RESUMEN
Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with a wide range of terrestrial plants, including rice. However, the mechanisms underlying the initiation of AM symbiosis are yet to be elucidated, particularly in nonleguminous plants. We previously demonstrated that chitin elicitor receptor kinase 1 (OsCERK1), a lysin motif receptor-like kinase essential for chitin-triggered immunity, also plays a key role in AM symbiosis in rice. However, the mechanisms underlying the regulation of switching between immunity and symbiosis by OsCERK1 are yet to be fully elucidated. SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK)/DOES NOT MAKE INFECTIONS 2 (DMI2) is a leucine-rich repeat receptor-like kinase associated with both root nodule symbiosis and AM symbiosis in legumes. The homolog of SYMRK in rice, OsSYMRK, has a shorter form than that in legumes because OsSYMRK lacks a malectin-like domain (MLD). The MLD reportedly contributes to symbiosis in Lotus japonicus; however, the contribution of OsSYMRK to AM symbiosis in rice remains unclear. Phylogenetic analyses indicated that the MLD of SYMRK/DMI2 is widely conserved even in mosses and ferns but absent in commelinids, including rice. To understand the function of OsSYMRK, we produced an Ossymrk knockout mutant using genome editing technology. AM colonization was mostly abolished in Ossymrk with a more severe phenotype than Oscerk1. Ca2+ spiking against chitin tetramer was also diminished in Ossymrk. In contrast, comparable defense responses against chitin heptamer to the wild type were observed in Ossymrk. Bimolecular fluorescence complementation studies demonstrating an interaction between OsSYMRK and OsCERK1 indicate that OsSYMRK may play an important role in switching from immunity to symbiosis through the interaction with OsCERK1 in rice.
Asunto(s)
Micorrizas , Oryza , Simbiosis/genética , Oryza/fisiología , Filogenia , Micorrizas/fisiología , Fosfotransferasas/genética , Quitina , Proteínas de Plantas/genéticaRESUMEN
Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.
Asunto(s)
Quitina/metabolismo , Estomas de Plantas/inmunología , Estomas de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Quitina/fisiología , Quitosano/metabolismo , Hongos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
KEY MESSAGE: The appropriate combination of fluorescent probes enabled the simultaneous visualization of callose deposition and plasma membrane in living Arabidopsis and can be useful for the cell biological study of papilla formation in plants. Localized callose deposition at the site of fungal infection is a central part of papilla formation, which creates a barrier between the host plasma membrane and the cell wall and plays an important role in preventing the penetration of fungal hyphae into the host cells. Using chitin-induced callose deposition as a model system, we examined suitable conditions for the simultaneous visualization of callose deposition and plasma membrane dynamics in living Arabidopsis cotyledons. We found that aniline blue fluorochrome (ABF) for callose staining selectively interferes with FM dyes for membrane visualization depending on the structure of the latter compounds and the proper combination of these fluorescent dyes and staining conditions is a key for successful live-cell imaging. The established conditions enabled the live-cell imaging of chitin-induced callose deposition and host membrane systems. The established system/conditions would also be useful for the cell biological studies on the localized callose deposition in other stress/development-associated processes. The finding that the slight difference in the structure of FM dyes affects the interaction with another fluorescent dye, ABF, would also give useful suggestions for the studies where multiple fluorescent dyes are utilized for live-cell imaging.
