<i>Solanum bulbocastanum</i> nucleotide‐binding leucine‐rich repeat receptor evolution reveals functional variants and critical residues in Rpi‐blb1/RB

Host-pathogen co-evolution shapes resistance (R) proteins and their recognition of pathogen avirulence factors. However, little attention has been paid to naturally occurring genetic diversity in R genes. In this study, 12 Solanum bulbocastanum accessions from the Commonwealth Potato Collection were screened for resistance to Phytophthora infestans, identifying 11 resistant and one susceptible accession. Targeted enrichment sequencing of nucleotide-binding leucine-rich repeat (NLR) genes using RenSeq, followed by diagnostic RenSeq (dRenSeq) analysis, revealed that all accessions except 7650 contained Rpi-blb1/RB variants. Variants in accessions 7641 and 7648 were non-functional, while three novel functional variants were identified. Cloning and functional analysis of Rpi-blb1/RB variants assessed their recognition of the avirulence factor IPI-O1. Three variants were functional, conferring resistance to P. infestans. Variants in accessions 7644 and 7647 also recognized IPI-O4, confirmed in transgenic potatoes. Analysis of a non-functional variant in S. bulbocastanum accession 7648 identified amino acid Ser347 in the nucleotide-binding (NB-ARC) domain as critical for cell-death initiation following IPI-O1 recognition. Predictions from the FunFOLD2 protein-ligand interaction model suggested that Ser347 is essential for ATP binding, suggesting potential inhibition on pentameric resistosome assembly. Western blot analysis revealed that the mutation of Ser347 to Asn markedly compromises the Rpi-blb1/RB protein stability, and co-immunoprecipitation assay further confirmed that this mutation severely disrupts the self-association of CCNB, thereby preventing Rpi-blb1/RB activation. Consistently, substituting Asn347 with serine restored function, underscoring its key role in Rpi-blb1/RB activity. Cell biology experiments demonstrated that Rpi-blb1/RB relocalizes to the plasma membrane in response to IPI-O1. This relocalization depends on Ser347, further supporting the idea that its mutation affects resistosome formation, impairing resistance. This study provides an in-depth functional analysis of natural Rpi-blb1/RB diversity, offering insights into NLR protein evolution and resistance mechanisms in potatoes.