Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level

Background: Leguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness. Results: Rhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing similar to 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogenfixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants. Conclusions: Alfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.