Genomic Characterization of Plant Immune Receptor Genes Reveals the Role of Alfalfa MsRLK725 in Salt Tolerance and Disease Resistance.

Plant immune receptor proteins, including pattern recognition receptors and nucleotide-binding domains and leucine-rich repeat sequence receptors (NLRs), are essential for various aspects of plant growth and development, including microbial sensing and immune responses. However, a comprehensive and systematic understanding of immune-related gene families in alfalfa plants is lacking. This study aimed to identify and characterize these genes in alfalfa to fill this knowledge gap. We identified 736 receptor-like kinase (RLK), 407 receptor-like protein (RLP), and 870 NLR genes in the alfalfa genome and analyzed their structural and genetic variation. Phylogenetic analysis classified RLKs, RLPs, and NLRs into 22, 21, and 3 subgroups, respectively. Chromosomal location and synteny analyses revealed that single-gene duplication events contributed to the evolution of these genes. Expression profiling, promoter cis-acting elements, and associated network analyses demonstrated their critical roles in plant growth and stress prevention. Specifically, the alfalfa RLK gene (MsRLK725) was selected for functional validation due to its numerous genetic variants and significant upregulation under biotic stress. Overexpression of MsRLK725 in Nicotiana benthamiana enhanced resistance to Rhizoctonia solani and Phoma medicaginis, and the hairy root transient transformation system improved alfalfa's salt tolerance. Our findings offer valuable insights for future research on using immune-related genes in alfalfa, highlighting their potential in molecular breeding for stress tolerance and disease resistance.