Four receptor‐like cytoplasmic kinases regulate development and immunity in rice

Receptor‐like cytoplasmic kinases (RLCKs) represent a large family of proteins in plants. However, few RLCKs have been well characterized. Here, we report the functional characterization of four rice RLCKs – OsRLCK57, OsRLCK107, OsRLCK118 and OsRLCK176 from subfamily VII. These OsRLCKs interact with the rice brassinosteroid receptor, OsBRI1 in yeast cell, but not the XA21 immune receptor. Transgenic lines silenced for each of these genes have enlarged leaf angles and are hypersensitive to brassinolide treatment compared to wild type rice. Transgenic plants silenced for OsRLCK57 had significantly fewer tillers and reduced panicle secondary branching, and lines silenced for OsRLCK107 and OsRLCK118 produce fewer seeds. Silencing of these genes decreased Xa21 gene expression and compromised XA21‐mediated immunity to Xanthomonas oryzae pv. oryzae. Our study demonstrates that these OsRLCKs negatively regulate BR signalling, while positively regulating immune responses by contributing to the expression of the immune receptor XA21.     

The Receptor-Like Cytoplasmic Kinase OsRLCK102 Regulates XA21-Mediated Immunity and Plant Development in Rice

Receptor-like cytoplasmic kinases (RLCKs) belong to a large subgroup of kinases that play pivotal roles in plant development and in protecting plants from various stresses. Here, we report the isolation and characterization of rice OsRLCK102, from the OsRLCK VII subgroup. Silencing of OsRLCK102 compromised receptor kinase XA21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) but did not affect plant basal resistance to Xoo or Magnaporthe oryzae (M. oryzae). Plants with silenced OsRLCK102 exhibit architecture alterations, including reduced plant height, enlarged angle of the lamina joint, decreased rates of seed setting and enhanced sensitivity to hormone brassinolide (BR). Collectively, our study reveals that OsRLCK102 positively regulates XA21-mediated immunity and negatively regulates rice development through BR signaling in rice.     

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity

Microbe‐associated molecular pattern (MAMP)‐triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif‐containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor‐like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN‐triggered immunity in rice. Silencing of OsCERK1 suppressed PGN‐induced (and chitin‐induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor‐binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand‐induced heterooligomeric receptor complexes. Furthermore, the receptor‐like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.     

Three LysM effectors of Zymoseptoria tritici collectively disarm chitin-triggered plant immunity

Chitin is a major structural component of fungal cell walls and acts as a microbe-associated molecular pattern (MAMP) that, on recognition by a plant host, triggers the activation of immune responses. To avoid the activation of these responses, the Septoria tritici blotch (STB) pathogen of wheat, Zymoseptoria tritici, secretes LysM effector proteins. Previously, the LysM effectors Mg1LysM and Mg3LysM were shown to protect fungal hyphae against host chitinases. Furthermore, Mg3LysM, but not Mg1LysM, was shown to suppress chitin-induced reactive oxygen species (ROS) production. Whereas initially a third LysM effector gene was disregarded as a presumed pseudogene, we now provide functional data to show that this gene also encodes a LysM effector, named Mgx1LysM, that is functional during wheat colonization. While Mg3LysM confers a major contribution to Z. tritici virulence, Mgx1LysM and Mg1LysM contribute to Z. tritici virulence with smaller effects. All three LysM effectors display partial functional redundancy. We furthermore demonstrate that Mgx1LysM binds chitin, suppresses the chitin-induced ROS burst, and is able to protect fungal hyphae against chitinase hydrolysis. Finally, we demonstrate that Mgx1LysM is able to undergo chitin-induced polymerization. Collectively, our data show that Z. tritici utilizes three LysM effectors to disarm chitin-triggered wheat immunity.     

Activation of immune receptor Rx1 triggers distinct immune responses culminating in cell death after 4 hours

Intracellular nucleotide-binding leucine-rich repeat (NLR)-type immune receptors are a fundamental part of plant immune systems. As infection occurs at foci, activation of immune responses is typically non-uniform and non-synchronized, hampering the systematic dissection of their cellular effects and determining their phasing. We investigated the potato NLR Rx1 using the CESSNA (Controlled Expression of effectors for Synchronized and Systemic NLR Activation) platform. CESSNA-mediated Potato virus X coat protein (CP) expression allowed the monitoring of Rx1-mediated immune responses in a quantitative and reproducible manner. Rx1 was found to trigger a reactive oxygen species (ROS) burst and ion leakage within 1 h and a change in autofluorescence within 2 h after the induction of CP production. After 2 h, HIN1 expression was increased and single-stranded DNA (ssDNA) damage and loss of cellular integrity became apparent, followed by double-stranded DNA (dsDNA) damage after 3 h and increased PR-1a, LOX, ERF1 and AOX1B expression and cell death at 4 h. Nuclear exclusion of Rx1 resulted in increased basal levels of ROS and permitted Rx1 activation by an Rx1-breaking CP variant. In contrast, nuclear-targeted Rx1 showed diminished basal ROS levels, and only avirulent CP could trigger a compromised ROS production. Both nuclear-excluded and nuclear-targeted Rx1 triggered a delayed ion leakage compared with non-modified Rx1, suggesting that ion leakage and ROS production originate from distinct signalling pathways. This work offers novel insights into the influence of Rx1 localization on its activity, and the interplay between Rx1-triggered processes.     

A rice fungal MAMP‐responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis

Plants recognize potential microbial pathogens through microbial‐associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti‐microbial secondary metabolites. Mitogen‐activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor‐induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4^DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4^DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4^DD‐induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP‐triggered defense responses.     

The Cotton Wall-Associated Kinase GhWAK7A Mediates Responses to Fungal Wilt Pathogens by Complexing with the Chitin Sensory Receptors

Plant receptor-like kinases (RLKs) are important players in response to pathogen infections. Verticillium and Fusarium wilts, caused by Verticillium dahliae (Vd) and Fusarium oxysporum f. sp vasinfectum (Fov), respectively, are among the most devastating diseases in cotton (Gossypium spp). To understand the cotton response to these soil-borne fungal pathogens, we performed a genome-wide in silico characterization and functional screen of diverse RLKs for their involvement in cotton wilt diseases. We identified Gossypium hirsutum GhWAK7A, a wall-associated kinase, that positively regulates cotton response to both Vd and Fov infections. Chitin, the major constituent of the fungal cell wall, is perceived by lysin-motif-containing RLKs (LYKs/CERK1), leading to the activation of plant defense against fungal pathogens. A conserved chitin sensing and signaling system is present in cotton, including chitin-induced GhLYK5-GhCERK1 dimerization and phosphorylation, and contributes to cotton defense against Vd and Fov. Importantly, GhWAK7A directly interacts with both GhLYK5 and GhCERK1 and promotes chitin-induced GhLYK5-GhCERK1 dimerization. GhWAK7A phosphorylates GhLYK5, which itself does not have kinase activity, but requires phosphorylation for its function. Consequently, GhWAK7A plays a crucial role in chitin-induced responses. Thus, our data reveal GhWAK7A as an important component in cotton response to fungal wilt pathogens by complexing with the chitin receptors.     

The receptor-like cytoplasmic kinase BSR1 mediates chitin-induced defense signaling in rice cells

Broad-Spectrum Resistance 1 (BSR1) encodes a rice receptor-like cytoplasmic kinase, and enhances disease resistance when overexpressed. Rice plants overexpressing BSR1 are highly resistant to diverse pathogens, including rice blast fungus. However, the mechanism responsible for this resistance has not been fully characterized. To analyze the BSR1 function, BSR1-knockout (BSR1-KO) plants were generated using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Experiments using suspension-cultured cells revealed that defense responses including H₂O₂ production (i.e. oxidative burst) and expression of defense-related genes induced by autoclaved conidia of the rice blast fungus significantly decreased in BSR1-KO cells. Furthermore, a treatment with chitin oligomers which function as microbe-associated molecular patterns (MAMPs) of the rice blast fungus resulted in considerably suppressed defense responses in BSR1-KO cells. These results suggest that BSR1 is important for the rice innate immunity triggered by the perception of chitin.     

Rice ubiquitin-conjugating enzyme OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a

Ubc13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity still remain largely unknown. Here, we used molecular biological, pathological, biochemical, and genetic approaches to evaluate the roles of rice OsUbc13 in response to pathogens. The OsUbc13-RNA interference (RNAi) lines with lesion mimic phenotypes displayed a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in defence-related genes expression or hormones as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Strikingly, OsUbc13 directly interacts with OsSnRK1a, which is the α catalytic subunit of SnRK1 (sucrose non-fermenting-1-related protein kinase-1) and acts as a positive regulator of broad-spectrum disease resistance in rice. In the OsUbc13-RNAi plants, although the protein level of OsSnRK1a did not change, its activity and ABA sensitivity were obviously enhanced, and the K63-linked polyubiquitination was weaker than that of wild-type Dongjin (DJ). Overexpression of the deubiquitinase-encoding gene OsOTUB1.1 produced similar effects with inhibition of OsUbc13 in affecting immunity responses, M. oryzae resistance, OsSnRK1a ubiquitination, and OsSnRK1a activity. Furthermore, re-interfering with OsSnRK1a in one OsUbc13-RNAi line (Ri-3) partially restored its M. oryzae resistance to a level between those of Ri-3 and DJ. Our data demonstrate OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.     

Recognition of N-acetylchitooligosaccharide elicitor by rice protoplasts

The response by rice protoplasts to N-acetylchitooligosaccharide elicitor was examined by monitoring the production of reactive oxygen species (ROS), and the expression of the two early-responsive genes, EL2 and EL3. Freshly prepared rice protoplasts produced a high level of ROS in the absence of the elicitor, and did not show further increase of the ROS generation in response to N-acetylchitooligosaccharide elicitor. By incubating protoplasts for 1 d, the background level decreased and the induction of ROS production and the induction of mRNAs for the two genes were observed. The structural requirements of N-acetylchitooligosaccharides for elicitor-activity, as well as the effects of inhibitors of protein kinase (K-252a), protein phosphatase (calyculin A) and protein synthesis (cycloheximide) on the ROS production and gene expression were very similar to those observed in suspension-cultured rice cells, indicating that rice protoplasts retain the machinery for the recognition of, and initial signaling from, N-acetylchitooligosaccharide elicitor.     

SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.     

Effector-mediated suppression of chitin-triggered immunity by magnaporthe oryzae is necessary for rice blast disease

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.     

Reactive Oxygen Species Regulate Caspase-11 Expression and Activation of the Non-canonical NLRP3 Inflammasome during Enteric Pathogen Infection

Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3- and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway.     

The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst

Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca²⁺) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca²⁺ burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca²⁺-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca²⁺ burst, supporting its role as a negative regulator of BIK1.     

沉默GmATG10导致大豆免疫反应的激活

自噬途径是真核生物中普遍存在的物质降解及循环利用的保守机制,在真核生物的生长发育以及免疫反应等方面起着至关重要的作用。而ATG10在自噬体(autophagosomes)的形成过程中起着非常重要的作用。为探讨大豆(Glycine max) ATG10在免疫防御反应中的功能,本研究采用大豆豆荚斑驳病毒(bean pod mottle virus,BPMV)诱导的基因沉默技术(virus-induced gene silencing,VIGS)成功地在大豆中同时沉默ATG10的两个同源基因(GmATG10a和GmATG10b);通过黑暗诱导的碳饥饿处理以及GmATG8积累水平的Western blotting分析证明,同时沉默GmATG10a/10b可导致大豆叶片出现自噬缺陷;抗病性鉴定与激酶分析证明沉默GmATG10a/10b可通过负调控Gm MPK3/6激活而参与免疫反应,是大豆免疫反应的负调控因子。     

Expression of the chimeric receptor between the chitin elicitor receptor CEBiP and the receptor-like protein kinase Pi-d2 leads to enhanced responses to the chitin elicitor and disease resistance against Magnaporthe oryzae in rice

We previously reported that rice plants expressing the chimeric receptor consisting of rice chitin oligosaccharides binding protein (CEBiP) and the intracellular protein kinase region of Xa21, which confers resistance to rice bacterial blight, showed enhanced cellular responses to a chitin elicitor N-acetylchitoheptaose and increased resistance to the rice blast fungus Magnaporthe oryzae. Here, we investigated whether CEBiP fused with another type of receptor-like protein kinase (RLK) also functions as a chimeric receptor. Fusion proteins CRPis consist of CEBiP and the intracellular protein kinase region of a true resistance gene Pi-d2. Transgenic rice expressing a CRPi showed enhanced cellular responses specifically to N-acetylchitoheptaose in cultured cells and increased levels of disease resistance against M. oryzae in plants. These responses depended on the amino acid sequences predicted to be essential for the protein kinase activity of CRPi. The structure of the transmembrane domain in CRPi affected the protein accumulation, cellular responses, and disease resistance in transgenic rice. These results suggest that the chimeric receptor consisting of CEBiP and Pi-d2 functions as a receptor for chitin oligosaccharides and CEBiP-based chimeric receptors fused with other RLKs may also act as functional receptors.     

SYK regulates macrophage MHC-II expression via activation of autophagy in response to oxidized LDL

Adaptive immunity, which plays an important role in the development of atherosclerosis, is mediated by major histocompatibility complex (MHC)-dependent antigen presentation. In atherosclerotic lesions, macrophages constitute an important class of antigen-presenting cells that activate adaptive immune responses to oxidized low-density lipoprotein (OxLDL). It has been reported that autophagy regulates adaptive immune responses by enhancing antigen presentation to MHC class II (MHC-II). In a previous study, we have demonstrated that SYK (spleen tyrosine kinase) regulates generation of reactive oxygen species (ROS) and activation of MAPK8/JNK1 in macrophages. Because ROS and MAPK8 are known to regulate autophagy, in this study we investigated the role of SYK in autophagy, MHC-II expression and adaptive immune response to OxLDL. We demonstrate that OxLDL induces autophagosome formation, MHC-II expression, and phosphorylation of SYK in macrophages. Gene knockout and pharmacological inhibitors of NOX2 and MAPK8 reduced OxLDL-induced autophagy. Using bone marrow-derived macrophages isolated from wild-type and myeloid-specific SYK knockout mice, we demonstrate that SYK regulates OxLDL-induced ROS generation, MAPK8 activation, BECN1-BCL2 dissociation, autophagosome formation and presentation of OxLDL-derived antigens to CD4⁺ T cells. ldlr^−/− syk^−/− mice fed a high-fat diet produced lower levels of IgG to malondialdehyde (MDA)-LDL, malondialdehyde-acetaldehyde (MAA)-LDL, and OxLDL compared to ldlr^−/− mice. These results provide new insights into the mechanisms by which SYK regulates MHC-II expression via autophagy in macrophages and may contribute to regulation of adaptive immune responses in atherosclerosis.