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 an important strategy for sessile plants to acquire nitrogen and phosphorus from the soil. Root exudate plays a key role in shaping the rhizosphere microbiome. Depending on their needs for nitrogen or phosphorus, plants can adjust the composition of root exudate to attract the appropriate microbes. Flavonoids, a group of secondary metabolites, have been well studied for their role in shaping the root microbiome, particularly in mediating root nodule symbiosis in legumes. However, the mechanism by which plants regulate the absorption of microbe-mediated nitrogen and phosphorus remains unclear. Here, we show that the Medicago truncatula phosphate starvation response regulatory network SPX1/3-PHR2 controls flavonoid biosynthesis to recruit nitrogen-fixing microbes for nitrogen acquisition. Nitrogen-fixing microbes, including rhizobia, were fewer recruited in the rhizosphere of the spx1spx3 double mutant. This was caused by lower flavonoid levels in the root exudate compared to wild-type plants R108. Further results indicate that the control of flavonoid biosynthesis is exerted via PHR2, the interacting transcription factor of SPX1/3. Under phosphate-limiting conditions, PHR2 suppresses the expression of flavonoid biosynthetic genes to reduce root nodule symbiosis levels. Under phosphate-sufficient conditions, the interaction between SPX1/3 and PHR2 releases this suppression, thereby promoting root nodule symbiosis. We further showed that PHR2 can bind to the promoter regions of flavonoid biosynthetic genes in yeast. We propose that the SPX1/3-PHR2 network can modulate root nodule-dependent nitrogen acquisition in response to phosphate levels. Thus, the SPX1/3-PHR2 module contributes to maintaining a balance in microbe-mediated nitrogen and phosphorus acquisition for optimal plant growth.