Improvement of Resistance to Rice Blast and Bacterial Blight by CRISPR/Cas9-Mediated Mutagenesis of OsERF922 and Xa41 in Rice

Rice blast and bacterial blight are two major diseases that seriously threaten rice production. Developing rice germplasm with enhanced resistance to multiple diseases while maintaining favorable agronomic traits is essential for sustainable breeding. In this study, two rice landraces from Motuo County, Xizang Autonomous Region, China, Benglinba and Gare, were used to simultaneously edit OsERF922 and Xa41 using a structurally optimized dual-target CRISPR/Cas9 vector, pRGEB32-2T. A total of 32 and 28 T0 transgenic plants were generated in the Benglinba and Gare backgrounds, respectively. Targeted mutagenesis generated eight homozygous oserf922 mutants and three homozygous xa41 mutants in Benglinba, and four and five homozygous mutants in Gare. Twelve double homozygous mutant lines (nine Benglinba and three Gare) were selected for further analysis. Disease resistance assays showed that these double mutants exhibited significantly enhanced resistance to the rice blast fungus strain GDYJ7 and the bacterial blight pathogen strain GDXO-1, with markedly reduced lesion size or lesion length compared with wild-type plants (p < 0.001, Student’s t-test). Importantly, three independent T-DNA-free double mutant lines from each genetic background displayed no significant differences from their corresponding wild types in major agronomic traits, including plant height, effective panicle number, panicle length, seed-setting rate, or thousand-grain weight (p > 0.05). Grain quality parameters, such as brown rice rate, milled rice rate, amylose content, and gel consistency, were also unaffected. Overall, this study generated rice materials with enhanced resistance to rice blast and bacterial blight while maintaining elite agronomic and quality traits, providing valuable germplasm resources and a feasible strategy for the precise improvement of disease resistance in rice landraces from Xizang Autonomous Region.