Investigating the involvement of cytoskeletal proteins MreB and FtsZ in the origin of legume-rhizobial symbiosis

Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants, but they don't carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homologue from pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small ineffective nodules on A. sinicus (Nod+ Fix-), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA-seq analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ1 and ftsZ2. Unlike the ubiquitous FtsZ1, FtsZ2 was commonly found in Rhizobium, Sinorhizobium and Mesorhizobium, but not in closely related non-symbiotic species. Bacterial Two-Hybrid analysis revealed that MreB interacts with FtsZ1 and FtsZ2, which are targeted by the host-derived NCR peptides. Significantly, MreB mutation D283A disrupted the protein-protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ1/FtsZ2 cause the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of Rhizobiales and subsequent acquisition of ftsZ2 are critical evolutionary steps leading to legume-rhizobial symbiosis.