Identification of two novel rice S genes through combination of association and transcription analyses with gene-editing technology.

Traditional rice blast resistance breeding largely depends on utilizing typical resistance (R) genes. However, the lack of durable R genes has prompted rice breeders to find new resistance resources. Susceptibility (S) genes are potential new targets for resistance genetic engineering using genome-editing technologies, but identifying them is still challenging. Here, through the integration of genome-wide association study (GWAS) and transcriptional analysis, we identified two genes, RNG1 and RNG3, whose polymorphisms in 3'-untranslated regions (3'-UTR) affected their expression variations. These polymorphisms could serve as molecular markers to identify rice blast-resistant accessions. Editing the 3'-UTRs using CRISPR/Cas9 technology affected the expression levels of two genes, which were positively associated with rice blast susceptibility. Knocking out either RNG1 or RNG3 in rice enhanced the rice blast and bacterial blight resistance, without impacting critical agronomic traits. RNG1 and RNG3 have two major genotypes in diverse rice germplasms. The frequency of the resistance genotype of these two genes significantly increased from landrace rice to modern cultivars. The obvious selective sweep flanking RNG3 suggested it has been artificially selected in modern rice breeding. These results provide new targets for S gene identification and open avenues for developing novel rice blast-resistant materials.