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.

Conferring drought resistant traits to crops is one of the major aims of current breeding programs in response to global climate changes. We previously showed that exogenous application of acetic acid to roots of various plants could induce increased survivability under subsequent drought stress conditions, but details of the metabolism of exogenously applied acetic acid, and the nature of signals induced by its application, have not been unveiled. In this study, we show that rice rapidly induces jasmonate signaling upon application of acetic acid, resulting in physiological changes similar to those seen under drought. The major metabolite of the exogenously applied acetic acid in xylem sap was determined as glutamine—a common and abundant component of xylem sap—indicating that acetic acid is not the direct agent inducing the observed physiological responses in shoots. Expression of drought-responsive genes in shoot under subsequent drought conditions was attenuated by acetic acid treatment. These data suggest that acetic acid activates root-to-shoot jasmonate signals that partially overlap with those induced by drought, thereby conferring an acclimated state on shoots prior to subsequent drought.

Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T 0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T 1 generation. These results show that many Oryza species can be transformed by using modified immatureembryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

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.