Asunto(s)
Arabidopsis/citología , Membrana Celular/metabolismo , Colorantes Fluorescentes/metabolismo , Glucanos/metabolismo , Células Vegetales/metabolismo , Compuestos de Anilina/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Quitina/metabolismo , Quitina/farmacología , Cotiledón/citología , Cotiledón/metabolismo , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Mutación , Proteínas Serina-Treonina Quinasas/genética , Compuestos de Piridinio/química , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismoRESUMEN
While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Quitina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fosforilación/genética , Fosforilación/fisiología , Inmunidad de la Planta/genética , Inmunidad de la Planta/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Lysin motif (LysM) receptor-like kinase CERK1 is a co-receptor essential for plant immune responses against carbohydrate microbe-associated molecular patterns (MAMPs). Concerning the immediate downstream signaling components of CERK1, receptor-like cytoplasmic kinases such as PBL27 and other RLCK VII members have been reported to regulate immune responses positively. In this study, we report that a novel CERK1-interacting E3 ubiquitin ligase, PUB4, is also involved in the regulation of MAMP-triggered immune responses. Knockout of PUB4 resulted in the alteration of chitin-induced defense responses, indicating that PUB4 positively regulates reactive oxygen species generation and callose deposition but negatively regulates MAPK activation and defense gene expression. On the other hand, detailed analyses of a double knockout mutant of pub4 and sid2, a mutant of salicylic acid (SA) synthesis pathway, showed that the contradictory phenotype of the pub4 mutant was actually caused by abnormal accumulation of SA in this mutant and that PUB4 is a positive regulator of immune responses. The present and recent findings on the role of PUB4 indicate that PUB4 is a unique E3 ubiquitin ligase involved in the regulation of both plant immunity and growth/development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Enfermedades de las Plantas , Inmunidad de la Planta/genética , Inmunidad de la Planta/fisiología , Transducción de Señal/fisiología , Ubiquitina/metabolismoRESUMEN
Plant cell surface receptor-like kinases (RLKs) mediate the signals from microbe-associated molecular patterns (MAMPs) that induce immune responses. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, is a common MAMP perceived by animals and plants; however, the plant receptors/co-receptors are unknown except for LORE, a bulb-type lectin S-domain RLK (B-lectin SD1-RLK) in Arabidopsis. OsCERK1 is a multifunctional RLK in rice that contains lysin motifs (LysMs) and is essential for the perception of chitin, a fungal MAMP, and peptidoglycan, a bacterial MAMP. Here, we analyzed the relevance of OsCERK1 to LPS perception in rice. Using OsCERK1-knockout mutants (oscerk1), we evaluated hydrogen peroxide (H2 O2 ) production and gene expression after LPS treatment. We also examined the LPS response in knockout mutants for the B-lectin SD1-RLK genes in rice and for all LysM-protein genes in Arabidopsis. Compared with wild-type rice cells, LPS responses in oscerk1 cells were mostly diminished. By contrast, rice lines mutated in either of three B-lectin SD1-RLK genes and Arabidopsis lines mutated in the LysM-protein genes responded normally to LPS. From these results, we conclude that OsCERK1 is an LPS receptor/co-receptor and that the LPS perception systems of rice and Arabidopsis are significantly different.
Asunto(s)
Lipopolisacáridos/farmacología , Oryza/inmunología , Inmunidad de la Planta/efectos de los fármacos , Proteínas de Plantas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Mutación/genética , Oryza/efectos de los fármacos , Células Vegetales/efectos de los fármacos , Células Vegetales/metabolismo , Proteínas de Plantas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified in AM fungal exudates and shown to activate nuclear calcium (Ca2+ ) spiking in the rice root epidermis. An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca2+ signaling following root treatment with either crude fungal exudates or purified Myc-COs. Compared with wild-type rice, Ca2+ spiking responses to AM fungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs. These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.
Asunto(s)
Quitina/metabolismo , Micorrizas/metabolismo , Oryza/microbiología , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Calcio/metabolismo , Técnicas de Inactivación de Genes , Mutación , Micorrizas/fisiología , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Proteínas Serina-Treonina Quinasas/genética , Transducción de SeñalRESUMEN
Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile122 in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)8 induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)8-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNß1,4GlcNAc)4, which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1.
