Genome-wide identification of Brassinosteroid insensitive 1-associated receptor kinase 1 genes and expression analysis in response to pathogen infection in cucumber (Cucumis sativus L.).

BACKGROUND: BAK1 (Brassinosteroid insensitive 1-associated receptor kinase 1) plays an important role in disease resistance in plants. However, the function of BAK1 family in cucumber and the decisive genes for disease-resistance remain elusive. RESULTS: Here, we identified 27 CsBAK1s in cucumber, and classified them into five subgroups based on phylogenetic analysis and gene structure. CsBAK1s in the same subgroup shared the similar motifs, but different gene structures. Cis-elements analysis revealed that CsBAK1s might respond to various stress and growth regulation. Three segmentally duplicated pairwise genes were identified in cucumber. In addition, Ka/Ks analysis indicated that CsBAK1s were under positive selection during evolution. Tissue expression profile showed that most CsBAK1s in Subgroup II and IV showed constitutive expression, members in other subgroups showed tissue-specific expression. To further explore whether CsBAK1s were involved in the resistance to pathogens, the expression patterns of CsBAK1s to five pathogens (gummy stem blight, powdery mildew, downy mildew, grey mildew, and fusarium wilt) reveled that different CsBAK1s had specific roles in different pathogen infections. The expression of CsBAK1-14 was induced/repressed significantly by five pathogens, CsBAK1-14 might play an important role in disease resistance in cucumber. CONCLUSIONS: 27 BAK1 genes were identified in cucumber from a full perspective, which have important functions in pathogen infection. Our study provided a theoretical basis to further clarify the function of BAK1s to disease resistance in cucumber.

Understanding and manipulating the recognition of necrosis-inducing secreted protein 1 (NIS1) by BRI1-associated receptor kinase 1 (BAK1).

Necrosis-inducing secreted protein 1 (NIS1) is a core effector of Ascomycota and Basidiomycota fungi. They inhibit the immune responses of host plants mainly through interaction with the multi-functional coreceptor BRI1-associated receptor kinase 1 (BAK1). However, the structural mechanism of the NIS1 family and how they are recognized by BAK1 are unknown. Herein, we report the first crystal structure of the NIS1 family protein, the Magnaporthe oryzae NIS1 (MoNIS1), analyze the recognition mechanism of NIS1s by BAK1, and explore regulation of the NIS1-BAK1 interaction by a chemical compound. MoNIS1 exists as a ß barrel formed by eight ß strands, a folding mode that has not been reported. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) assay suggested that ß4-ß5 loop and ß5 strand of MoNIS1 participate in OsBAK1 interaction, which was supported by further single-point mutational assays. For OsBAK1, HDX-MS assay suggested four regions involved in MoNIS1 interaction. Additionally, we identified a compound that blocks MoNIS1-OsBAK1 interaction in vitro and inhibits the virulence of M. oryzae on rice. Collectively, we determined the first structure of NIS1 family effectors, presented the recognition mechanism of NIS1 by BAK1, and showed that blocking NIS1-BAK1 interaction could be a new target for fungicide development.

SERK3A and SERK3B could be S-nitrosylated and enhance the salt resistance in tomato seedlings.

Salinity hinders plant growth and development, resulting in reduced crop yields and diminished crop quality. Nitric oxide (NO) and brassinolides (BR) are plant growth regulators that coordinate a plethora of plant physiological responses. Nonetheless, the way in which these factors interact to affect salt tolerance is not well understood. BR is perceived by the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and its co-receptor BRI1-associated kinase 1 (BAK1) to form the receptor complex, eventually inducing BR-regulated responses. To response stress, a wide range of NO-mediated protein modifications is undergone in eukaryotic cells. Here, we showed that BR participated in NO-enhanced salt tolerance of tomato seedlings (Solanum lycopersicum cv. Micro-Tom) and NO may activate BR signaling under salt stress, which was related to NO-mediated S-nitrosylation. Further, in vitro and in vivo results suggested that BAK1 (SERK3A and SERK3B) was S-nitrosylated, which was inhibited under salt condition and enhanced by NO. Accordingly, knockdown of SERK3A and SERK3B reduced the S-nitrosylation of BAK1 and resulted in a compromised BR response, thereby abolishing NO-induced salt tolerance. Besides, we provided evidence for the interaction between BRI1 and SERK3A/SERK3B. Meanwhile, NO enhanced BRI1-SERK3A/SERK3B interaction. These results imply that NO-mediated S-nitrosylation of BAK1 enhances the interaction BRI1-BAK1, facilitating BR response and subsequently improving salt tolerance in tomato. Our findings illustrate a mechanism by which redox signaling and BR signaling coordinate plant growth in response to abiotic stress.

Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

Association mechanism between Arabidopsis immune coreceptor BAK1 and Pseudomonas syringae effector HopF2.

Brassinosteroid activated kinase 1 (BAK1) is a cell-surface coreceptor which plays multiple roles in innate immunity of plants. HopF2 is an effector secreted by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 into Arabidopsis and suppresses host immune system through interaction with BAK1 as well as its downstream kinase MKK5. The association mechanism of HopF2 to BAK1 remains unclear, which prohibits our understanding and subsequent interfering of their interaction for pathogen management. Herein, we found the kinase domain of BAK1 (BAK1-KD) is sufficient for HopF2 association. With a combination of hydrogen/deuterium exchange mass spectrometry and mutational assays, we found a region of BAK1-KD N-lobe and a region of HopF2 head subdomain are critical for intermolecular interaction, which is also supported by unbiased protein-protein docking with ClusPro and kinase activity assay. Collectively, this research presents the interaction mechanism between Arabidopsis BAK1 and P. syringae HopF2, which could pave the way for bactericide development that blocking the functioning of HopF2 toward BAK1.

Cellooligomer/CELLOOLIGOMER RECEPTOR KINASE1 Signaling Exhibits Crosstalk with PAMP-Triggered Immune Responses and Sugar Metabolism in Arabidopsis Roots.

The degradation of cellulose generates cellooligomers, which function as damage-associated molecular patterns and activate immune and cell wall repair responses via the CELLOOLIGOMER RECEPTOR KINASE1 (CORK1). The most active cellooligomer for the induction of downstream responses is cellotriose, while cellobiose is around 100 times less effective. These short-chain cellooligomers are also metabolized after uptake into the cells. In this study, we demonstrate that CORK1 is mainly expressed in the vascular tissue of the upper, fully developed part of the roots. Cellooligomer/CORK1-induced responses interfere with chitin-triggered immune responses and are influenced by BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE1 and the receptor kinase FERONIA. The pathway also controls sugar transporter and metabolism genes and the phosphorylation state of these proteins. Furthermore, cellotriose-induced ROS production and WRKY30/40 expression are controlled by the sugar transporters SUCROSE-PROTON SYMPORTER1, SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER11 (SWEET11), and SWEET12. Our data demonstrate that cellooligomer/CORK1 signaling is integrated into the pattern recognition receptor network and coupled to the primary sugar metabolism in Arabidopsis roots.

A novel lectin receptor kinase gene, AtG-LecRK-I.2, enhances bacterial pathogen resistance through regulation of stomatal immunity in Arabidopsis.

The S-locus lectin receptor kinases (G-LecRKs) have been suggested as receptors for microbe/damage-associated molecular patterns (MAMPs/DAMPs) and to be involved in the pathogen defense responses, but the functions of most G-LecRKs in biotic stress response have not been characterized. Here, we identified a member of this family, G-LecRK-I.2, that positively regulates flg22- and Pseudomonas syringae pv. tomato (Pst) DC3000-induced stomatal closure. G-LecRK-I.2 was rapidly phosphorylated under flg22 treatment and could interact with the FLS2/BAK1 complex. Two T-DNA insertion lines, glecrk-i.2-1 and glecrk-i.2-2, had lower levels of reactive oxygen species (ROS) and nitric oxide (NO) production in guard cells, as compared with the wild-type Col-0, under Pst DC3000 infection. Also, the immunity marker genes CBP60g and PR1 were induced at lower levels under Pst DC3000 hrcC- infection in glecrk-i.2-1 and glecrk-i.2-2. The GUS reporter system also revealed that G-LecRK-I.2 was expressed only in guard cells. We also found that G-LecRK-I.2 could interact H+-ATPase AHA1 to regulate H+-ATPase activity in the guard cells. Taken together, our results show that G-LecRK-I.2 plays an important role in regulating stomatal closure under flg22 and Pst DC3000 treatments and in ROS and NO signaling specifically in guard cells.

