Location: Dale Bumpers National Rice Research Center
Project Number: 6028-21000-012-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2022
End Date: Aug 31, 2025
Objective:
1. Determine the function of the ABC (ATP-binding cassette) tranporter gene OsABC9 in resistance to Rhizoctonia solani using the CRISPR-Cas9 gene editing method.
2. Identify new resisance genes in highly resistant cultivar IR8 as a part of rice diversity panel 1 (RPD1) using genetic and CRISPR-Cas9 gene editing methods.
Approach:
Rice sheath blight is one of the most economically significant rice diseases in the U.S. and other rice growing countries. The disease causes significant grain yield (up to 50%) and quality losses. It is a soilborne disease caused by the fungus Rhizoctonia solani. There is a lack of genetic resistance to Rhizoctonia solani although tremendous efforts have been made to screen for resistance cultivars in the last three decades. Recently, the Cooperator's lab found that the so-called “Green Revolution” cultivar IR8 confers a higher level of resistance to Rhizoctonia solani than the resistant control cultivar Jasmine 85. In addition, the ARS researcher's lab reported that the ATP (adenosine triphosphate)-binding cassette transporter gene OsABC9 is a candidate quantitative trait loci (QTL) gene against Rhizoctonia solani. Our overall objective is to identify and analyze the candidate genes to the sheath blight fungal pathogen Rhizoctonia solani.
To confirm the function of the sheath blight candidate gene OsABC9, we will design two sgRNAs based on the genomic sequence of OsABC9 on chromosome 9. The CRISPR-Cas9 construct will be transformed into rice calli and homozgouys transgenic plants with deletions in the gene will be selected. Their resistance to Rhizoctonia solani will be tested in both growth chambers and greenhouses with multiple isolates.
An F2 mapping population will be generated from a cross between the resistant cultivar IR8 and susceptible cultivar Lemont. About 200 F2 plants will be inoculated with an Rhizoctonia solani isolate and DNA will be isolated from the inoculated plants. High-throughput sequencing-based mapping method QTL-Seq will be used to idenitfy the major QTLs in IR8. Candidate genes will be confirmed in a large F2 population. CRISPR-Cas9 gene editing method will be used to confirm the function of the candidate genes in resistance to Rhizoctonia solani. The CRISPR-Cas9 constructs of the candidate genes will be tranaformed rice calli and the derived treangenic line will be used for sheath blight inoculation.
Expected results: About 20 OsABC9 knockout lines will be produced for sheath blight inoculation and an F2 population of the IR8xLemont cross will be produced for QTL-seq mapping in the first year of this project.
