Cyclophilin A-mediated Cis/trans Isomerization Modulates RIN4 to Control Intracellular Rhizobial Infection in Legumes

In most legume-rhizobium symbioses, rhizobial colonization occurs through host-derived intracellular infection threads, which enable rhizobial recruitment while presumably modulating the host immune system to prevent rejection. To investigate post-translational regulation of immune responses during rhizobial infection, we focused on Cyclophilin A (CyPA), a peptidyl-prolyl cis/trans isomerase. The model legume Lotus japonicus encodes three canonical CyPA genes. Using CRISPR/Cas9 mutagenesis, structural modeling, and phylogenomics, we characterized LjCyPA1 as essential for normal intracellular infection by compatible rhizobia. A gain-of-function LjCyPA1 variant in a soybean cultivar promoted symbiosis with both compatible and incompatible rhizobia. Functional association between LjCyPA1 and the immune hub protein LjRIN4 is essential for symbiosis. The putative cis-LjRIN4 promoted intracellular rhizobial infection, while the putative trans-LjRIN4 suppressed it. LjCyPA1 and LjRIN4 acted in concert with the rhizobial type III secretion system (T3SS), highlighting a cooperative role between host and symbiont in facilitating infection. Our results contribute to the understanding of how legumes accept symbiotic partners while balancing immune responses.