Recent advances in understanding the regulatory mechanism of plasma membrane H+-ATPase through the brassinosteroid signaling pathway.

The polyhydroxylated steroid phytohormone brassinosteroids (BRs) control many aspects of plant growth, development and responses to environmental changes. Plasma membrane (PM) H+-ATPase, the well-known PM proton pump, is a central regulator in plant physiology, which mediates not only plant growth and development, but also adaptation to stresses. Recent studies highlight that PM H+-ATPase is at least partly regulated via the BR signaling. Firstly, the BR cell surface receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and multiple key components of BR signaling directly or indirectly influence PM H+-ATPase activity. Secondly, the SMALL AUXIN UP RNA (SAUR) gene family physically interacts with BRI1 to enhance organ development of Arabidopsis by activating PM H+-ATPase. Thirdly, RNA-sequencing (RNA-seq) assays showed that the expression of some SAUR genes is upregulated under the light or sucrose conditions, which is related to the phosphorylation state of the penultimate residue of PM H+-ATPase in a time-course manner. In this review, we describe the structural and functional features of PM H+-ATPase, and summarize recent progress toward understanding the regulatory mechanism of PM H+-ATPase by BRs, and briefly introduce how PM H+-ATPase activity is modulated by its own biterminal regions and the post-translational modifications.

SERKs and NIKs: Coreceptors or signaling hubs in a complex crosstalk between growth and defense?

SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES (SERKs) and NUCLEAR SHUTTLE PROTEIN-INTERACTING KINASES (NIKs) belong to superfamily II of leucine-rich repeat receptor-like kinases, which share cytosolic kinase conservation and a similar ectodomain configuration. SERKs have been extensively demonstrated to function as coreceptors of receptor-like kinases, which sense biotic or developmental signals to initiate specific responses. NIKs, on the other hand, have emerged as downstream components in signaling cascades, not functioning as coreceptors but rather serving as hubs that converge information from both biotic and abiotic signals, resulting in a unified response. Like SERKs, NIKs play a crucial role as information spreaders in plant cells, forming hubs of high centrality. However, unlike SERKs, which function as coreceptors and assemble paired receptor-specific responses, NIKs employ a shared signaling circuit to transduce diverse biotic and abiotic signals into the same physiological response. Therefore, this review highlights the concept of signaling hubs that differ from coreceptors in signaling pathways.

The prodomain of Arabidopsis metacaspase 2 positively regulates immune signaling mediated by pattern-recognition receptors.

Metacaspases (MCs) are structural homologs of mammalian caspases found in plants, fungi, and protozoa. Type-I MCs carry an N-terminal prodomain, the function of which is unclear. Through genetic analysis of Arabidopsis mc2-1, a T-DNA insertion mutant of MC2, we demonstrated that the prodomain of metacaspase 2 (MC2) promotes immune signaling mediated by pattern-recognition receptors (PRRs). In mc2-1, immune responses are constitutively activated. The receptor-like kinases (RLKs) BAK1/BKK1 and SOBIR1 are required for the autoimmune phenotype of mc2-1, suggesting that immune signaling mediated by the receptor-like protein (RLP)-type PRRs is activated in mc2-1. A suppressor screen identified multiple mutations in the first exon of MC2, which suppress the autoimmunity in mc2-1. Further analysis revealed that the T-DNA insertion at the end of exon 1 of MC2 causes elevated expression of the MC2 prodomain, and overexpression of the MC2 prodomain in wild-type (WT) plants results in the activation of immune responses. The MC2 prodomain interacts with BIR1, which inhibits RLP-mediated immune signaling by interacting with BAK1, suggesting that the MC2 prodomain promotes plant defense responses by interfering with the function of BIR1. Our study uncovers an unexpected function of the prodomain of a MC in plant immunity.

BTL2 phospho-switch surveils plant immunity.

BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) is a co-receptor involved in the recognition of pattern-associated molecular patterns (PAMPs) via plasma membrane-localized pattern recognition receptors (PRRs). Absence of BAK1/SERK4 leads to the activation of autoimmunity in plants. Yu et al. recently showed that BAK-TO-LIFE 2 (BTL2) is required for the surveillance of BAK1/SERK4 integrity to maintain immune homeostasis.