Asunto(s)
Quitina/química , Oryza/inmunología , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Epítopos/inmunología , Ligandos , Lisina/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligosacáridos/química , Oryza/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Homología de Secuencia de Aminoácido , NicotianaRESUMEN
In legume-specific rhizobial symbiosis, host plants perceive rhizobial signal molecules, Nod factors, by a pair of LysM receptor-like kinases, NFR1/LYK3 and NFR5/NFP, and activate symbiotic responses through the downstream signaling components also required for arbuscular mycorrhizal (AM) symbiosis. Recently, the rice NFR1/LYK3 ortholog, OsCERK1, was shown to play crucial roles for AM symbiosis. On the other hand, the roles of the NFR5/NFP ortholog in rice have not been elucidated, while it has been shown that NFR5/NFP orthologs, Parasponia PaNFR5 and tomato SlRLK10, engage in AM symbiosis. OsCERK1 also triggers immune responses in combination with a receptor partner, OsCEBiP, against fungal or bacterial infection, thus regulating opposite responses against symbiotic and pathogenic microbes. However, it has not been elucidated how OsCERK1 switches these opposite functions. Here, we analyzed the function of the rice NFR5/NFP ortholog, OsNFR5/OsRLK2, as a possible candidate of the OsCERK1 partner for symbiotic signaling. Inoculation of AM fungi induced the expression of OsNFR5 in the rice root, and the chimeric receptor consisting of the extracellular domain of LjNFR5 and the intracellular domain of OsNFR5 complemented the Ljnfr5 mutant for rhizobial symbiosis, indicating that the intracellular kinase domain of OsNFR5 could activate symbiotic signaling in Lotus japonicus. Although these data suggested the possible involvement of OsNFR5 in AM symbiosis, osnfr5 knockout mutants were colonized by AM fungi similar to the wild-type rice. These observations suggested several possibilities including the presence of functionally redundant genes other than OsNFR5 or involvement of novel ligands, which do not require OsNFR5 for recognition.
Asunto(s)
Micorrizas/fisiología , Oryza/enzimología , Oryza/microbiología , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Simbiosis , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Genes de Plantas , Prueba de Complementación Genética , Lotus/metabolismo , Mutación/genética , Oryza/genética , Fenotipo , Filogenia , Proteínas de Plantas/genética , Nodulación de la Raíz de la Planta/genética , Proteínas Quinasas/genética , Multimerización de Proteína , Homología de Secuencia de Aminoácido , Simbiosis/genéticaRESUMEN
Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Quitina/farmacología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Treonina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Mutación , Fosforilación/efectos de los fármacos , Inmunidad de la Planta/efectos de los fármacos , Plantas Modificadas Genéticamente , Dominios Proteicos , Transducción de Señal/efectos de los fármacosRESUMEN
Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/enzimología , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/inmunología , Transducción de Señal , Alternaria/fisiología , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Quitina/metabolismo , Técnicas de Inactivación de Genes , Glucanos/metabolismo , Modelos Biológicos , Fosforilación , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/fisiología , Plantas Modificadas Genéticamente , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Reconocimiento de Patrones , Especificidad por Sustrato , Nicotiana/enzimología , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/fisiologíaRESUMEN
OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. Green fluorescent protein-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.
Asunto(s)
Quitina/metabolismo , Magnaporthe/fisiología , Oryza/enzimología , Peptidoglicano/metabolismo , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/inmunología , Secuencias de Aminoácidos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Marcación de Gen , Genes Reporteros , Peróxido de Hidrógeno/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Oryza/genética , Oryza/inmunología , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Transducción de SeñalRESUMEN
CEBiP, a plasma membrane-localized glycoprotein of rice, directly binds with chitin elicitors (CE), and has been identified as a receptor for CE by using CEBiP-RNAi rice cells. To further clarify the function of CEBiP, we produced CEBiP-disrupted rice plants by applying an efficient Agrobacterium-mediated gene-targeting system based on homologous recombination, which has recently been developed for rice. Homologous recombination occurred at the CEBiP locus in ~0.5 % of the positive/negative selected calli. In the self-pollinated next generation, it was confirmed that the first exon of CEBiP was replaced with the hygromycin selection cassette as designed, and that the expression of CEBiP was completely deficient in homozygous cebip lines. Affinity-labeling analysis using biotinylated N-acetylchitooctaose demonstrated that CEBiP is the major CE-binding protein in rice cultured cells and leaves, which was consistent with the result that the response to CE in cebip cells was greatly diminished. Nevertheless, we observed a significant decrease in disease resistance against Magnaporthe oryzae, the causal agent of rice blast disease, only when the cebip leaf sheaths were inoculated with a weakly virulent strain, suggesting that CE perception during the infection process of M. oryzae is limited. The response to peptidoglycan and lipopolysaccharides in cebip cells was not affected, strongly suggesting that CEBiP is a CE-specific receptor.
