Small Extra-Large GTPase-like proteins influence rhizobial symbiosis in Lotus japonicus

Plants possess a unique class of heterotrimeric Gα subunits called extra-large GTPases (XLGs) which contribute to numerous developmental and stress responses. In addition to the canonical Gα domain, XLGs have an uncharacterized N-terminal domain and functions that are distinct from conventional Gα subunits. In this study, we identified homologs of XLG3 in Lotus japonicus responsive to rhizobial and mycorrhizal symbiosis. However, these proteins were approximately one-third the size of conventional XLGs and only aligned to the N-terminal domain, containing a putative nuclear localization signal and a cysteine-rich domain of unknown function. Multiple sequence alignment and phylogenetic analysis determined these small XLGs (SXLGs) did not share domains with other mono– or heterotrimeric G-protein classes and exhibited a pattern of duplication and neofunctionalization typical of genes involved in symbiotic signaling pathways. Transient expression of LjSXLG s in tobacco demonstrated their potential for localization to the plasma membrane, nucleus, and nucleolus. Analysis of L. japonicus sxlg2 mutants revealed transient impairment of immature nodule formation in a destructive experimental setup and inhibition of infection events in a nutrient-limited non-destructive experimental setup, with a delayed onset of established infection events and a potential impact on nodule maturation rate. Additionally, sxlg2 mutants showed a potential impairment of the root growth response in N-limited conditions. We discuss the potential utility of SXLGs in better understanding the evolution of XLGs and their possible function as transcriptional regulators, as well as the likelihood SXLGs are involved in the establishment of rhizobial and mycorrhizal symbioses through influencing membrane reorganization, such as during infection thread development.