BAK-up: the receptor kinase BAK-TO-LIFE 2 enhances immunity when BAK1 is lacking.

BRI1-ASSOCIATED KINASE 1 (BAK1/SERK3) and its closest homolog BAK1-LIKE 1 (BKK1/SERK4) are leucine-rich repeat receptor kinases (LRR-RKs) belonging to the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family. They act as co-receptors of various other LRR-RKs and participate in multiple signaling events by complexing and transphosphorylating ligand-binding receptors. Initially identified as the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) co-receptor, BAK1 also functions in plant immunity by interacting with pattern recognition receptors. Mutations in BAK1 and BKK1 cause severely stunted growth and cell death, characterized as autoimmune cell death. Several factors play a role in this type of cell death, including RKs and components of effector-triggered immunity (ETI) signaling pathways, glycosylation factors, ER quality control components, nuclear trafficking components, ion channels, and Nod-like receptors (NLRs). The Shan lab has recently discovered a novel RK BAK-TO-LIFE 2 (BTL2) that interacts with BAK1 and triggers cell death in the absence of BAK1 and BKK1. This RK compensates for the loss of BAK1-mediated pattern-triggered immunity (PTI) by activating phytocytokine-mediated immune and cell death responses.

A novel pyroptosis-related signature predicts prognosis and indicates immunotherapy in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has been recognized as a frequently occurring oral malignant tumor. Pyroptosis plays an extremely important role in the occurrence and development of cancer, but the role of pyroptosis in OSCC remains unclear. METHODS: OSCC-related data were obtained from the TCGA and GEO databases. A PSscore risk model was constructed through LASSO regression analysis. The GEO database was utilized as the validation set of the model. The "ESTIMATE" and "CIBERSORT" algorithms were utilized to additionally evaluate the relationship between the immune cell score and PSscore. TIDE and IPS algorithms were used to assess patient response to immunotherapy. In addition, Western blot analysis and MTT assay was used to further validate key genes. RESULTS: Comprehensive bioinformatics analysis showed that a low-PSscore had a significant survival advantage, richer immune cell infiltration, more active immune-related pathways, higher TME scores, and lower tumor purity. The results of TIDE and IPS analysis indicated that the high-PSscore group had higher immune escape potential and was less sensitive to immunotherapy. In contrast, the low-PSscore group patients might be more sensitive to PD1 and CTLA4 + PD1 immunotherapy. Univariate and multivariate COX results indicated that PSscore was an independent prognostic factor in OSCC patients. Another important finding is that BAK1 is a potential target of OSCC and is related to the Nod-like receptor signaling pathway. Knockdown of BAK1 can significantly reduce the proliferation of OSCC cells. CONCLUSION: The PSscore model could be utilized as a powerful prognostic indicator and can help in the development of new immunotherapies.

PSW1, an LRR receptor kinase, regulates pod size in peanut.

Pod size is a key agronomic trait that greatly determines peanut yield, the regulatory genes and molecular mechanisms that controlling peanut pod size are still unclear. Here, we used quantitative trait locus analysis to identify a peanut pod size regulator, POD SIZE/WEIGHT1 (PSW1), and characterized the associated gene and protein. PSW1 encoded leucine-rich repeat receptor-like kinase (LRR-RLK) and positively regulated pod stemness. Mechanistically, this allele harbouring a 12-bp insertion in the promoter and a point mutation in the coding region of PSW1 causing a serine-to-isoleucine (S618I) substitution substantially increased mRNA abundance and the binding affinity of PSW1 for BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Notably, PSW1HapII (super-large pod allele of PSW1) expression led to up-regulation of a positive regulator of pod stemness PLETHORA 1 (PLT1), thereby resulting in larger pod size. Moreover, overexpression of PSW1HapII increased seed/fruit size in multiple plant species. Our work thus discovers a conserved function of PSW1 that controls pod size and provides a valuable genetic resource for breeding high-yield crops.

Hsa_circ_0002348 regulates trophoblast proliferation and apoptosis through miR-126-3p/BAK1 axis in preeclampsia.

