Functional and genomic characterization of a Tsw resistance-breaking tomato spotted wilt virus isolate reveals diversification of the NSs avirulence determinant

The dominant immune receptors encoded by Tsw in pepper and Sw-5b in tomato provide effective resistance against tomato spotted wilt virus (TSWV), but their durability is threatened by resistance-breaking variants. Whereas several substitutions in the viral movement protein NSm have been associated with Sw-5b resistance breakdown, the molecular variation underlying escape from Tsw -mediated recognition remains poorly resolved. Here, we combined whole-genome sequencing, phylogenetic analysis, transient expression of NSs, host infectivity assays, population-level sequence analysis, and structural modeling to investigate three pepper-associated TSWV isolates collected from geographically distinct regions of Texas, USA. One isolate from Bushland, designated HTPepRB, overcame Tsw -mediated resistance. Complete genome sequencing revealed that all three isolates belonged to the North American TSWV lineage and shared high nucleotide similarity with previously reported U.S. isolates. Despite high overall sequence conservation, HTPepRB occupied a distinct phylogenetic position across the L, M, and S segments relative to the Uvalde and College Station isolates. Transient expression of HTPepRB NSs failed to induce a Tsw-dependent hypersensitive response, whereas NSs proteins from the Uvalde and College Station isolates retained avirulence activity. Consistent with these observations, infectivity assays demonstrated that HTPepRB successfully overcame Tsw -mediated resistance in pepper but remained unable to overcome Sw-5b -mediated resistance in tomato. Population-level analyses of the key pathogenicity determinants NSs and NSm revealed contrasting evolutionary patterns. Whereas NSm remained highly conserved, NSs exhibited substantially greater sequence diversification, particularly among Bushland isolates, which contained several region-specific substitutions and clustered with previously reported resistance-breaking populations. These findings demonstrate greater sequence flexibility in NSs than in NSm among the sampled populations and support the possibility that escape from Tsw -mediated recognition can arise through more than one NSs sequence background. Collectively, the study links loss of Tsw-dependent NSs recognition with a validated resistance-breaking phenotype and identifies NSs sequence variation that can guide future functional dissection of Tsw–NSs recognition and resistance breeding.