Distinct Evolutionary Constraints Shape TNL and CNL Immune Receptors in Wild and Cultivated Tomato

Nucleotide-binding site leucine-rich repeat (NLR, historically termed NBS-LRR) proteins are among the most rapidly evolving components of plant innate immunity, yet comparative analyses of NLR subfamily diversification between wild and cultivated accessions of tomato (Solanum lycopersicum) have remained limited. Here, we compared the NLR gene family across three representative tomato accessions-the wild species S. chilense and the cultivated tomatoes S. lycopersicum Heinz1706 (large-fruited) and S. lycopersicum LA1464 (cherry tomato)-by integrating genome-wide identification, phylogenetic reconstruction, motif analysis, orthologous clustering, selection-pressure assessment, and expression profiling. We identified 220, 223, and 245 candidate NLR genes in S. chilense, Heinz1706, and LA1464, respectively, and classified them into TIR-type (TNL) and coiled-coil-type (CNL) subfamilies at a consistent ratio of approximately 1:5 across all three accessions. Motif analysis revealed pronounced structural divergence between TNL and CNL proteins, most notably in the region corresponding to the second conserved motif of the NB-ARC domain. Orthologous clustering identified 67 gene clusters shared among the three accessions; within these, TNL genes showed greater sequence divergence (higher Ka and Ks values) than CNL genes but significantly lower Ka/Ks ratios (ω), indicating stronger purifying selection despite their greater raw divergence. Expression profiling further showed that CNL genes were more broadly expressed across tissues and were induced by multiple pathogen-associated molecular patterns, whereas TNL gene expression was more spatially restricted and preferentially induced by effector-related treatments. These results indicate that the TNL and CNL subfamilies of the tomato NLR repertoire have followed distinct evolutionary trajectories and regulatory strategies. As these conclusions are based on comparative genomic and transcriptomic evidence from three accessions rather than on functional validation, they should be regarded as hypotheses; nonetheless, they offer candidate genomic resources and a comparative framework that may inform future disease-resistance breeding in tomato.