Dasong Chen’s research while affiliated with Huazhong Agricultural University and other places

The lipid transport protein (LTP) product of the AsE246 gene of Chinese milk vetch (Astragalus sinicus) contributes to the transport of plant-synthesized lipids to the symbiosome membranes (SMs) that are required for nodule organogenesis in this legume. However, the mechanisms used by nodule-specific LTPs remain unknown. In this study, a functional protein in the DnaJ-like family, designated AsDJL1, was identified and shown to interact with AsE246. Immunofluorescence showed that AsDJL1 was expressed in infection threads (ITs) and in nodule cells and that it co-localized with rhizobium, and an immunoelectron microscopy assay localized the protein to SMs. Via co-transformation into Nicotiana benthamiana cells, AsDJL1 and AsE246 displayed subcellular co-localization in the cells of this heterologous host. Co-immunoprecipitation assays confirmed that AsDJL1 interacted with AsE246 in nodules. The essential interacting region of AsDJL1 was determined to be the zinc finger domain at its C-terminus. Chinese milk vetch plants transfected with AsDJL1-RNAi had significantly decreased numbers of ITs, nodule primordia and nodules as well as reduced (by 83%) nodule nitrogenase activity compared with the controls. By contrast, AsDJL1 overexpression led to increased nodule fresh weight and nitrogenase activity. RNAi-AsDJL1 also significantly affected the abundance of lipids, especially digalactosyldiacylglycerol, in early-infected roots and transgenic nodules. Taken together, the results of this study provide insights into the symbiotic functions of AsDJL1, which may participate in lipid transport to SMs and play an essential role in rhizobial infection and nodule organogenesis.

In the Rhizobium-Legume symbiosis, the nodulation outer protein P (NopP) effector is one of the key regulators for rhizobial infection and nodule organogenesis. However, the molecular mechanism through which host legume plants sense NopP remains largely unknown. Here, we constructed an nopP deletion mutant of Mesorhizobium huakuii and found that nopP negatively regulates nodulation on Chinese milk vetch (Astragalus sinicus). Screening for NopP interacting proteins in host plants using the yeast 2-hybrid system identified NopP interacting protein 43 (AsNIP43), which encodes a G-type receptor-like kinase (LecRLK). The B-lectin domain at the N terminus of AsNIP43 was essential in mediating its interaction with NopP, which was confirmed in vitro and in vivo. Subcellular localization, co-localization, and gene expression analyses showed that AsNIP43 and NopP function tightly associated with earlier infection events. RNA interference (RNAi) knockdown of AsNIP43 expression by hairy root transformation led to decreased nodule formation. AsNIP43 plays a positive role in symbiosis, which was further verified in the model legume Medicago truncatula. Transcriptome analysis indicated that MtRLK (a homolog of AsNIP43 in M. truncatula) may function to affect defense gene expression and thus to regulate early nodulation. Taken together, we show that LecRLK AsNIP43 is a legume host target that interacts with rhizobia effector NopP is essential for rhizobial infection and nodulation.

Rhizobia can infect legumes and induce the coordinated expression of symbiosis and defense genes for the establishment of mutualistic symbiosis. Numerous studies have elucidated the molecular interactions between rhizobia and host plants, which are associated with Nod factor, exopolysaccharide, and T3SS effector proteins. However, there have been relatively few reports about how the host plant recognizes the outer membrane proteins (OMPs) of rhizobia to mediate symbiotic nodulation. In our previous work, a gene (Mhopa22) encoding an OMP was identified in Mesorhizobium huakuii 7653R, whose homologous genes are widely distributed in Rhizobiales. In this study, a germin-like protein GLP1 interacting with Mhopa22 was identified in Astragalus sinicus. RNA interference of AsGLP1 resulted in a decrease in nodule number, whereas overexpression of AsGLP1 increased the number of nodules in the hairy roots of A. sinicus. Consistent symbiotic phenotypes were identified in Medicago truncatula with MtGLPx (refer to medtr7g111240.1, the isogeny of AsGLP1) overexpression or Tnt1 mutant (glpx-1) in symbiosis with Sinorhizobium meliloti 1021. The glpx-1 mutant displayed hyperinfection and the formation of more infection threads but a decrease in root nodules. RNA sequencing analysis showed that many differentially expressed genes were involved in hormone signaling and symbiosis. Taken together, AsGLP1 and its homology play an essential role in mediating the early symbiotic process through interacting with the OMPs of rhizobia. IMPORTANCE This study is the first report to characterize a legume host plant protein to sense and interact with an outer membrane protein (OMP) of rhizobia. It can be speculated that GLP1 plays an essential role to mediate early symbiotic process through interacting with OMPs of rhizobia. The results provide deeper understanding and novel insights into the molecular interactive mechanism of a legume symbiosis signaling pathway in recognition with rhizobial OMPs. Our findings may also provide a new perspective to improve the symbiotic compatibility and nodulation of legume.

