Structural Alteration of Rice Pectin Affects Cell Wall Mechanical Strength and Pathogenicity of the Rice Blast Fungus Under Weak Light Conditions.

Pectin, a component of the plant cell wall, is involved in cell adhesion and environmental adaptations. We generated OsPG-FOX rice lines with little pectin due to overexpression of the gene encoding a pectin-degrading enzyme [polygalacturonase (PG)]. Overexpression of OsPG2 in rice under weak light conditions increased the activity of PG, which increased the degradation of pectin in the cell wall, thereby reducing adhesion. Under weak light conditions, the overexpression of OsPG decreased the pectin content and cell adhesion, resulting in abnormally large intercellular gaps and facilitating invasion by the rice blast fungus. OsPG2-FOX plants had weaker mechanical properties and greater sensitivity to biotic stresses than wild-type (WT) plants. However, the expression levels of disease resistance genes in non-infected leaves of OsPG2-FOX were more than twice as high as those of the WT and the intensity of disease symptoms was reduced, compared with the WT. Under normal light conditions, overexpression of OsPG2 decreased the pectin content, but did not affect cell adhesion and sensitivity to biotic stresses. Therefore, PG plays a role in regulating intercellular adhesion and the response to biotic stresses in rice.

Magnaporthe oryzae Glycine-Rich Secretion Protein, Rbf1 Critically Participates in Pathogenicity through the Focal Formation of the Biotrophic Interfacial Complex.

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, Required-for-Focal-BIC-Formation 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.

OsCERK1 plays a crucial role in the lipopolysaccharide-induced immune response of rice.

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.

Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon.

Rhizoctonia solani is a soil-borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)-dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear. We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone-induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity. Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA-deficient NahG transgenic rice and the sheath blight-resistant B. distachyon accessions, Bd3-1 and Gaz-4, which activate SA-dependent signaling on inoculation. Our findings suggest a hemi-biotrophic nature of R. solani, which can be targeted by SA-dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

OsTGAP1 is responsible for JA-inducible diterpenoid phytoalexin biosynthesis in rice roots with biological impacts on allelopathic interaction.

Phytocassanes and momilactones are known as major diterpenoid phytoalexins (DPs), characterized by abundant production and antimicrobial activity, and their biosynthetic genes are clustered in rice genomes. The basic leucine zipper transcription factor OsTGAP1 is known to act as a regulator of the coordinated production of DPs in cultured rice cells, but in planta functions of OsTGAP1 remain largely unknown. Here, we present evidence on the biological function of OsTGAP1 in planta. In wild-type plants, OsTGAP1 is abundantly expressed in roots compared with that in shoots. Moreover, the inductive expression of OsTGAP1 under jasmonic acid (JA) treatment was only observed in a root-specific manner consistent with the JA-inducible expressions of DP biosynthetic genes in roots. In reverse genetic approaches on OsTGAP1-overexpressing and OsTGAP1-knockdown plants, expressions of the biosynthetic genes relevant for DP accumulation were found to be remarkably increased and decreased, respectively. Reporter analysis in planta revealed that OsTGAP1 activated the promoters of OsDXS3 and momilactone biosynthetic gene OsKSL4, presumably through binding to the TGACGT motif. Furthermore, cocultivation experiments with barnyard grass suggested that the allelopathic effect of knockdown and overexpression of OsTGAP1 was significantly changed compared with the controls. These results demonstrate that OsTGAP1 positively regulates DP accumulation via the transcriptional regulation of DP biosynthetic genes in rice roots, and this is indispensable for maintaining allelopathic interactions with paddy weeds by regulating the production of specialized metabolites like momilactones.

The elicitor-responsive gene for a GRAS family protein, CIGR2, suppresses cell death in rice inoculated with rice blast fungus via activation of a heat shock transcription factor, OsHsf23.

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.

Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae.

Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map-based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST-OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower-related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild-type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast-induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.

Targeted gene disruption of OsCERK1 reveals its indispensable role in chitin perception and involvement in the peptidoglycan response and immunity in rice.

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.

OsNAC111, a blast disease-responsive transcription factor in rice, positively regulates the expression of defense-related genes.

