Regulatory divergence of duplicated VIH genes links phosphate signaling to nodule development in Medicago truncatula.
Primary metabolites and their derivatives often serve as intracellular signals. Inositol pyrophosphates are central regulators of phosphate signaling, but their roles in legume nodulation remain poorly understood. Here, we identify two conserved Vip1 Homolog/Diphosphoinositol Pentakisphosphate Kinase genes, MtVIH1 and MtVIH2, in Medicago truncatula. Biochemical analyses show that the kinase domains of both MtVIH1 and MtVIH2 retain PP-InsP kinase activity. However, transcriptomic and expression analyses reveal regulatory divergence between the duplicated genes, with MtVIH2 being preferentially induced during nodulation and co-expressed with the phosphate starvation marker Mt4. Silencing MtVIH2 reduces nodule formation, nitrogenase activity, and mature nodule marker expression, and alters extractable inorganic Pi accumulation in nodules. Consistently, CRISPR/Cas9-mediated editing of MtVIH2 reproduces these defects, whereas Mtvih1-cas9 shows much weaker effects and Mtvih1/2-cas9 edited roots display phenotypes comparable to Mtvih2-cas9. These results demonstrate that regulatory divergence between duplicated VIH genes is associated with a predominant role of MtVIH2 in nodule development. Promoter analysis and luciferase assays reveal that a P1BS element contributes to MtPHR1-mediated activation of the MtVIH2 promoter. Together, these findings reveal regulatory divergence of duplicated VIH genes and suggest that conserved phosphate-responsive mechanisms contribute to nodule development and nitrogen fixation in M. truncatula.