BACKGROUND: Preeclampsia is a common pregnancy complication characterized by high blood pressure and damage to organs. Abnormal placenta and vascular function can lead to preeclampsia. Accumulating evidence has suggested a potential link between circular RNAs (circRNAs) and preeclampsia. As a placenta and endothelial-expressed circRNA, hsa_circ_0002348, may be promising to be the novel molecular target for preeclampsia. However, the function and mechanism of hsa_circ_0002348 in preeclampsia has not been elucidated. MATERIALS AND METHODS: An overlap analysis of two circRNA profiles from placenta and endothelial cells was used to identify a functionally unknown circRNA, hsa_circ_0002348. Quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) were used to detect its expression in the trophoblast cells and placental tissues. The mouse model of lipopolysaccharide (LPS)-induced preeclampsia was established to determine the in vivo role of hsa_circ_0002348. RNA immunoprecipitation (RIP), Luciferase reporter assay, qRT-PCR, western blot, gain- and loss-of-function and rescue experiments were conducted to uncover the role of hsa_circ_0002348 and its interaction with miR-126-3p and BAK1 in regulating trophoblast proliferation and apoptosis. Fluorescence in situ hybridization (FISH) and Immunohistochemistry (IHC) were performed to examine the expression of miR-126-3p and BAK1 in mice and human placentas, respectively. RESULTS: Hsa_circ_0002348 was significantly increased in the preeclampsia placentas, and positively correlated with the severity of preeclampsia patients' clinical manifestations. Its overexpression exacerbated preeclampsia-like features in the mouse model of LPS-induced preeclampsia. Functionally, hsa_circ_0002348 was found to inhibit trophoblast proliferation and promote trophoblast apoptosis. Mechanistically, hsa_circ_0002348, as an endogenous miR-126-3p sponge, upregulated the expression of BAK1. Additionally, both hsa_circ_0002348 knockdown and miR-126-3p overexpression enhanced the mammalian target of rapamycin (mTOR) and ERK1/2 signaling pathway. CONCLUSIONS: Hsa_circ_0002348 might be a novel regulator of trophoblast proliferation and apoptosis through miR-126-3p/BAK1 axis in preeclampsia, which may serve as a potential target for detecting and treating preeclampsia.

Investigation of the Apoptosis Inducing and ß-catenin Silencing by Tetradentate Schiff Base Zinc(II) Complex on the T-47D Breast Cancer Cells.

INTRODUCTION: Several mechanisms are known for the anticancer effects of cisplatin. However, its most wellknown function involves binding to DNA and activating the DNA damage response. METHODS: Despite its good effects, the treatment process often leads to chemoresistance and affects the mechanisms that support cell survival, such as pathways that promote cell growth, apoptosis, DNA damage repair, and endocytosis. For this reason, we investigated the effects of a new metal complex (tetradentate Schiff base zinc(II) complex) on breast cancer cells (T-47D). We evaluated its effect on cytotoxicity, apoptosis, and drug resistance in comparison to cisplatin. RESULTS: The results of the MTT test showed that tetradentate Schiff base zinc(II) complex has good cytotoxicity compared to cisplatin. The IC50 values for the [Zn(SB)]Cl2 complex and cisplatin after 72 h of exposure were equal to 42.1 and 276.1 µM, respectively. Real-time PCR assay confirmed that the [Zn(SB)]Cl2 complex activated the mitochondrial pathway of apoptosis and increased the expression of Bak1 and caspase-3 genes significantly compared to cisplatin. More importantly, the [Zn(SB)]Cl2 was able to reduce the expression of the ß-catenin gene, which plays a role in drug resistance, by 0.011 compared to the control. CONCLUSION: Therefore, we can hope for this new complex because, without the help of any ß-catenin silencing agent, it was able to inhibit the drug resistance in the T-47D cell line that overexpresses the ß-catenin gene.

Comprehensive Analysis of BR Receptor Expression under Hormone Treatment in the Rubber Tree (Hevea brasiliensis Muell. Arg.).

