Fine-Tuned Immune Antagonism and Nodule-Specific Cysteine-Rich Peptides Govern the Symbiotic Specificity Between Alfalfa Cultivars and Ensifer meliloti

Alfalfa expresses significantly distinct sets of genes in response to infection with different rhizobial strains at the below species level (i.e., biotype or strain). However, differences in the transcriptomic profiles of two alfalfa cultivars nodulated by a single rhizobium strain have been largely unexamined. In this study, comparative RNA-seq analysis of two alfalfa cultivars, Medicago sativa cv. Gannong No. 3 (G3) and cv. Gannong No. 9 (G9) inoculated with one Ensifer meliloti strain LL2, with varying symbiotic performance, was conducted, followed by hub gene interaction network construction based on weighted gene co-expression network analysis (WGCNA). The G9-LL2 symbiotic system showed better nodule formation, nitrogen fixation, and growth characteristics than the G3-LL2 system. Compared with the non-inoculated control, the LL2-inoculated G9 plants (10,053) produced more differentially expressed genes (DEGs) than the LL2-inoculated G3 plants (7112). A group of 227 genes displayed completely distinguished expression in G9 (6.63 < log(2)(FC) < 15.45) and G3 (- 3.05 < log(2)(FC) < 12.05), which are primarily involved in encoding nodule-specific cysteine-rich peptides (NCRs), nodulin, and leghemoglobin. Although genes with predicted roles in nitrogen metabolism were primarily upregulated and almost all of those in ubiquitin-mediated proteolysis and plant-pathogen interaction were suppressed, interestingly, a consistently higher expression level measured by log(2)(FC) was observed in G9 plants. Hub gene interaction networks showed that NCRs, late nodulin, and genes related to plant immunity (TIR-NBS-LRR, defensin, thioredoxin, thionine, and polygalacturonase) regulate other genes at the source node positions. After successful initiation of nodulation in both alfalfa cultivars G3 and G9 by E. meliloti strain LL2, G9 achieved preferable outcomes of rhizobia-alfalfa symbiosis by equilibrating the antagonism and compatibility of plant immunity. It elevated PTI, suppressed defense and ETI, and enhanced nitrogen fixation and utilization efficiency by inducing the expression of genes encoding NIN, NFH1, LysM-RLK, LRP, NCRs, nodulin, and leghemoglobin. Hub genes were predominantly associated with highly specific rhizobia-alfalfa symbiosis positively governed by NCRs and fine-tuned immune antagonism, comprising NCRs, late nodulin, and TIR-NBS-LRR. These findings provide insights into the genetic mechanisms underlying the modification and efficient utilization of semi-compatible and incompatible rhizobial resources.

Automated summary

This study compared the transcriptomic profiles of two alfalfa cultivars inoculated with the same rhizobium strain LL2 and found that the G9 cultivar showed better nodule formation, nitrogen fixation, and growth characteristics than the G3 cultivar. Differential expression analysis revealed distinct gene expression patterns between G9 and G3, with a larger number of differentially expressed genes in G9. These genes are primarily involved in encoding nodule-specific peptides, nodulin, and leghemoglobin. Furthermore, hub gene interaction network analysis identified the important roles of NCRs, late nodulin, and genes related to plant immunity in regulating other genes.