Asunto(s)
Quitina/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Prueba de Complementación Genética , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/metabolismo , Magnaporthe/fisiología , Mutación , Oligosacáridos/metabolismo , Oligosacáridos/farmacología , Oryza/genética , Oryza/microbiología , Peptidoglicano/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica , Interferencia de ARN , Receptores de Reconocimiento de Patrones/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.
Asunto(s)
Quitina/metabolismo , Micorrizas/fisiología , Oryza/fisiología , Proteínas de Plantas/metabolismo , Simbiosis , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Quitina/inmunología , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Lotus/genética , Datos de Secuencia Molecular , Mutación , Oryza/inmunología , Oryza/microbiología , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Rhizobium/fisiología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Resistencia a la Enfermedad/fisiología , Magnaporthe/fisiología , Oryza/inmunología , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Células Cultivadas , Quitina/metabolismo , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Mutación , Oligosacáridos/metabolismo , Oryza/genética , Oryza/microbiología , Oryza/fisiología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , TransgenesRESUMEN
Plant cell wall plays important roles in the regulation of plant growth/development and affects the quality of plant-derived food and industrial materials. On the other hand, genetic variability of cell wall structure within a plant species has not been well understood. Here we show that the endosperm cell walls, including both starchy endosperm and aleurone layer, of rice grains with various genetic backgrounds are clearly classified into two groups depending on the presence/absence of ß-1,4-linked glucomannan. All-or-none distribution of the glucomannan accumulation among rice varieties is very different from the varietal differences of arabinoxylan content in wheat and barley, which showed continuous distributions. Immunoelectron microscopic observation suggested that the glucomannan was synthesized in the early stage of endosperm development, but the synthesis was down-regulated during the secondary thickening process associated with the differentiation of aleurone layer. Significant amount of glucomannan in the cell walls of the glucomannan-positive varieties, i.e., 10% or more of the starchy endosperm cell walls, and its close association with the cellulose microfibril suggested possible effects on the physicochemical/biochemical properties of these cell walls. Comparative genomic analysis indicated the presence of striking differences between OsCslA12 genes of glucomannan-positive and negative rice varieties, Kitaake and Nipponbare, which seems to explain the all-or-none glucomannan cell wall trait in the rice varieties. Identification of the gene responsible for the glucomannan accumulation could lead the way to clarify the effect of the accumulation of glucomannan on the agronomic traits of rice by using genetic approaches.
RESUMEN
Nitrogen-fixing symbiosis between legumes and rhizobia is initiated by the recognition of rhizobial Nod factors (NFs) by host plants. NFs are diversely modified derivatives of chitin oligosaccharide, a fungal elicitor that induces defense responses in plants. Recent evidence has shown that both NFs and chitin elicitors are recognized by structurally related LysM receptor kinases. Transcriptome analyses of Lotus japonicus roots indicated that NFs not only activate symbiosis genes but also transiently activate defense-related genes through NF receptors. Conversely, chitin oligosaccharides were able to activate symbiosis genes independently of NF receptors. Analyses using chimeric genes consisting of the LysM receptor domain of a Lotus japonicus NF receptor, NFR1, and the kinase domain of an Arabidopsis chitin receptor, CERK1, demonstrated that substitution of a portion of the αEF helix in CERK1 with the amino acid sequence YAQ from the corresponding region of NFR1 enables L. japonicus nfr1 mutants to establish symbiosis with Mesorhizobium loti. We also showed that the kinase domains of two Lotus japonicus LysM receptor kinases, Lys6 and Lys7, which also possess the YAQ sequence, suppress the symbiotic defect of nfr1. These results strongly suggest that, in addition to adaptation of extracellular LysM domains to NFs, limited alterations in the kinase domain of chitin receptors have played a crucial role in shifting the intracellular signaling to symbiosis from defense responses, thus constituting one of the key genetic events in the evolution of root nodule symbiosis in legume plants.