Brassinosteroids (BRs) are important for plant growth and development, with BRI1 and BAK1 kinases playing an important role in BR signal transduction. Latex from rubber trees is crucial for industry, medicine and defense use. Therefore, it is beneficial to characterize and analyze HbBRI1 and HbBAK1 genes to improve the quality of the resources obtained from Hevea brasiliensis (rubber tree). Based on bioinformatics predictions and rubber tree database, five HbBRI1s with four HbBAK1s were identified and named HbBRI1~HbBRL3 and HbBAK1a~HbBAK1d, respectively, which were clustered in two groups. HbBRI1 genes, except for HbBRL3, exclusively contain introns, which is convenient for responding to external factors, whereas HbBAK1b/c/d contain 10 introns and 11 exons, and HbBAK1a contains eight introns. Multiple sequence analysis showed that HbBRI1s include typical domains of the BRI1 kinase, indicating that HbBRI1s belong to BRI1. HbBAK1s that possess LRR and STK_BAK1_like domains illustrate that HbBAK1s belong to the BAK1 kinase. BRI1 and BAK1 play an important role in regulating plant hormone signal transduction. Analysis of the cis-element of all HbBRI1 and HbBAK1 genes identified hormone response, light regulation and abiotic stress elements in the promoters of HbBRI1s and HbBAK1s. The results of tissue expression patterns indicate that HbBRL1/2/3/4 and HbBAK1a/b/c are highly expressed in the flower, especially HbBRL2-1. The expression of HbBRL3 is extremely high in the stem, and the expression of HbBAK1d is extremely high in the root. Expression profiles with different hormones show that HbBRI1 and HbBAK1 genes are extremely induced by different hormone stimulates. These results provide theoretical foundations for further research on the functions of BR receptors, especially in response to hormone signals in the rubber tree.

Hydathode immunity protects the Arabidopsis leaf vasculature against colonization by bacterial pathogens.

Plants prevent disease by passively and actively protecting potential entry routes against invading microbes. For example, the plant immune system actively guards roots, wounds, and stomata. How plants prevent vascular disease upon bacterial entry via guttation fluids excreted from specialized glands at the leaf margin remains largely unknown. These so-called hydathodes release xylem sap when root pressure is too high. By studying hydathode colonization by both hydathode-adapted (Xanthomonas campestris pv. campestris) and non-adapted pathogenic bacteria (Pseudomonas syringae pv. tomato) in immunocompromised Arabidopsis mutants, we show that the immune hubs BAK1 and EDS1-PAD4-ADR1 restrict bacterial multiplication in hydathodes. Both immune hubs effectively confine bacterial pathogens to hydathodes and lower the number of successful escape events of an hydathode-adapted pathogen toward the xylem. A second layer of defense, which is dependent on the plant hormones' pipecolic acid and to a lesser extent on salicylic acid, reduces the vascular spread of the pathogen. Thus, besides glands, hydathodes represent a potent first line of defense against leaf-invading microbes.

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.

Deletion of Bak1 alleviates microglial necroptosis and neuroinflammation after experimental subarachnoid hemorrhage.

Microglial necroptosis exacerbates neurodegenerative diseases, central nervous system (CNS) injury, and demonstrates a proinflammatory process, but its contribution to subarachnoid hemorrhage (SAH) is poorly characterized. BCL-2 homologous antagonist-killer protein (Bak1), a critical regulatory molecule of endogenous apoptosis, can be involved in the pathologic process of necroptosis by regulating mitochondrial permeability. In this study, we revealed microglia undergo necroptosis after SAH in vivo and vitro. Western blot revealed that Bak1 was elevated at 24 h after SAH. Knocked down of Bak1 by adeno-associated virus attenuates microglial necroptosis, alleviates neuroinflammation, and improves neurologic function after SAH in mice. Furthermore, oxyhemoglobin (10 µM) induced necroptosis in BV2 microglia, increasing Bak1 expression and mediating proinflammatory phenotype transformation, exacerbating oxidative stress and neuroinflammation. Abrogating BV2 Bak1 could reduce necroptosis by down-regulating the expression of phosphorylated pseudokinase mixed lineage kinase domain-like protein (p-MLKL), then down-regulating proinflammatory phenotype gene expression. RNA-Seq showed that disrupting BV2 Bak1 down-regulates multiple immune and inflammatory pathways and ameliorates cell injury by elevating thrombospondin 1 (THBS1) expression. In summary, we identified a critical regulatory role for Bak1 in microglial necroptosis and neuroinflammation after SAH. Bak1 is expected to be a potential target for the treatment strategy of SAH.