Protein domain-specific approach for identifying resistance gene analogs in Vigna species: A model for broad application in plant defense

Background : Resistance gene analogs ( RGA s) play a crucial role in recognizing plant pathogens and initiating inducible defense mechanisms. However, traditional RGA identification methods using degenerate primers targeting conserved motifs, such as the P-loop and GLPLAL , exhibit limitations in sensitivity and scope. Methods and Results : To address these challenges, a Protein Domain-Specific (PDS) qPCR primer strategy was developed, targeting the entire NB-ARC domain, including key motifs across R genes. Sixteen PDS qPCR primers were designed, optimized using multiplex strategies, and validated through qPCR melt curve analysis. Of these, six primers demonstrated effectiveness in differentiating functional RGA s based on differential gene expression analysis (2 ^-ΔΔCT ) in three Vigna species infected with mungbean yellow mosaic India virus (MYMIV). The study revealed the absence of NB-ARC mediated defense in black gram, while green gram and rice bean exhibited elevated expression. Additionally, a universal functional domain-specific region within R genes was identified across legume species. Conclusions : This novel PDS qPCR approach eliminates the need for degenerate primers, offering a superior alternative for resistance gene identification. By characterizing functional domain-specific regions within R genes, this method enhances our understanding of RGA -pathogen interactions. These findings have significant implications for resistance breeding and disease management in legumes.