BRASSINOSTEROID-SIGNALING KINASE 1 modulates OPEN STOMATA 1 phosphorylation and contributes to stomatal closure and plant immunity.

Stomatal movement plays a critical role in plant immunity by limiting the entry of pathogens. OPEN STOMATA 1 (OST1) is a key component that mediates stomatal closure in plants, however, how OST1 functions in response to pathogens is not well understood. RECEPTOR-LIKE KINASE 902 (RLK902) phosphorylates BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1) and positively modulates plant resistance. In this study, by a genome-wide phosphorylation analysis, we found that the phosphorylation of BSK1 and OST1 was missing in the rlk902 mutant compared with the wild-type plants, indicating a potential connection between the RLK902-BSK1 module and OST1-mediated stomatal closure. We showed that RLK902 and BSK1 contribute to stomatal immunity, as the stomatal closure induced by the bacterial pathogen Pto DC3000 was impaired in rlk902 and bsk1-1 mutants. Stomatal immunity mediated by RLK902 was dependent on BSK1 phosphorylation at Ser230, a key phosphorylation site for BSK1 functions. Several phosphorylation sites of OST1 were important for RLK902- and BSK1-mediated stomatal immunity. Interestingly, the phosphorylation of Ser171 and Ser175 in OST1 contributed to the stomatal immunity mediated by RLK902 but not by BSK1, while phosphorylation of OST1 at Ser29 and Thr176 residues was critical for BSK1-mediated stomatal immunity. Taken together, these results indicate that RLK902 and BSK1 contribute to disease resistance via OST1-mediated stomatal closure. This work revealed a new function of BSK1 in activating stomatal immunity, and the role of RLK902-BSK1 and OST1 module in regulating pathogen-induced stomatal movement.

A complex receptor locus confers responsiveness to necrosis and ethylene-inducing like peptides in Brassica napus.

Brassica crops are susceptible to diseases which can be mitigated by breeding for resistance. MAMPs (microbe-associated molecular patterns) are conserved molecules of pathogens that elicit host defences known as pattern-triggered immunity (PTI). Necrosis and Ethylene-inducing peptide 1-like proteins (NLPs) are MAMPs found in a wide range of phytopathogens. We studied the response to BcNEP2, a representative NLP from Botrytis cinerea, and showed that it contributes to disease resistance in Brassica napus. To map regions conferring NLP response, we used the production of reactive oxygen species (ROS) induced during PTI across a population of diverse B. napus accessions for associative transcriptomics (AT), and bulk segregant analysis (BSA) on DNA pools created from a cross of NLP-responsive and non-responsive lines. In silico mapping with AT identified two peaks for NLP responsiveness on chromosomes A04 and C05 whereas the BSA identified one peak on A04. BSA delimited the region for NLP-responsiveness to 3 Mbp, containing ~245 genes on the Darmor-bzh reference genome and four co-segregating KASP markers were identified. The same pipeline with the ZS11 genome confirmed the highest-associated region on chromosome A04. Comparative BLAST analysis revealed unannotated clusters of receptor-like protein (RLP) homologues on ZS11 chromosome A04. However, no specific RLP homologue conferring NLP response could be identified. Our results also suggest that BR-SIGNALLING KINASE1 may be involved with modulating the NLP response. Overall, we demonstrate that responsiveness to NLP contributes to disease resistance in B. napus and define the associated genomic location. These results can have practical application in crop improvement.

A Nicotiana benthamiana receptor-like kinase regulates Phytophthora resistance by coupling with BAK1 to enhance elicitin-triggered immunity.

Cell-surface-localized leucine-rich-repeat receptor-like kinases (LRR-RLKs) are crucial for plant immunity. Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain (ECD), whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs. Here, we identified LRR-RLK regulators using high-throughput tobacco rattle virus (TRV)-based gene silencing in the model plant Nicotiana benthamiana. We used the cell-death phenotype caused by INF1, an oomycete elicitin that induces pattern-triggered immunity, as an indicator. By screening 33 small LRR-RLKs (≤6 LRRs) of unknown function, we identified ELICITIN INSENSITIVE RLK 1 (NbEIR1) as a positive regulator of INF1-induced immunity and oomycete resistance. Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly compromised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phytophthora capsici. NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1 (NbBAK1) and a downstream component, BRASSINOSTEROID-SIGNALING KINASE 1 (NbBSK1). NbBSK1 also contributes to INF1-induced defense and P. capsici resistance. Upon INF1 treatment, NbEIR1 was released from NbBAK1 and NbBSK1 in vivo. Moreover, the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1. We also showed that NbEIR1 regulates flg22-induced immunity and associates with its receptor, FLAGELLIN SENSING 2 (NbFLS2). Collectively, our results suggest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity. NbBSK1-NbEIR1 association is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.

The role of N-myristoylation in homeostasis of brassinosteroid signaling kinase 1.

