Unveiling evolutionary drivers and functional diversity of the NBS-LRR gene in pepper using a T2T pan-NLRome

NBS-LRR (NLR) genes are crucial for plant disease resistance. However, the evolutionary drivers and functional diversity of the NLR gene family in pepper have not yet been systematically elucidated. Through constructing a pan-NLRome from 11 Telomere-to-Telomere (T2T) pepper genomes and conducting integrated multi-omics analyses, we revealed the evolutionary patterns and functional differentiation of the NLR gene family. We characterized the composition of the pepper NLR family, identifying 4,789 genes grouped into 227 orthogroups, with 32.6% core and 67.4% dispensable genes highlighting substantial intraspecific diversity within a conserved framework. Mechanistically, gene family expansion was primarily driven by dispersed duplication (61.5%), whereas LTR retrotransposons (LTR-RTs), particularly the Ale subgroup, were significantly associated with dispensable genes ( p = 0.01021), facilitating cross-chromosomal gene flow and copy number variation as exemplified by transposon-mediated duplications at the BS2 locus. Furthermore, structural predictions revealed that point mutations in Pepper mild mottle virus (PMMoV) resistance genes induce substantial conformational rearrangements in the LRR domain, increasing its opening angle from 25° to 84.6°, which directly modulates effector binding and explains the observed differences in resistance spectra. Expression profiling under stress conditions showed that core subgroups SF_25 and SF_35 are specifically induced by ethylene (ET) and Pepper Mottle Virus (PepMoV), emphasizing the hub function of ET signaling in coordinating defense responses. These findings elucidate the dynamic evolution and functional diversity of the NLR gene family, providing critical genetic resources and a theoretical foundation for disease resistance breeding in pepper.