Positional cloning of a peanut CC-NBS-LRR gene, AhRRS6, confers resistance to Ralstonia solanacearum

Bacterial wilt, caused by <i>Ralstonia solanacearum</i>, is a destructive disease with no effective chemical control, severely affecting global crop production. This study applied BSR-seq on 581 recombinant inbred lines (RILs), combined with linkage mapping, to identify resistance quantitative trait loci (QTL). Illumina sequencing yielded 189.6 Gb of data, identifying 70,035 high-quality SNPs from 55,840 genes. Two resistance loci were mapped on chromosome 12: a novel 1.11 Mb QTL and an adjacent 1.03 Mb region. Five CC-NBS-LRR-type resistance candidate genes were identified. The <i>AhRRS6</i> alleles were cloned, and three allele-specific SNP markers were developed and validated across peanut breeding varieties. Additionally, 3,851 differentially expressed genes were detected, including key resistance-related genes. Transgenic <i>AhRRS6y</i> conferred strong resistance to <i>R. solanacearum</i>, while <i>AhRRS6x</i> caused susceptibility in both <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>. These alleles differentially regulated genes in HR, ETI, and PTI pathways, particularly affecting <i>NbPDF1.2</i> and <i>NbNDR1</i>. <i>AhRRS6y</i> expression reduced oxidative damage, indicated by lower malondialdehyde and higher ascorbate peroxidase activity. This work provides critical genetic resources for breeding bacterial wilt-resistant peanut varieties and enhances the mechanistic understanding of plant immune responses.