MAIN CONCLUSION: The N-myristoylation is required for BSK1 proper plasma membrane targeting and protein turnover. Brassinosteroid (BR) signaling kinase 1 (BSK1), with a myristoylation site at its N-terminus to anchor at plasma membrane (PM), is involved in BR-regulated plant growth and flg22-triggered immunity responses. However, little is known about the role of N-myristoylation in BSK1 protein homeostasis. Here, we revealed that N-myristoylation is critical to the PM targeting and protein stability of BSK1. The N-myristoylation-deficient mutant BSK1G2A mainly distributed in the cytoplasm and retained in the endoplasmic reticulum. We further found that the BSK1G2A proteins were unstable and degraded through ATG8e-labled autophagic pathway. This study provides a new insight into the regulation of plant protein homeostasis.

The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling.

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine-rich repeat receptor-like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per µg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.

BRASSINOSTEROID-SIGNALING KINASE1 associates with and is required for cysteine protease RESPONSE TO DEHYDRATION 19-mediated disease resistance in Arabidopsis.

The receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALING KINASE1 (BSK1) interacts with pattern recognition receptor (PRR) FLAGELLIN SENSING2 (FLS2) and positively regulates plant innate immunity in Arabidopsis thaliana. However, the molecular components involved in BSK1-mediated immune signaling remain largely unknown. To further explore the molecular mechanism underlying BSK1-mediated disease resistance, we screened two cysteine proteases, RESPONSE TO DEHYDRATION 19 (RD19) and RD19-LIKE 2 (RDL2), as BSK1-binding partners. Overexpression of RD19, but not RDL2, displayed an autoimmune phenotype, presenting programmed cell death and enhanced resistance to multiple pathogens. Interestingly, RD19-mediated immune activation depends on BSK1, as knockout of BSK1 in RD19-overexpressing plants rescued their autoimmunity and abolished the increased resistance. Furthermore, we found that BSK1 plays a positive role in maintaining RD19 protein abundance in Arabidopsis. Our results provide new insights into BSK1-mediated immune signaling and reveal a potential mechanism by which BSK1 stabilizes RD19 to promote effective immune output.

Dynamic spatial reorganization of BSK1 complexes in the plasma membrane underpins signal-specific activation for growth and immunity.

Growth and immunity are opposing processes that compete for cellular resources, and proper resource allocation is crucial for plant survival. BSK1 plays a key role in the regulation of both growth and immunity by associating with BRI1 and FLS2, respectively. However, it remains unclear how two antagonistic signals co-opt BSK1 to induce signal-specific activation. Here we show that the dynamic spatial reorganization of BSK1 within the plasma membrane underlies the mechanism of signal-specific activation for growth or immunity. Resting BSK1 localizes to membrane rafts as complexes. Unlike BSK1-associated FLS2 and BRI1, flg22 or exogenous brassinosteroid (BR) treatment did not decrease BSK1 levels at the plasma membrane (PM) but rather induced BSK1 multimerization and dissociation from FLS2/BSK1 or BRI1/BSK1, respectively. Moreover, flg22-activated BSK1 translocated from membrane rafts to non-membrane-raft regions, whereas BR-activated BSK1 remained in membrane rafts. When applied together with flg22, BR suppressed various flg22-induced BSK1 activities such as BSK1 dissociation from FLS2/BSK1, BSK1 interaction with MAPKKK5, and BSK translocation together with MAPKKK5. Taken together, this study provides a unique insight into how the precise control of BSK1 spatiotemporal organization regulates the signaling specificity to balance plant growth and immunity.

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.

BSK1, a receptor-like cytoplasmic kinase, involved in both BR signaling and innate immunity in Arabidopsis.

Molecular interaction between powdery mildew fungi and Arabidopsis has been widely used as a model system to study plant immunity. Arabidopsis EDR2 (enhanced disease resistance 2) is a well characterized negative regulator in powdery mildew resistance and mildew-induced cell death. Recently, we showed that a mutation in BSK1 (br-signaling kinase 1), suppressed edr2-mediated disease resistance. (1) And the bsk1-1 single mutant displayed enhanced susceptibility to multiple pathogens, indicating that BSK1 plays important roles in plant immunity. BSK1 is a receptor-like cytoplasmic kinase and localizes on plasma membrane; loss of the membrane localization signaling disrupts BSK1 functions in edr2-mediated resistance. Significantly, BSK1 physically associates with the PAMP receptor FLS2 (flagellin sensing 2) and is required by FLS2-mediated ROS burst. (1) Here we show that disruption of BSK1 membrane localization affects the BSK1-FLS2 interactions, suggesting the membrane association of BSK1 is important for both edr2-mediated signaling and the BSK1-FLS2 complex formation. Previously, it was shown that BSK1 is a substrate of the brassinosteroid (BR) receptor BRI1 (brassinosteroid insensitive 1) and plays critical roles in BR signaling. (2) Further exploration of signaling transductions downstream of BSK1-FLS2 complex will not only shed new light on how BSK1 regulates plant immunity, but may also help to dissect the connections between plant growth and defense.