Characterization of age-dependent disease resistance in rice and application to production of disease resistant plants.
In rice plant, the mechanism of disease resistance including age-related resistance and the restriction of pathogen growth has not been well elucidated. In this study, to establish the basic techniques for generating suitable transgenic rice plants resistant to some major pathogens, I examined the mechanism of disease resistance at each developmental stage of rice plant.
Chapter 1: Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field. The thionins purified from seeds and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens. In this study, we found that no endogenous rice thionin genes alone are enough for the resistance to 2 major seed-transmitted phytopathogenic bacteria, such as Burkholderia (B.) plantarii and B. glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria. Thus we isolated thionin genes from oat, and one of them was overexpressed in rice plant. When wild-type rice seeds were germinated with these bacteria, all seedlings were wilted with severe blight. In the seedling infected with B. plantarii, bacterial staining was intensively marked around stomata and intercellular spaces. However transgenic rice seedlings accumulating a high level of oat thionin in cell wall grew almost normally with bacterial staining only on the surface of stomata. These results indicated that the oat thionin effectively works in rice plants against the phytopathogenic bacterial attack.
Chapter 2: The role of ethylene (ET) in resistance to infection with Magnaporthe grisea (blast fungus) in rice (Oryza sativa L.) is poorly understood. To study it, we quantified ET levels after the inoculation using young rice plants at 4-leaf stage of cv. Nipponbare (WT) and its isogenic plant (IL7) which contains Pi-i resistance gene to blast fungus race 003. Small necrotic lesions by hypersensitive reaction (HRLs) were formed at 42 to 72 hours post inoculation (hpi) in resistant IL7 leaves, and whitish expanding lesions (ELs) at 96 hpi in susceptible WT leaves. Notable was the enhanced ET emission at 48 hpi accompanied by increased ACC levels and highly elevated ACC oxidase (ACO) activity in IL7 leaves, while only an enhanced ACC increase at 96 hpi in WT leaves. Among six ACS (ACC synthase) and seven ACO genes found in the rice genome, OsACS2 was transiently expressed at 48 hpi in IL7 and 96 hpi in WT, and OsACO7 was at 48 hpi in IL7. Treatment with an inhibitor for ACS, aminooxyacetic acid (AOA), suppressed the enhanced ET emission at 48 hpi in IL7 resulting ELs instead of HRLs. Exogenously supplied ACC compromised the AOA-induced breakdown of the resistance in IL7, and treatment with 1-MCP and STS, the inhibitors of ET action, did not suppress the resistance. These findings suggest the importance of ET biosynthesis, consequently the coproduct, cyanide for HR-accompanied resistance to blast fungus in young rice plants and the contribution of induced OsACS2 and OsACO7 gene expression to it.
Chapter 3: Probenazole (PBZ) is the active ingredient of Olyzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Olyzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pretreatment of inoculated 4th leaves of young wild-type rice plants at 4-leaf stage with PBZ did not influence the development of whitish expanding lesions in the susceptible interaction without the accumulation of salicylic acid (SA) and PR proteins. However, PBZ pretreatment increased accumulation of SA and PR proteins in the 8th leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction-type lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.