Plants respond to pathogen attack by transcriptionally regulating defense-related genes via various types of transcription factors. We identified a transcription factor in rice, OsNAC111, belonging to the TERN subgroup of the NAC family that was transcriptionally upregulated after rice blast fungus (Magnaporthe oryzae) inoculation. OsNAC111 was localized in the nucleus of rice cells and had transcriptional activation activity in yeast and rice cells. Transgenic rice plants overexpressing OsNAC111 showed increased resistance to the rice blast fungus. In OsNAC111-overexpressing plants, the expression of several defense-related genes, including pathogenesis-related (PR) genes, was constitutively high compared with the control. These genes all showed blast disease-responsive expression in leaves. Among them, two chitinase genes and one ß-1,3-glucanase gene showed reduced expression in transgenic rice plants in which OsNAC111 function was suppressed by a chimeric repressor (OsNAC111-SRDX). OsNAC111 activated transcription from the promoters of the chitinase and ß-1,3-glucanase genes in rice cells. In addition, brown pigmentation at the infection sites, a defense response of rice cells to the blast fungus, was lowered in OsNAC111-SRDX plants at the early infection stage. These results indicate that OsNAC111 positively regulates the expression of a specific set of PR genes in the disease response and contributes to disease resistance.

CEBiP is the major chitin oligomer-binding protein in rice and plays a main role in the perception of chitin oligomers.

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.

Surface α-1,3-glucan facilitates fungal stealth infection by interfering with innate immunity in plants.

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant's defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.

Ubiquitin ligase EL5 maintains the viability of root meristems by influencing cytokinin-mediated nitrogen effects in rice.

Root formation is dependent on meristematic activity and is influenced by nitrogen supply. We have previously shown that ubiquitin ligase, EL5, in rice (Oryza sativa) is involved in the maintenance of root meristematic viability. When mutant EL5 protein is overexpressed to dominantly inhibit the endogenous EL5 function in rice, primordial and meristematic necrosis ia observed. Here, we analysed the cause of root cell death in transgenic rice plants (mEL5) overexpressing EL5V162A, which encodes a partly inactive ubiquitin ligase. The mEL5 mutants showed increased sensitivity to nitrogen that was reflected in the inhibition of root formation. Treatment of mEL5 with nitrate or nitrite caused meristematic cell death accompanied by browning. Transcriptome profiling of whole roots exhibited overlaps between nitrite-responsive genes in non-transgenic (NT) rice plants and genes with altered basal expression levels in mEL5. Phytohormone profiling of whole roots revealed that nitrite treatment increased cytokinin levels, but mEL5 constitutively contained more cytokinin than NT plants and showed increased sensitivity to exogenous cytokinin. More superoxide was detected in mEL5 roots after treatment with nitrite or cytokinin, and treatment with an inhibitor of superoxide production prevented mEL5 roots from both nitrite- and cytokinin-induced meristematic cell death. These results indicate a nitrogen-triggered pathway that leads to changes in root formation through the production of cytokinin and superoxide, on which EL5 acts to prevent meristematic cell death.

Reverse-genetic approach to verify physiological roles of rice phytoalexins: characterization of a knockdown mutant of OsCPS4 phytoalexin biosynthetic gene in rice.

A variety of labdane-related diterpenoids, including phytocassanes, oryzalexins and momilactones, were identified as phytoalexins in rice (Oryza sativa L.). Momilactone B was also isolated as an allelochemical exuded from rice roots. The biosynthetic genes of these phytoalexins have been identified, including six labdane-related diterpene cyclase genes such as OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8 and OsKSL10. Here we identified an OsCPS4 knockdown mutant, cps4-tos, by screening Tos17 mutant lines using polymerase chain reaction. OsCPS4 encodes a syn-copalyl diphosphate synthase responsible for momilactones and oryzalexin S biosynthesis. Because Tos17 was inserted into the third intron of OsCPS4, the mature OsCPS4 mRNA was detected in the cps4-tos mutant as well as the wild type. Nevertheless, mature OsCPS4 transcript levels in the cps4-tos mutant were about one sixth those in the wild type. The cps4-tos mutant was more susceptible to rice blast fungus than the wild type, possibly due to lower levels of momilactones and oryzalexin S in the mutant. Moreover, co-cultivation experiments suggested that the allelopathic effect of cps4-tos against some kinds of lowland weeds was significantly lower than that of the wild type, probably because of lower momilactone content exuded from cps4-tos roots. A reverse-genetic strategy using the cps4-tos mutant showed the possible roles of momilactones not only as phytoalexins but also as allelopathic substances.

