Natural variation in the atypical resistance gene Ptr confers broad-spectrum neck blast resistance in rice

Neck blast (NB), caused by Magnaporthe oryzae, infects rice (Oryza sativa L.) and reduces yield. Knowledge of NB resistance remains limited due to the lack of reliable resistance evaluation methods. Here, we applied a newly established neck injection method and performed a genome-wide association study (GWAS) on 335 diverse accessions from the 3K Rice Genomes Project to identify loci associated with NB resistance. We detected a significant association on chromosome 12 (qNB1) explaining 15%-18% of the phenotypic variations caused by a highly virulent blast isolate (M64-1-3-9-1). Linkage disequilibrium analysis refined this region to a 42.3-kb interval containing Ptr, a known gene for leaf blast resistance. We found that two Ptr allelic variants, Ptr_a and Ptr_c, both harboring the variant A/G (Lys879) in the last exon (Chr12:10,833,400), are associated with NB resistance. IR64 and the CO39 near-isogenic line (NIL) (IRBLta2-Pi[CO]), both harboring Ptr_a, were resistant, whereas CRISPR-Cas9 knockout of Ptr_a in IR64 caused susceptibility to M64-1-3-9-1 and IK81-25 isolates. Furthermore, the CO39 near-isogenic line (IRBLta2-Pi[CO]) and Lijiangxintuanheigu monogenic line (IRBLta2-Pi) harboring the Ptr_a allele exhibited resistance to 75% (six of eight isolates) and 85% (17 of 20 isolates) of Philippine differential blast isolates, respectively. Haplotype analysis revealed a superior haplotype containing two major single-nucleotide polymorphism (SNPs; A/G and A/C at Chr12:10,833,400 and Chr12:10,845,095, respectively) and occurring in 83% of International Rice Research Institute (IRRI) elite indica breeding lines. These SNPs can be used to select NB-resistant genotypes with an accuracy of 86%. Our findings identify Ptr_a as a major contributor to NB resistance and provide a valuable genetic resource for developing blast-resistant rice.