Accumulation of phosphatidylinositol 4,5‐bisphosphate inhibits the excessive infection of rhizobia in <i>Lotus japonicus</i>

During the symbiosis of legumes with nitrogen-fixing bacteria, collectively called rhizobia, suppression of excessive rhizobial infection by host plants is important to maximize the benefits of symbiotic nitrogen fixation. However, the molecular mechanism involved in the suppression remains relatively poorly understood. We performed LC-MS and RNA-Seq analysis using rhizobia-infected Lotus japonicus roots and investigated the role of phosphatidylinositol (PI) and phosphatidylinositol phosphates (PIPs) in the symbiosis. Phosphatidylinositol transfer protein (PITP)-like proteins 4 (PLP4), phosphatidylinositol 3-phosphate 5-kinase 4 (PIP5K4), and PIP5K6 mutants, which are involved in the vesicular transport of lipids and phosphorylation of PIPs, were used to show the involvement of the signaling of PI and PIPs. Accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] during rhizobial infection was examined by a fluorescent marker 1×TUBBY-C (TUBBY). We found that PI signaling-related genes were upregulated, and the amount of PIP₂ increased in L. japonicus roots during rhizobial infection. In the PLP4, PIP5K4, and PIP5K6 mutants, rhizobial infection increased, while PIP₂ accumulation failed. Furthermore, the observation of PI(4,5)P₂ in rhizobia-infected roots revealed that ectopic accumulation was closely related to the suppression of rhizobial infection. Our findings indicate that the accumulation of PI(4,5)P₂, mediated by PLP and PIP5Ks, suppresses excessive rhizobial infection in the root epidermis and cortex, leading to the optimal number of nodules.