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.

OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus.

WRKY transcription factors form a large family of plant-specific transcription factors and participate in plant defense responses either as positive or negative regulators. In this study, we comprehensively analyzed the role of one of the group IIa WRKY transcription factors in rice, OsWRKY28, in the regulation of basal defense responses to a compatible race of the rice blast fungus Magnaporthe oryzae, strain Ina86-137. The expression analyses of the group IIa WRKY transcription factors in rice revealed that OsWRKY28, together with OsWRKY71, exhibit an early-induced expression prior to the late-induced expressions of OsWRKY62 and OsWRKY76. The GFP-OsWRKY28 fusion protein localized mainly in the nuclei of onion epidermal cells, and the maltose-binding protein-fused OsWRKY28 recombinant protein specifically bound to W-box elements. A transient reporter gene assay clearly showed that OsWRKY28 functions as a transcriptional repressor. Overexpression of OsWRKY28 in rice plants resulted in enhanced susceptibility to Ina86-137. Finally, transcriptome analysis revealed that the induction of several defense-related genes in the wild type after Ina86-137 infection was counteracted in OsWRKY28-overexpressing rice plants. These results strongly suggest that OsWRKY28 is a negative regulator of basal defense responses against Ina86-137 and acts as a modulator to maintain the responses at an appropriate level by attenuating the activation of defense-related gene expression levels.

WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.

OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

OsJAR1 contributes mainly to biosynthesis of the stress-induced jasmonoyl-isoleucine involved in defense responses in rice.

Jasmonate plays key roles in plant growth and stress responses, as in defense against pathogen attack. Jasmonoyl-isoleucine (JA-Ile), a major active form of jasmonates, is thought to play a pivotal role in plant defense responses, but the involvement of JA-Ile in rice defense responses, including phytoalexin production, remains largely unknown. Here we found that OsJAR1 contributes mainly to stress-induced JA-Ile production by the use of an osjar1 Tos17 mutant. The osjar1 mutant was impaired in JA-induced expression of JA-responsive genes and phytoalexin production, and these defects were restored genetically. Endogenous JA-Ile was indispensable to the production of a flavonoid phytoalexin, sakuranetin, but not to that of diterpenoid phytoalexins in response to heavy metal stress and the rice blast fungus. The osjar1 mutant was also found to be more susceptible to the blast fungus than the parental wild type. These results suggest that JA-Ile production makes a contribution to rice defense responses with a great impact on stress-induced sakuranetin production.

XopR, a type III effector secreted by Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular pattern-triggered immunity in Arabidopsis thaliana.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. The XopR protein, secreted into plant cells through the type III secretion apparatus, is widely conserved in xanthomonads and is predicted to play important roles in bacterial pathogenicity. Here, we examined the function of XopR by constructing transgenic Arabidopsis thaliana plants expressing it under control of the dexamethasone (DEX)-inducible promoter. In the transgenic plants treated with DEX, slightly delayed growth and variegation on leaves were observed. Induction of four microbe-associated molecular pattern (MAMP)-specific early-defense genes by a nonpathogenic X. campestris pv. campestris hrcC deletion mutant were strongly suppressed in the XopR-expressing plants. XopR expression also reduced the deposition of callose, an immune response induced by flg22. When transiently expressed in Nicotiana benthamiana, a XopR::Citrine fusion gene product localized to the plasma membrane. The deletion of XopR in X. oryzae pv. oryzae resulted in reduced pathogenicity on host rice plants. Collectively, these results suggest that XopR inhibits basal defense responses in plants rapidly after MAMP recognition.

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus Magnaporthe oryzae.

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.

Perception of the chitin oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice.

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.