Genetic identification of Pid3-1 and its regulatory role in promoting blast resistance in rice

Rice blast is a destructive rice disease caused by the fungus Magnaporthe oryzae. Here, we identified a resistance gene from the rice cultivar Wanhui 66 which is resistant to the rice blast Guy11 isolate. Genetic mapping positioned a blast resistance locus to chromosome 6. Employing map-based cloning approach ultimately mapped the novel blast resistance locus to a genomic region of 117 kb that contains the Pid3 gene. Candidate gene prediction and cDNA sequencing indicate that the target resistance gene in the Wanhui 66 is allelic to Pid3, thus it was designated Pid3-1. Further analysis showed that the Pid3-1 has 3 nucleotide substitutions, resulting in 3 amino acid substitutions in the Pid3-1 protein, which significantly affect the structure of the Pid3-1 protein as indicated by the 3D structure simulation. The CRISPR/Cas9 system was employed to generate a Pid3-1 knockout mutants that confirmed that the Wanhui 66 resistant phenotype is controlled by Pid3-1. A molecular marker, Indel-6-34, cosegregates with Pid3-1 was identified that could have a great impact on rice breeding against blast disease resistance.