Identification of a candidate rice blast resistance gene, <i>Pior4</i>(t), in an introgression line of <i>Oryza rufipogon</i> 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 <i>Pior4</i>(t), associated with leaf and panicle blasts in an introgression line carrying the chromosome 4 segment of wild rice (<i>Oryza rufipogon</i> Griff.) backcrossed with the cultivar 'Nipponbare' (<i>Oryza sativa</i> L.). Mapping analysis based on leaf blast severity confirmed that <i>Pior4</i>(t) was localized in the 177-kb NBS-LRR cluster region. To identify the <i>Pior4</i>(t) sequence, mutant lines were generated by knocking out a candidate NBS-LRR gene in a homozygous line carrying <i>Pior4</i>(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. <i>Pior4</i>(t) showed homology to <i>Pi63</i> in the resistant cultivar 'Kahei', and an NBS-LRR gene in the resistant cultivar 'Mine-haruka' carrying <i>Pi39</i>(t). These results suggest that the NBS-LRR gene is a candidate gene of <i>Pior4</i>(t) and is present on the long arm of chromosome 4.