Overexpression of the Endoplasmic Reticulum Chaperone BiP3 Regulates XA21-Mediated Innate Immunity in Rice

Recognition of pathogen-associated molecular patterns by pattern recognition receptors (PRRs) activates the innate immune response. Although PRR-mediated signaling events are critical to the survival of plants and animals, secretion and localization of PRRs have not yet been clearly elucidated. Here we report the in vivo interaction of the endoplasmic reticulum (ER) chaperone BiP3 with the rice XA21 PRR, which confers resistance to the Gram negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). We show that XA21 is glycosylated and is primarily localized to the ER and also to the plasma membrane (PM). In BiP3-overexpressing rice plants, XA21-mediated immunity is compromised, XA21 stability is significantly decreased, and XA21 proteolytic cleavage is inhibited. BiP3 overexpression does not affect the general rice defense response, cell death or brassinolide-induced responses. These results indicate that BiP3 regulates XA21 protein stability and processing and that this regulation is critical for resistance to Xoo.     

Big Roles of Small Kinases： The Complex Functions of Receptor-Like Cytoplasmic Kinases in Plant Immunity and Development

Plants have evolved a large number of receptor-like cytoplasmic kinases （RLCKs） that often functionally and physically associate with receptor-like kinases （RLKs） to modulate plant growth, development and immune responses. Without any apparent extracellular domain, RLCKs relay intracellular signaling often via RLK complex-mediated transphosphorylation events. Recent advances have suggested essential roles of diverse RLCKs in concert with RLKs in regulating various cellular and physiological responses. We summarize here the complex roles of RLCKs in mediating plant immune responses and growth regulation, and discuss specific and overlapping functions of RLCKs in transducing diverse signaling pathways.     

植物类受体胞质激酶的结构和功能

本文介绍植物类受体胞质激酶的结构及其在植物的抗病、抗逆、生长发育、自交不亲和、油菜素内酯信号转导等方面的功能。     

The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity

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Receptor-Like Kinases in Plant Innate Immunity

Plants employ a highly effective surveillance system to detect potential pathogens, which is critical for the success of land plants in an environment surrounded by numerous microbes. Recent efforts have led to the identification of a number of immune receptors and components of immune receptor complexes. It is now clear that receptor-like kinases （RLKs） and receptor-like proteins （RLPs） are key pattern-recognition receptors （PRRs） for microbe- and plant-derived molecular patterns that are associated with pathogen invasion. RLKs and RLPs involved in immune signaling belong to large gene families in plants and have undergone lineage specific expansion. Molecular evolution and population studies on phytopathogenic molecular signatures and their receptors have provided crucial insight into the co-evolution between plants and pathogens.     

Identification, Expression and Functional Analysis of a Receptor-like Cytoplasmic Kinase, OsRLCK1, in Rice

Pollination involves a series of complex cellular interactions and signal transduction events. Numerous reports have suggested a central role for protein kinases in pollen germination and pollen tube growth and a large number of receptor-like kinases have been detected exclusively in pollen in higher plants. However, few are well characterized, especially for the receptor-like cytoplasmic kinases. Here we report a receptor-like kinase gene, OsRLCK1, which belongs to the receptor-like cytoplasmic kinase Ⅷ subfamily. Real-time quantitative polymerase chain reaction analysis and whole mount RNA in situ hybridization showed that OsRLCK1 is a pollen-specific gene and expressed only in the mature pollen. When expressed in the onion epidermal cells, the OsRLCK1-GFP fusion protein was diffused throughout the cell, indicating its cytoplasmic and nuclear localization. The Maltose Binding Protein-OsRLCK1 recombinant protein was found to be capable of autophosphorylation on threonine residue, showing that it encodes a functional kinase. These results suggest that OsRLCK1 is likely to play a role in a signaling pathway associated with pollen performance during pollination in rice.     

