The Medicago SPX1/3-PHR2 network relays phosphate signaling to orchestrate root nodulation-dependent nitrogen acquisition by controlling flavonoid biosynthesis

The formation of symbiotic associations with rhizospheric microbes is a key strategy by which sessile plants acquire nitrogen and phosphorus from the soil. Root exudates play a central role in shaping the rhizosphere microbiome, and plants can adjust exudate composition in response to their nitrogen or phosphorus demands to recruit appropriate microbial partners. Flavonoids, a major class of secondary metabolites, have been extensively studied for their roles in shaping the root microbiome, particularly in mediating root nodule symbiosis in legumes. However, the mechanisms by which plants coordinate microbe-mediated nitrogen and phosphorus acquisition remain poorly understood. Here, we demonstrate that the Medicago truncatula phosphate starvation response regulatory network SPX1/3-PHR2 controls flavonoid biosynthesis to recruit nitrogen-fixing microbes for nitrogen acquisition. The abundance of nitrogen-fixing microbes, including rhizobia, is reduced in the rhizosphere of the spx1 spx3 double mutant. This reduction is associated with decreased flavonoid levels in root exudates compared with wild-type R108 plants. Further analyses indicate that regulation of flavonoid biosynthesis is mediated by PHR2, a transcription factor that interacts with SPX1/3. Under phosphate-limiting conditions, PHR2 suppresses the expression of flavonoid biosynthetic genes, thereby reducing root nodule symbiosis. By contrast, under phosphate-sufficient conditions, interaction between SPX1/3 and PHR2 relieves this repression, promoting root nodule symbiosis. We further show that PHR2 binds to the promoter regions of flavonoid biosynthetic genes in yeast. Together, these findings support a model in which the SPX1/3-PHR2 network modulates root nodule-dependent nitrogen acquisition in response to phosphate availability. Thus, the SPX1/3-PHR2 module balances microbe-mediated nitrogen and phosphorus acquisition to support optimal plant growth.