Integrated proteomic and transcriptomic analyses reveal that the Rj4-mediated immunity network restricts soybean-rhizobia symbiosis

Legumes-rhizobia symbiosis has high specificity regulated by a specific class of genes, such as Rj4. Rj4 encodes a thaumatin-like protein belonging to the PR-5 family that restricts soybean from nodulation with many strains of Bradyrhizobium elkanii. How Rj4, a member of broad-spectrum resistance family, specifically regulates nodulation remains unclear. To uncover the molecular mechanism of Rj4, current study integrated transcriptome and proteome to analyze the downstream regulatory pathways and key genes mediated by Rj4, through investigating the gene expression and protein abundance in the roots of soybean BARC2 (Rj4/Rj4) after 0, 6 and 24 h post inoculation (hpi) with B. elkanii USDA61. The results showed that a total of 1660 differentially expressed genes (DEGs) and 2633 differentially abundance proteins (DAPs) were identified, in which resistance-related genes and symbiosis-related genes showed opposite expression trend. The key symbiosis-related genes, such as ENOD55, PUB1, and NFP, were up-regulated at 6 hpi but down-regulated at 24 hpi compared with control group. Conversely, the expression of most plant immunity related genes exhibited an initial decrease at 6 hpi followed by an increase at 24 hpi, suggested Rj4 restricted host plant from nodulation in early process through induction of plant immunity related genes and pathways. The key genes involved in plant immunity included pattern-triggered immunity (PTI)-related genes, such as pattern recognition receptors FLS2 and EFR, mitogen-activated protein kinases (MAPK) signaling pathway MPKs, calcium ion related genes CNGCs, CaMCML, and ROS related genes RBOH, as well as effector-triggered immunity (ETI) related resistance (R) genes RPM1 and Ptis, plant hormone signal transduction related genes JAZ and TGA, and flavonoid and isoflavonoid biosynthesis gene Cyps. We validated 8 DEGs via qRT-PCR, showing consistent trends with RNA-Seq (Spearman r > 0.9). These findings provide new insights into how plant immunity inhibits legume-rhizobial symbiosis in the Rj4-mediated regulatory network, and provide key candidate genes to study legume-rhizobial nodulation specificity for future research.