elicitor ͉ host-pathogen interaction ͉ N-acetylchitooligosaccharides ͉ pathogen-associated molecular patterns H igher plants have the ability to initiate various defense reactions such as hypersensitive responses, production of phytoalexins and antimicrobial proteins, and reinforcement of cell walls when they are infected by various pathogens (1, 2). They can distinguish self and non-self, or detect specific pathogens, through the perception of signal molecules (elicitors) mostly generated͞secreted from pathogens. Fragments of cell surface macromolecules typical of microorganisms such as cell wall polysaccharides, secreted proteins, as well as a flagella protein, often serve as a potent elicitor to induce defense reactions. They are classified as ''general elicitors'' that are commonly found in various microorganisms and induce defense responses in a wide range of plant species. Perception of general elicitors has been thought to play an important role in the basic resistance, or nonhost resistance, of plants to most potential pathogens. It has also been emerged in recent years that the defense systems mediated by the perception of these ''general elicitors'' have a considerable similarity with mammalian innate immunity, in the recognition of pathogen-associated molecular patterns as well as the molecules involved in the perception and transduction of these signal molecules (3).Chitin oligosaccharides (N-acetylchitooligosaccharides) are a representative general elicitor inducing defense responses in a wide range of plant cells including both monocots and dicots (4). Chitin oligosaccharides were reported to induce defense responses also in mammalian and insect cells (4, 5). Interestingly, specific modifications of chitin oligosaccharides by fatty acids, sulfate, or some sugars, generate ''Nod factors'' that induce nodulation in legume roots in the symbiotic interaction with rhizobial bacteria (6). Thus, the recognition of chitin oligosaccharides and related compounds seems to play a fundamental role in the establishment of basal resistance to potential pathogens in plants and in some cases the symbiotic relationships between leguminous plants and rhizobial bacteria.Concerning to the receptor for chitin oligosaccharide elicitor, we previously identified a high-affinity binding protein for this elicitor in the plasma membrane of rice cells by affinity labeling (7). Similar binding proteins were also detected in various plant cells that could respond to the elicitor (8,9). Correlation between the presence of the binding proteins and the elicitor responsiveness of these cells, correlation between the binding specificity and the preference of the structure of chitin oligosaccharides in defense responses, strongly indicated that the binding proteins function as a receptor, or a part of receptor complex, for chitin oligosaccharide elicitor. Here we report the purification of this chitin oligosaccharide elicitor-binding protein (hereafter designated as CEBiP), cloning of the corresponding cDNA and its functional characte...

Chitin is a major molecular pattern for various fungi, and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. A plasma membrane glycoprotein, CEBiP (chitin elicitor binding protein) and a receptor kinase, CERK1 (chitin elicitor receptor kinase) (also known as LysM-RLK1), were identified as critical components for chitin signaling in rice and Arabidopsis, respectively. However, it is not known whether each plant species requires both of these two types of molecules for chitin signaling, nor the relationships between these molecules in membrane signaling. We report here that rice cells require a LysM receptor-like kinase, OsCERK1, in addition to CEBiP, for chitin signaling. Knockdown of OsCERK1 resulted in marked suppression of the defense responses induced by chitin oligosaccharides, indicating that OsCERK1 is essential for chitin signaling in rice. The results of a yeast two-hybrid assay indicated that both CEBiP and OsCERK1 have the potential to form hetero- or homo-oligomers. Immunoprecipitation using a membrane preparation from rice cells treated with chitin oligosaccharides suggested the ligand-induced formation of a receptor complex containing both CEBiP and OsCERK1. Blue native PAGE and chemical cross-linking experiments also suggested that a major portion of CEBiP exists as homo-oligomers even in the absence of chitin oligosaccharides.

The biological role of a secretory catalase of the rice blast fungus Magnaporthe oryzae was studied. The internal amino acid sequences of the partially purified catalase in the culture filtrate enabled us to identify its encoding gene as a catalase-peroxidase gene, CPXB, among four putative genes for catalase or catalase-peroxidase in M. oryzae. Knockout of the gene drastically reduced the level of catalase activity in the culture filtrate and supernatant of conidial suspension (SCS), and increased the sensitivity to exogenously added H₂O₂ compared with control strains, suggesting that CPXB is the major gene encoding the secretory catalase and confers resistance to H₂O₂ in hyphae. In the mutant, the rate of appressoria that induced accumulation of H₂O₂ in epidermal cells of the leaf sheath increased and infection at early stages was delayed; however, the formation of lesions in the leaf blade was not affected compared with the control strain. These phenotypes were complimented by reintroducing the putative coding regions of CPXB driven by a constitutive promoter. These results suggest that CPXB plays a role in fungal defense against H₂O₂ accumulated in epidermal cells of rice at the early stage of infection but not in pathogenicity of M. oryzae.

Magnaporthe oryzae, the fungus causing rice blast disease, should contend with host innate immunity to develop invasive hyphae (IH) within living host cells. However, molecular strategies to establish the biotrophic interactions are largely unknown. Here, we report the biological function of a M. oryzae-specific gene, R equired-for-Focal- B IC- F ormation 1 (RBF1). RBF1 expression was induced in appressoria and IH only when the fungus was inoculated to living plant tissues. Long-term successive imaging of live cell fluorescence revealed that the expression of RBF1 was upregulated each time the fungus crossed a host cell wall. Like other symplastic effector proteins of the rice blast fungus, Rbf1 accumulated in the biotrophic interfacial complex (BIC) and was translocated into the rice cytoplasm. RBF1-knockout mutants (Δrbf1) were severely deficient in their virulence to rice leaves, but were capable of proliferating in abscisic acid-treated or salicylic acid-deficient rice plants. In rice leaves, Δrbf1 inoculation caused necrosis and induced defense-related gene expression, which led to a higher level of diterpenoid phytoalexin accumulation than the wild-type fungus did. Δrbf1 showed unusual differentiation of IH and dispersal of the normally BIC-focused effectors around the short primary hypha and the first bulbous cell. In the Δrbf1-invaded cells, symplastic effectors were still translocated into rice cells but with a lower efficiency. These data indicate that RBF1 is a virulence gene essential for the focal BIC formation, which is critical for the rice blast fungus to suppress host immune responses.

We show that a rice GRAS family protein, CIGR2, is a bonafide transcriptional activator, and through this function, targets the B-type heat shock protein-encoding gene OsHsf23 (Os09g0456800). CIGR2 (Os07g0583600) is an N-acetylchitooligosaccharide elicitor-responsive gene whose activity, through the direct transcriptional control of OsHsf23, is required for mediating hypersensitive cell death activation during pathogen infection. RNAi lines of CIGR2 and OsHsf23 similarly exhibited the higher level of granulation in the epidermal cells of leaf sheath inoculated with an avirulent isolate of rice blast fungus. Interestingly, we did not observe altered levels of resistance, suggesting that CIGR2 suppresses excessive cell death in the incompatible interaction with blast fungus via activation of OsHsf23.