Asunto(s)
Lotus/enzimología , Mesorhizobium/fisiología , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Quitina/metabolismo , Evolución Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/fisiología , Prueba de Complementación Genética , Lipopolisacáridos/metabolismo , Lotus/genética , Lotus/microbiología , Mesorhizobium/genética , Datos de Secuencia Molecular , Fijación del Nitrógeno/fisiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Nódulos de las Raíces de las Plantas/citología , Nódulos de las Raíces de las Plantas/metabolismo , Alineación de Secuencia , Transducción de Señal , Simbiosis/genéticaRESUMEN
Chitin is a representative microbe-associated molecular pattern (MAMP) molecule for various fungi and induces immune responses in many plant species. It has been clarified that the chitin signaling in rice requires a receptor kinase OsCERK1 and a receptor-like protein (Os)CEBiP, which specifically binds chitin oligosaccharides. On the other hand, Arabidopsis requires a receptor kinase (At)CERK1 for chitin signaling but it is not clear whether the plant also requires a CEBiP-like molecule for chitin perception/signaling. To clarify the similarity/difference of the chitin receptor in these two model plants, we first characterized CEBiP homologs in Arabidopsis. Only one of three CEBiP homologs, AtCEBiP (LYM2), showed a high-affinity binding for chitin oligosaccharides similar to rice CEBiP. AtCEBiP also represented the major chitin-binding protein in the Arabidopsis membrane. However, the single/triple knockout (KO) mutants of Arabidopsis CEBiP homologs and the overexpressor of AtCEBiP showed chitin-induced defense responses similar to wild-type Arabidopsis, indicating that AtCEBiP is biochemically functional as a chitin-binding protein but does not contribute to signaling. Studies of the chitin binding properties of the ectodomains of At/OsCERK1 and the chimeric receptors consisting of ecto/cytosolic domains of these molecules indicated that AtCERK1 is sufficient for chitin perception by itself.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quitina/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Oryza/inmunología , Oryza/microbiología , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Transducción de Señal , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
In rice, the lysin motif (LysM) receptor-like kinase OsCERK1, originally identified as the essential molecule for chitin-triggered immunity, plays a key role in arbuscular mycorrhizal (AM) symbiosis. As we previously reported, although AM colonization was largely repressed at 2 weeks after inoculation (WAI), arbuscules were observed at 5 WAI in oscerk1 mutant. Conversely, most mutant plants that defect the common symbiosis signaling pathway exhibited no arbuscule formation. Concerning the reason for this characteristic phenotype of oscerk1, we speculated that OsRLK10, which is a putative paralog of OsCERK1, may have a redundant function in AM symbiosis. The protein sequences of these two genes are highly conserved and it is estimated that the gene duplication occurred 150 million years ago. Here we demonstrated that OsCERK2/OsRLK10 induced AM colonization and chitin-triggered reactive oxygen species production in oscerk1 knockout mutant as similar to OsCERK1. The oscerk2 mutant showed a slight but significant reduction of AM colonization at 5 WAI, indicating the contribution of OsCERK2 for AM symbiosis. However, the oscerk2;oscerk1 double-knockout mutant produced arbuscules at 5 WAI as similar to the oscerk1 mutant, indicating that the redundancy of OsCERK1 and OsCERK2 did not explain the mycorrhizal colonization in oscerk1 at 5 WAI. These results indicated that OsCERK2 has a potential to regulate both chitin-triggered immunity and AM symbiosis and at least partially contributes to AM symbiosis in rice though the contribution of OsCERK2 appears to be weaker than that of OsCERK1.
RESUMEN
Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M. oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M. oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases.