Genome-wide association mapping identifies GhGLR4.8 as a major regulator of basal immunity against Rhizoctonia solani in cotton

Resistance to the necrotrophic pathogen Rhizoctonia solani is a major bottleneck in cotton breeding, hindered by the lack of resistance germplasm, accurate phenotyping methods, and defined resistance genes. Here, we established a precise phenotyping system and performed a genome-wide association study (GWAS) on a natural population of Upland cotton ( Gossypium hirsutum ). A major resistance locus on chromosome D03, which explains 19.65% of the phenotypic variation, was identified. GhGLR4.8 , encoding a glutamate receptor-like protein, was pinpointed as the causal gene. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout significantly compromised resistance, whereas overexpression of the elite haplotype GhGLR4.8 Hap3 conferred robust protection. Functional characterization indicated that GhGLR4.8 promotes lignin deposition and strengthens cell wall structure while maintaining reactive oxygen species (ROS) balance. Loss-of-function mutants displayed excessive ROS accumulation accompanied by pronounced tissue maceration, suggesting that GhGLR4.8 restricts fungal invasion through coordinated regulation of ROS homeostasis. This study reports the first major resistance gene against R. solani in cotton, providing key insights into the balance between ROS signaling and structural defense, as well as a valuable genetic resource for molecular breeding to stabilize cotton yield.