Identification of a candidate rice blast resistance gene, Pior4(t), in an introgression line of Oryza rufipogon using CRISPR/Cas9-mediated genome editing.

Resistance breeding for rice blast is an economic strategy for protecting rice crops against this disease. Genes with nucleotide-binding site leucine-rich repeat (NBS-LRR) structures are known to contribute to disease resistance. Here, we identified a candidate resistance gene, named Pior4(t), associated with leaf and panicle blasts in an introgression line carrying the chromosome 4 segment of wild rice (Oryza rufipogon Griff.) backcrossed with the cultivar 'Nipponbare' (Oryza sativa L.). Mapping analysis based on leaf blast severity confirmed that Pior4(t) was localized in the 177-kb NBS-LRR cluster region. To identify the Pior4(t) sequence, mutant lines were generated by knocking out a candidate NBS-LRR gene in a homozygous line carrying Pior4(t), M18, using CRISPR/Cas9-mediated genome editing. Leaf blast resistance was lost in the mutant lines lacking the corresponding Os04g0620950 N-terminal sequence of the M18 line. The result suggested that the counterpart NBS-LRR gene in the M18 line is involved in resistance to leaf blast. Pior4(t) showed homology to Pi63 in the resistant cultivar 'Kahei', and an NBS-LRR gene in the resistant cultivar 'Mine-haruka' carrying Pi39(t). These results suggest that the NBS-LRR gene is a candidate gene of Pior4(t) and is present on the long arm of chromosome 4.