The multifunctional leucine-rich repeat receptor kinase BAK1 is implicated in Arabidopsis development and immunity

Plant receptor-like kinases (RLKs) are transmembrane proteins with putative N-terminal extracellular ligand-binding domains and C-terminal intracellular protein kinase domains. RLKs have been implicated in multiple physiological programs including plant development and immunity to microbial infection. Arabidopsis thaliana gene expression patterns support an important role of this class of proteins in biotic stress adaptation. Here, we provide a comprehensive survey of plant immunity-related RLK gene expression. We further document the role of the Arabidopsis Brassinosteroid Insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) in seemingly unrelated biological processes, such as plant development and immunity, and propose a role of this protein as an adaptor molecule that is required for proper functionality of numerous RLKs. This view is supported by the identification of an additional RLK, PEPR1, and its closest homolog, PEPR2 as BAK1-interacting RLKs.     

The multifaceted function of BAK1/SERK3: Plant immunity to pathogens and responses to insect herbivores

Almost a decade ago BRI1-associated kinase 1 (BAK1) was identified as a co-receptor of brassinosteroid (BR) insensitive 1 (BRI1), the receptor for BRs, which plays an essential role in transducing BR signaling to regulate plant development. BAK1 is also critical in resistance to various pathogens. BAK1 rapidly binds to certain receptors for pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) after the perception of pathogen elicitors and is required for the full elicitation of pathogen-induced defense responses, such as the activation of the mitogen-activated protein kinase 6 (MPK6) and production of reactive oxygen species. Thus, BAK1 functions in both BR signaling and PAMP-triggered immunity (PTI). Recently BAK1 was also found to play an important role in mediating defense responses against an insect herbivore (Manduca sexta) of Nicotiana attenuata. In this interaction, BAK1 positively modulates wound- or herbivore feeding-induced accumulation of jasmonic acid (JA) and JA-isoleucine (JA-Ile). This mini-review summarizes recent advances in our understanding of the functions of BAK1 in resistance to pathogens and herbivores.Key words: BAK1, defense, herbivore, immunity, insect, jasmonate, pathogen, wounding     

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana

Background

The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance.

Principal Findings

We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs.

Conclusions/Significance

We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.     

Using mutant alleles to determine the structure and function of leucine-rich repeat receptor-like kinases

The leucine-rich-repeat class of receptor-like kinase (RLK)-encoding genes represents the largest class of putative receptor-encoding genes in the Arabidopsis genome. The biological functions of several of these genes have been determined through genetic analysis. With dozens of mutant alleles described for various RLKs in Arabidopsis and other plants, comparisons of the mutations found in different receptors, as well as of structural features that are conserved between receptors, can provide insights into the common and/or divergent regulation and functions of these receptors.     

The ubiquitin pathway is required for innate immunity in Arabidopsis

Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain-of-function mutant suppressor of npr1-1 constitutive 1 ( snc1 ) carries a point mutation in a Resistance ( R )-gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1 -mediated constitutive resistance is modifier of snc1 5 ( mos5 ), which carries a 15-bp deletion in UBA1 , one of two ubiquitin-activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1 , suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1 -mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C-terminus of UBA1 might affect binding affinity of the downstream ubiquitin-conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1 -related R -genes RPP4 and RPP5 , are dispensable in snc1 -mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R-proteins.     

RLK7, a leucine-rich repeat receptor-like kinase, is required for proper germination speed and tolerance to oxidative stress in Arabidopsis thaliana

The leucine-rich repeat class of receptor-like kinase (LRR-RLKs) encoding genes represents the largest family of putative receptor genes in the Arabidopsis thaliana genome. However, very little is known about the range of biological process that they control. We present in this paper the functional characterization of RLK7 that has all the structural features of a receptor-like kinase of the plant-specific LRR type. To this end, we identified and characterized three independent T-DNA insertion mutants, constructed lines carrying truncated versions of this putative receptor, one lacking the cytoplasmic kinase domain (RLK7Δkin) and the other one lacking 14 LRR repeats (RLK7ΔLRR) and generated RLK7 overexpressing lines. We thus provide evidences that RLK7 is involved in the control of germination speed and the tolerance to oxidant stress. First, consistent with the expression kinetics of the RLK7 gene in the seeds, we found that all three mutants showed a delay in germination, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines displayed a phenotype of more precocious germination. Second, a non-hypothesis driven proteomic approach revealed that in the seedlings of the three T-DNA insertion lines, four enzymes directly or indirectly involved in reactive oxygen species detoxification, were significantly less abundant. Consistent with this finding, the three mutants were less tolerant than the wild type to a hydrogen peroxide treatment, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines presented the opposite phenotype.     

The BAM1/BAM2 receptor-like kinases are important regulators of Arabidopsis early anther development

Anther development involves the formation of several adjacent cell types required for normal male fertility. Only a few genes are known to be involved in early anther development, particularly in the establishment of these different cell layers. Arabidopsis thaliana BAM1 (for BARELY ANY MERISTEM) and BAM2 encode CLAVATA1-related Leu-rich repeat receptor-like kinases that appear to have redundant or overlapping functions. We characterized anther development in the bam1 bam2 flowers and found that bam1 bam2 anthers appear to be abnormal at a very early stage and lack the endothecium, middle, and tapetum layers. Analyses using molecular markers and cytological techniques of bam1 bam2 anthers revealed that cells interior to the epidermis acquire some characteristics of pollen mother cells (PMCs), suggesting defects in cell fate specification. The pollen mother-like cells degenerate before the completion of meiosis, suggesting that these cells are defective. In addition, the BAM1 and BAM2 expression pattern supports both an early role in promoting somatic cell fates and a subsequent function in the PMCs. Therefore, analysis of BAM1 and BAM2 revealed a cell-cell communication process important for early anther development, including aspects of cell division and differentiation. This finding may have implications for the evolution of multiple signaling pathways in specifying cell types for microsporogenesis.     

RPK1 and TOAD2 are two receptor-like kinases redundantly required for arabidopsis embryonic pattern formation

Although the basic plant body plan is established during embryogenesis, the molecular basis of embryonic patterning remains to be fully understood. We have identified two receptor-like kinases, RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and TOADSTOOL2 (TOAD2), required for Arabidopsis embryonic pattern formation. Genetic analysis indicates that RPK1 and TOAD2 have overlapping embryonic functions. The zygotic gene dosage of TOAD2 in an rpk1 background is of critical importance, suggesting that signaling mediated by RPK1 and TOAD2 must be above a threshold level for proper embryo development. The localization of RPK1 and TOAD2 translational fusions to GFP coupled with the analysis of cell-type-specific markers indicate that RPK1 and TOAD2 are redundantly required for both pattern formation along the radial axis and differentiation of the basal pole during early embryogenesis. We propose that RPK1 and TOAD2 receive intercellular signals and mediate intracellular responses that are necessary for embryonic pattern formation.     

Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1

Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.     

Receptor-like kinases and receptor-like proteins: keys to pathogen recognition and defense signaling in plant innate immunity

Plants have evolved multiple layers of defense against various pathogens in the environment. Receptor-like kinases/proteins (RLKs/RLPs) are on the front lines of the battle between plants and pathogens since they are present at the plasma membrane and perceive signature molecules from either the invading pathogen or damaged plant tissue. With a few notable exceptions, most RLKs/RLPs are positive regulators of plant innate immunity. In this review, we summarize recently discovered RLKs/RLPs that are involved in plant defense responses against various classes of pathogens.We also describe what is currently known about the mechanisms of RLK-mediated initiation of signaling via protein-protein interactions and phosphorylation.     

The Receptor Kinases BAK1/SERK4 Regulate Ca2+ Channel-Mediated Cellular Homeostasis for Cell Death Containment

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Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2

Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe‐associated molecular patterns (MAMPs) and/or damage‐associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP‐triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co‐receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well‐known class of DAMPs, are oligomers of α‐1,4‐linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG‐triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin‐related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG‐activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.     

Both the extracellular leucine-rich repeat domain and the kinase activity of FSL2 are required for flagellin binding and signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Nucleotide binding site/leucine-rich repeats,Pto-like and receptor-like kinases related to disease resistance in grapevine

Nucleotide Binding Site/Leucine-Rich Repeat (NBS-LRR) and Serine/Threonine Kinase (STK) genes are two of the known classes of resistance (R-) genes in plants, and occur in large multigene families. Systematic identification of genes for NBS-LRRs and STKs provides a means of access to genomic regions that may be involved in disease resistance. Here we present a picture of these two families of R-gene analogs (RGAs) in grape with the aim of developing a set of resistance-related sequence-tagged-site (STS) markers. One hundred and three NBS-LRR sequences were isolated. They included members of the CC (coiled-coil) and TIR (Toll-interleukin receptor) sub-classes. A comparative analysis with other angiosperm NBSs is provided. Fifty-three genes for receptor-like kinases (RLKs) with serine/threonine specificity were identified. RLK sequences formed a putative monophyletic group within the kinase superfamily. They were similar to both cytoplasmic RLKs, such as Pto, and RLKs with LRR, S-locus, lectin-like and thaumatin-like extracellular binding-domains. The latter resembled the products of the R-related genes Xa21, FLS2, Rlk10, SFR2, and PR5K. Forty-five reference RGAs were converted into STSs by using appropriately designed specific primers. RGA-STSs were present in diverse grape genotypes, and >85% of the primers were capable of amplifying the STSs across the taxa Vitis and Muscadinia. DNA sequence polymorphism among these RGAs was assessed by SSCP (single-strand conformation polymorphism) analysis in over 20 Vitis spp. Finally, 45 universal primers for grape RGAs are proposed that should permit tagging of R-related regions in any grape genome.Communicated by R. Hagemann     

The tyrosine‐sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner

The tyrosine‐sulfated peptides PSKα and PSY1 bind to specific leucine‐rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss‐of‐function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen‐associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild‐type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate‐dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, ‐2 and PSY1‐receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst‐1 mutants partially restore the defense‐related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth‐promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes.     

Unfolded protein response genes regulated by CED-1 are required for Caenorhabditis elegans innate immunity

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria.     

RPN1a, a 26S proteasome subunit, is required for innate immunity in Arabidopsis

Accumulating evidence shows that proper degradation of proteins that affect defense responses in a positive or negative manner is critical in plant immunity. However, the role of plant degradation systems such as the 26S proteasome in plant immunity is not well understood. Loss‐of‐function mutations in EDR2 (ENHANCED DISEASE RESISTANCE 2) lead to increased resistance to the adapted biotrophic powdery mildew pathogen Golovinomyces cichoracearum. To study the molecular interactions between powdery mildew pathogen and Arabidopsis, we performed a screen for suppressors of edr2 and found that mutation in the gene that encodes RPN1a, a subunit of the 26S proteasome, suppressed edr2‐associated disease resistance phenotypes. In addition, RPN1a is required for edr1‐ and pmr4‐mediated powdery mildew resistance and mildew‐induced cell death. Furthermore, we show that rpn1a displayed enhanced susceptibility to the fungal pathogen G. cichoracearum and to virulent and avirulent bacterial Pto DC3000 strains, which indicated that rpn1a has defects in basal defense and resistance (R) protein‐mediated defense. RPN1a–GFP localizes to both the nucleus and cytoplasm. Accumulation of RPN1a is affected by salicylic acid (SA) and the rpn1a mutant has defects in SA accumulation upon Pto DC3000 infection. Further analysis revealed that two other subunits of the 26S proteasome, RPT2a and RPN8a are also involved in edr2‐mediated disease resistance. Based on these results, we conclude that RPN1a is required for basal defense and R protein‐mediated defense. Our data provide evidence that some subunits of the 26S proteasome are involved in innate immunity in Arabidopsis.