Functional Study Of Rice Blast Resistance Associated Gene OsSRB11

Rice(Oryza sativa L.)is an important food crop for more than half of the world’s population.Rice blast,caused by Magnaporthe oryzae,is one of the most serious fungal diseases that threatens the world’s rice production.However,due to the diversity and rapid evolution of the physiological races of M.oryzae,rice blast resistant cultivars may be broken down few years after releasing to farmers.Therefore,continuously identification of new resistance genes in rice is a prerequisite for breeding new disease-resistant varieties and sustainable control of rice blast.OsSRB11 is a rice blast resistance-related gene,which was identified by genome-wide association analysis in a previously study of our lab.In this study,we conducted a preliminary research on its function.First,the gene CRISPR/Cas9 knockout and overexpression mutants were obtained,and the function of the OsSRB11 gene was determined based on the disease resistance phenotypes of the mutants and molecular experiments.Specific research results are as follows:1.The CRISPR/Cas9 gene editing vector p YB02-OsSRB11 and the overexpression vector p ROXs-HA-OsSRB11 were constructed.The OsSRB11 knockout and overexpression transgenic mutants in the Nipponbare background were obtained by using the agrobacterium-mediated transformation method.2.The genotype of the OsSRB11 homozygous knockout mutants E-11-2-2,E-9-1-2 and E-9-1-5 were confirmed via PCR.The expression level of the OsSRB11 gene in the overexpression mutants OE-4、OE-5、OE-10 was analyzed.These mutants were used in subsequent experiments.3.The punch and spray inoculation experiments showed that the knockout lines confered enhanced resistance to rice blast than the Nipponbare control,while the overexpressing lines conferred enhanced susceptibility to rice blast.Besides,the detection results of lesion area and fungal biomass are consistent with the phenotype identification results.The accumulation of reactive oxygen species(ROS)and the expression of disease resistance marker genes in the knockout and overexpression lines were further verified that were correlated with the phenotypes.4.Subcellular localization assay showed the OsSRB11 protein was localized in the rice chloroplast.In summary,the new rice blast resistance associated gene OsSRB11 was identified through genome-wide association analysis.By performing phenotypic and molecular functional analysis of this gene,we proved that OsSRB11 plays a negative regulatory role in rice blast resistance and provides a candidate gene for breeding of rice blast resistant varieties.