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Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation. Author summary: In legume-rhizobium symbiotic interactions, growth of the plant-made infection thread (IT) through epidermal and cortical root cells is led by nuclear movement. RPG is a gene that is present in nitrogen-fixing root nodule (NFN) clades and is specifically required for IT formation. In this study, we found that RPG and CERBERUS co-localize and interact in root hairs close to the nucleus after rhizobial inoculation. Both are essential for normal IT growth, both co-localize in the TGN/EE compartment and are required for endomembrane dynamics during IT formation. Our study sheds light on how the RPG-CERBERUS complex promotes IT elongation. [ABSTRACT FROM AUTHOR]

Summary: CERBERUS (also known as LIN) and VAPYRIN (VPY) are essential for infection of legumes by rhizobia and arbuscular mycorrhizal fungi (AMF). Medicago truncatula LIN (MtLIN) was reported to interact with MtVPY, but the significance of this interaction is unclear and the function of VPY in Lotus japonicus has not been studied.We demonstrate that CERBERUS has auto‐ubiquitination activity in vitro and is localized within distinct motile puncta in L. japonicus root hairs and in Nicotiana benthamiana leaves. CERBERUS colocalized with the trans‐Golgi network/early endosome markers.In L. japonicus, two VPY orthologs (LjVPY1 and LjVPY2) were identified. CERBERUS interacted with and colocalized with both LjVPY1 and LjVPY2. Co‐expression of CERBERUS with LjVPY1 or LjVPY2 in N. benthamiana led to increased protein levels of LjVPY1 and LjVPY2, which accumulated as mobile punctate bodies in the cytoplasm. Conversely, LjVPY2 protein levels decreased in cerberus roots after rhizobial inoculation. Mutant analysis indicates that LjVPY1 and LjVPY2 are required for rhizobial infection and colonization by AMF.Our data suggest that CERBERUS stabilizes LjVPY1 and LjVPY2 within the trans‐Golgi network/early endosome, where they might function to regulate endocytic trafficking and/or the formation or recycling of signaling complexes during rhizobial and AMF symbiosis. [ABSTRACT FROM AUTHOR]