacterial virulence on Arabidopsis thaliana plants lacking a functional RPS2 gene (rps2 mutant plants). To investigate the mechanisms underlying the virulence activity of AvrRpt2, we examined the phenotypes of transgenic A. thaliana rps2 seedlings constitutively expressing AvrRpt2. These seedlings exhibited phenotypes reminiscent of A. thaliana mutants with altered auxin physiology, including longer primary roots, increased number of lateral roots, and increased sensitivity to exogenous auxin. They also had increased levels of free indole acetic acid (IAA). The presence of AvrRpt2 also was correlated with a further increase in free IAA levels during infection with P. syringae pv. tomato strain DC3000 (PstDC3000). These results indicate that AvrRpt2 alters A. thaliana auxin physiology. Application of the auxin analog 1-naphthaleneacetic acid promoted disease symptom development in PstDC3000-infected plants, suggesting that elevated auxin levels within host tissue promote PstDC3000 virulence. Thus, AvrRpt2 may be among the virulence factors of P. syringae that modulate host auxin physiology to promote disease.

One for all: the receptor-associated kinase BAK1.

The plant receptor kinase BAK1/SERK3 has been identified as a partner of ligand-binding leucine-rich repeat receptor kinases, in particular the brassinosteroid receptor BRI1 and the immune receptor FLS2. BAK1 positively regulates BRI1 receptor function via physical interaction and transphosphorylation. Since its first description in 2001, several independent groups have discovered BAK1/SERK3 as a component of diverse processes, including brassinosteroid signaling, light responses, cell death, and plant innate immunity. Here, we summarize current knowledge of the functional repertoire of BAK1 and discuss how its multiple functions could be integrated, how receptor complexes are potentially formed and how specificity might be determined.

The Pseudomonas AvrPto Protein Is Differentially Recognized by Tomato and Tobacco and Is Localized to the Plant Plasma Membrane

The avrPto gene of Pseudomonas syringae pv tomato triggers race-specific resistance in tomato plants carrying Pto, a resistance gene encoding a protein kinase. When introduced into P. s. tabaci, avrPto triggers resistance in tobacco W38 plants that carry the corresponding R gene. The AvrPto protein is believed to be secreted into host cells through the bacterial type III secretion pathway, where it activates disease resistance in tomato by interacting with Pto. We report here the identification of two distinct regions in AvrPto that determine the recognition specificity of this protein in tomato and tobacco. Point mutations in the central region disrupted the avirulence activity in tomato but not in tobacco. Conversely, point mutations in the C-terminal region abolished the avirulence in tobacco but not in tomato. We further report that AvrPto was localized to the plasma membrane of plant cells. Disrupting the membrane association by mutating a putative myristoylation motif of AvrPto abolished the avirulence activity in both tomato and tobacco. These findings demonstrate that AvrPto is recognized differently by the R genes in tomato and tobacco and that the recognition of AvrPto probably is associated with the plasma membrane.

Elicitation and suppression of microbe-associated molecular pattern-triggered immunity in plant-microbe interactions

Recent studies have uncovered fascinating molecular mechanisms underlying plant–microbe interactions that coevolved dynamically. As in animals, the primary plant innate immunity is immediately triggered by the detection of common pathogen‐ or microbe‐associated molecular patterns (PAMPs/MAMPs). Different MAMPs are often perceived by distinct cell‐surface pattern‐recognition receptors (PRRs) and activate convergent intracellular signalling pathways in plant cells for broad‐spectrum immunity. Successful pathogens, however, have evolved multiple virulence factors to suppress MAMP‐triggered immunity. Specifically, diverse pathogenic bacteria have employed the type III secretion system to deliver a repertoire of virulence effector proteins to interfere with host immunity and promote pathogenesis. Plants challenged by pathogens have evolved the secondary plant innate immunity. In particular, some plants possess the specific intracellular disease resistance (R) proteins to effectively counteract virulence effectors of pathogens for effector‐triggered immunity. This potent but cultivar‐specific effector‐triggered immunity occurs rapidly with localized programmed cell death/hypersensitive response to limit pathogen proliferation and disease development. Remarkably, bacteria have further acquired virulence effectors to block effector‐triggered immunity. This review covers the latest findings in the dynamics of MAMP‐triggered immunity and its interception by virulence factors of pathogenic bacteria.

Bifurcation of Arabidopsis NLR Immune Signaling via Ca2+-Dependent Protein Kinases

Nucleotide-binding domain leucine-rich repeat (NLR) protein complexes sense infections and trigger robust immune responses in plants and humans. Activation of plant NLR resistance (R) proteins by pathogen effectors launches convergent immune responses, including programmed cell death (PCD), reactive oxygen species (ROS) production and transcriptional reprogramming with elusive mechanisms. Functional genomic and biochemical genetic screens identified six closely related Arabidopsis Ca2+-dependent protein kinases (CPKs) in mediating bifurcate immune responses activated by NLR proteins, RPS2 and RPM1. The dynamics of differential CPK1/2 activation by pathogen effectors controls the onset of cell death. Sustained CPK4/5/6/11 activation directly phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, to synergistically regulate transcriptional reprogramming crucial for NLR-dependent restriction of pathogen growth, whereas CPK1/2/4/11 phosphorylate plasma membrane-resident NADPH oxidases for ROS production. Our studies delineate bifurcation of complex signaling mechanisms downstream of NLR immune sensors mediated by the myriad action of CPKs with distinct substrate specificity and subcellular dynamics.