The Secreted Peptide PIP1 Amplifies Immunity through Receptor-Like Kinase 7

In plants, innate immune responses are initiated by plasma membrane-located pattern recognition receptors (PRRs) upon recognition of elicitors, including exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs). Arabidopsis thaliana produces more than 1000 secreted peptide candidates, but it has yet to be established whether any of these act as elicitors. Here we identified an A. thaliana gene family encoding precursors of PAMP-induced secreted peptides (prePIPs) through an in-silico approach. The expression of some members of the family, including prePIP1 and prePIP2, is induced by a variety of pathogens and elicitors. Subcellular localization and proteolytic processing analyses demonstrated that the prePIP1 product is secreted into extracellular spaces where it is cleaved at the C-terminus. Overexpression of prePIP1 and prePIP2, or exogenous application of PIP1 and PIP2 synthetic peptides corresponding to the C-terminal conserved regions in prePIP1 and prePIP2, enhanced immune responses and pathogen resistance in A. thaliana. Genetic and biochemical analyses suggested that the receptor-like kinase 7 (RLK7) functions as a receptor of PIP1. Once perceived by RLK7, PIP1 initiates overlapping and distinct immune signaling responses together with the DAMP PEP1. PIP1 and PEP1 cooperate in amplifying the immune responses triggered by the PAMP flagellin. Collectively, these studies provide significant insights into immune modulation by Arabidopsis endogenous secreted peptides.     

xopAC-triggered Immunity against Xanthomonas Depends on Arabidopsis Receptor-Like Cytoplasmic Kinase Genes PBL2 and RIPK

Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc.     

LYK4, a Lysin Motif Receptor-Like Kinase, Is Important for Chitin Signaling and Plant Innate Immunity in Arabidopsis

Chitin is commonly found in fungal cell walls and is one of the well-studied microbe/pathogen-associated molecular patterns. Previous studies showed that lysin motif (LysM)-containing proteins are essential for plant recognition of chitin, leading to the activation of plant innate immunity. In Arabidopsis (Arabidopsis thaliana), the LYK1/CERK1 (for LysM-containing receptor-like kinase1/chitin elicitor receptor kinase1) was shown to be essential for chitin recognition, whereas in rice (Oryza sativa), the LysM-containing protein, CEBiP (for chitin elicitor-binding protein), was shown to be involved in chitin recognition. Unlike LYK1/CERK1, CEBiP lacks an intracellular kinase domain. Arabidopsis possesses three CEBiP-like genes. Our data show that mutations in these genes, either singly or in combination, did not compromise the response to chitin treatment. Arabidopsis also contains five LYK genes. Analysis of mutations in LYK2, -3, -4, or -5 showed that LYK4 is also involved in chitin signaling. The lyk4 mutants showed reduced induction of chitin-responsive genes and diminished chitin-induced cytosolic calcium elevation as well as enhanced susceptibility to both the bacterial pathogen Pseudomonas syringae pv tomato DC3000 and the fungal pathogen Alternaria brassicicola, although these phenotypes were not as dramatic as that seen in the lyk1/cerk1 mutants. Similar to LYK1/CERK1, the LYK4 protein was also localized to the plasma membrane. Therefore, LYK4 may play a role in the chitin recognition receptor complex to assist chitin signal transduction and plant innate immunity.     

Evolutionary History and Stress Regulation of Plant Receptor-Like Kinase/Pelle Genes

Receptor-Like Kinase (RLK)/Pelle genes play roles ranging from growth regulation to defense response, and the dramatic expansion of this family has been postulated to be crucial for plant-specific adaptations. Despite this, little is known about the history of or the factors that contributed to the dramatic expansion of this gene family. In this study, we show that expansion coincided with the establishment of land plants and that RLK/Pelle subfamilies were established early in land plant evolution. The RLK/Pelle family expanded at a significantly higher rate than other kinases, due in large part to expansion of a few subfamilies by tandem duplication. Interestingly, these subfamilies tend to have members with known roles in defense response, suggesting that their rapid expansion was likely a consequence of adaptation to fast-evolving pathogens. Arabidopsis (Arabidopsis thaliana) expression data support the importance of RLK/Pelles in biotic stress response. We found that hundreds of RLK/Pelles are up-regulated by biotic stress. Furthermore, stress responsiveness is correlated with the degree of tandem duplication in RLK/Pelle subfamilies. Our findings suggest a link between stress response and tandem duplication and provide an explanation for why a large proportion of the RLK/Pelle gene family is found in tandem repeats. In addition, our findings provide a useful framework for potentially predicting RLK/Pelle stress functions based on knowledge of expansion pattern and duplication mechanism. Finally, we propose that the detection of highly variable molecular patterns associated with specific pathogens/parasites is the main reason for the up-regulation of hundreds of RLK/Pelles under biotic stress.     

Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase

Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases.Receptor-like kinases (RLKs) are a large group of kinases with a variable extracellular domain and a cytoplasmic kinase domain linked by a single transmembrane motif. RLKs have been shown to play diverse roles in regulating plant innate immunity as well as growth and development (Morillo and Tax, 2006). The extracellular domains of RLKs are believed to bind directly to ligands to perceive extracellular signals, whereas the cytoplasmic kinase domains transduce these signals into the cell. There are over 600 RLKs (Shiu and Bleecker, 2001) in Arabidopsis (Arabidopsis thaliana). The biological functions of most RLKs are unknown.Several RLKs have been identified to be receptors of microbe-associated molecular patterns (MAMPs). FLS2 and EFR are two well-characterized RLKs with extracellular Leu-rich repeats (LRRs) that recognize bacterial flagellin and translation elongation factor EF-Tu, respectively (Gomez-Gomez and Boller, 2000; Zipfel et al., 2006). BAK1 is also an RLK with extracellular LRRs. BAK1 seems to function as an adaptor protein for multiple RLKs including BRI1, FLS2, and BIR1 (Li et al., 2002; Nam and Li, 2002; Chinchilla et al., 2007; Heese et al., 2007; Gao et al., 2009). Interestingly, knocking out BIR1 activates cell death and defense responses mediated by another RLK, SOBIR1 (Gao et al., 2009). Recently, the rice (Oryza sativa) RLK Xa21 was also suggested to be a MAMP receptor. Xa21 recognizes a peptide derived from the secreted effector protein AvrXa21, which is conserved among different Xanthomonas species (Lee et al., 2009). Unlike FLS2, EFR, and Xa21, the putative receptor for chitin is an RLK with three extracellular LysM domains instead of LRRs that are required for the perception of chitin as well as resistance against bacterial pathogens (Miya et al., 2007; Wan et al., 2008; Gimenez-Ibanez et al., 2009).Perception of MAMPs by receptors leads to the rapid activation of mitogen-activated protein (MAP) kinases including MAP KINASE3 (MPK3), MPK4, and MPK6 (Boller and Felix, 2009). MAP KINASE SUBSTRATE1 (MKS1) was identified as an MPK4-interacting protein that positively regulates defense responses (Andreasson et al., 2005). Silencing of MKS1 compromises basal resistance to Pseudomonas syringae pv tomato DC3000, whereas overexpression of MKS1 leads to enhanced pathogen resistance. Activation of MAMP receptors also induces a number of responses such as oxidative burst, callose deposition, and increased salicylic acid synthesis (Boller and Felix, 2009). Defense responses induced by different MAMPs seem similar, suggesting that they may share common signaling components. Identification of the signaling components downstream of RLK receptors remains a major task in understanding MAMP-triggered immunity.Here we report the identification and characterization of suppressor of npr1-1, constitutive4-1D (snc4-1D), a gain-of-function mutant of an atypical RLK that is autoactivated by a mutation in its kinase domain. The snc4-1D mutant plants constitutively express defense maker genes PR1, PR2, and PDF1.2, and display enhanced resistance to Hyaloperonospora arabidopsidis (H. a.) Noco2. Epistasis analysis showed that snc4-1D-mediated defense responses are dependent on multiple factors including NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), MKS1, and OPR3.     

The Monocot-Specific Receptor-like Kinase SDS2 Controls Cell Death and Immunity in Rice

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The Receptor-Like Kinase SIT1 Mediates Salt Sensitivity by Activating MAPK3/6 and Regulating Ethylene Homeostasis in Rice

High salinity causes growth inhibition and shoot bleaching in plants that do not tolerate high salt (glycophytes), including most crops. The molecules affected directly by salt and linking the extracellular stimulus to intracellular responses remain largely unknown. Here, we demonstrate that rice (Oryza sativa) Salt Intolerance 1 (SIT1), a lectin receptor-like kinase expressed mainly in root epidermal cells, mediates salt sensitivity. NaCl rapidly activates SIT1, and in the presence of salt, as SIT1 kinase activity increased, plant survival decreased. Rice MPK3 and MPK6 function as the downstream effectors of SIT1. SIT1 phosphorylates MPK3 and 6, and their activation by salt requires SIT1. SIT1 mediates ethylene production and salt-induced ethylene signaling. SIT1 promotes accumulation of reactive oxygen species (ROS), leading to growth inhibition and plant death under salt stress, which occurred in an MPK3/6- and ethylene signaling-dependent manner in Arabidopsis thaliana. Our findings demonstrate the existence of a SIT1-MPK3/6 cascade that mediates salt sensitivity by affecting ROS and ethylene homeostasis and signaling. These results provide important information for engineering salt-tolerant crops.