Unlocking the epigenetic potential of stress tolerance in rice

Research Project

Project/Area Number	23KF0288
Research Category	Grant-in-Aid for JSPS Fellows
Allocation Type	Multi-year Fund
Section	外国
Review Section	Basic Section 44030:Plant molecular biology and physiology-related
Research Institution	Okinawa Institute of Science and Technology Graduate University
Principal Investigator	佐瀬英俊沖縄科学技術大学院大学, 植物エピジェネティクスユニット, 教授 (70510006)
Co-Investigator(Kenkyū-buntansha)	FURCI LEONARDO 沖縄科学技術大学院大学, 植物エピジェネティクスユニット, 外国人特別研究員
Project Period (FY)	2023-11-15 – 2026-03-31
Project Status	Granted (Fiscal Year 2023)
Budget Amount *help	¥2,400,000 (Direct Cost: ¥2,400,000) Fiscal Year 2025: ¥800,000 (Direct Cost: ¥800,000) Fiscal Year 2024: ¥1,200,000 (Direct Cost: ¥1,200,000) Fiscal Year 2023: ¥400,000 (Direct Cost: ¥400,000)
Keywords	Plant / Epigenome / rice / Pathogen response
Outline of Research at the Start	Biotic stresses by pests and diseases are a major constraint on global crop yields, and account for 20-40% of crop losses yearly. In this study, DNA methylation in the rice genome will be exploited for crop protection purposes and obtain broad-spectrum protection against biotic and abiotic stresses.
Outline of Annual Research Achievements	We obtained transgenic rice plants for scalable DNA hypomethylation. We demonstrated estradiol-dependent activation of AtROS1 in rice using RT-PCR. Preliminary results using ELISA-based antibody quantification showed global DNA hypo-methylation in one rice sample treated with the highest concentration of estradiol. Analysis of DNA methylation level at previously identified regions though Chop-PCR confirmed DNA hypomethylation in estradiol-treated rice plants. These regions have previously been shown to dynamically change their DNA methylation status in response to abiotic stress. DNA hypomethylation was detected 2.5 weeks after initial estradiol treatment, indicating meta-stability of DNA hypomethylation during vegetative growth within the same generation. We completed an additional project on the role of RNA N6-adenine methylation in resistance against H. arabidopsidis infection in Arabidopsis.
Current Status of Research Progress	Current Status of Research Progress 1: Research has progressed more than it was originally planned. Reason The project indicated three separated work packages (WP). WP1 involved obtaining transgenic rice plants (T0) and validating AtROS1 accumulation and DNA de-methylation following estradiol treatment. Most tasks from WP1 have now been completed. AtROS1 activation followed estradiol treatment was confirmed through RT-PCR. DNA hypomethylation 2.5 weeks after estradiol treatment was assessed globally using ELISA-based colorimetric analysis, and at specific regions through Chop-PCR. More experiments will be carried on to further validate the activity of the vector.. The project is now moving forward into WP2, and WP3 in few months. We have obtained natural isolates of rice pathogens M. oryzae and X. oryzae, and successfully propagated them in preparation for WP2.
Strategy for Future Research Activity	The project will now move into WP2, which will involve: a) isolating homozygous transgenic rice plants containing the XVE:ROS1 vector (T1), to be treated with various estradiol concentrations; b) producing transgenic plants displaying trans-generational meta-stable DNA hypomethylation. Both types of plant lines will be screened for resistance against pathogens M. oryzae and X. oryzae, as well as excess salt and drought stresses. Once resistant plant will be identified, the project will move to WP3: further characterization of causal epialleles. If AtROS1-dependant DNA methylation displays trans-generational meta-stability, transgenic plants will be backcrossed to wild-type nipponbare to generate epigenetic recombinant inbred rice lines for further epialleles mapping.