Symbioses between legumes and rhizobia require establishment of the plant-derived symbiosome membrane, which surrounds the rhizobia and accommodates the symbionts by providing an interface for nutrient and signal exchange. The host cytoskeleton and endomembrane trafficking systems play central roles in the formation of a functional symbiotic interface for rhizobia endosymbiosis; however, the underlying mechanisms remain largely unknown. Here we demonstrate that the nodulation-specific kinesin-like calmodulin-binding protein (nKCBP), a plant-specific microtubule-based kinesin motor, controls central vacuole morphogenesis in symbiotic cells in Medicago truncatula. Phylogenetic analysis further indicated that nKCBP duplication occurs solely in legumes of the clade that form symbiosomes. Knockout of nKCBP results in central vacuole deficiency, defective symbiosomes and abolished nitrogen fixation. nKCBP decorates linear particles along microtubules, and crosslinks microtubules with the actin cytoskeleton, to control central vacuole formation by modulating vacuolar vesicle fusion in symbiotic cells. Together, our findings reveal that rhizobia co-opted nKCBP to achieve symbiotic interface formation by regulating cytoskeletal assembly and central vacuole morphogenesis during nodule development.

Plant non-specific lipid transfer proteins (nsLTPs) are involved in a number of biological processes including root nodule symbiosis. However, the role of nsLTPs in legume-rhizobium symbiosis remains poorly understood, and no rhizobia proteins that interact with nsLTPs have been reported to date. In this study, we used a bacteria two-hybrid system and identified the HtpG protein from Mesorhizobium huakuii that interacts with the nsLTP AsE246. The interaction between HtpG and AsE246 was confirmed by far-western blotting and bimolecular fluorescence complementation. Our results indicated that the HSP90 domain of HtpG mediates the HtpG-AsE246 interaction. Immunofluorescence assay showed that HtpG was co-localized with AsE246 in infected nodule cells and symbiosome membranes. Expression of the htpG gene was relatively higher in young nodules and was highly expressed in the infection zones. Further investigaton showed that htpG expression affects lipid abundance and profiles in root nodules and plays an essential role in nodule development and nitrogen fixation. Our findings provide further insights into the functional mechanisms behind the transport of symbiosome lipids via nsLTPs in root nodules.

In higher plants, the 12-oxo-phytodienoic acid reductase (OPR) family mainly consists of two subgroups. OPR3-like enzymes belong to subgroup II, and they were proved to be key functional enzymes participating in jasmonic acid (JA) biosynthesis, while functions of OPR1-like enzymes classified into subgroup I remain largely unclear, particularly in symbiosis. This study identified and functionally characterized a gene encoding 12-oxophytodienoate reductase in Astragalus sinicus. Sequence homology analysis indicated that this gene encodes an OPR1-like enzyme. It was found that the gene expression of AsOPR1 was upregulated after inoculation with Mesorhizobium huakuii 7653R. Subcellular localization showed that in uninfected host plant cells, AsOPR1 was localized in the amyloplast, a differentiated form of plastid; while in the infected cells, AsOPR1 co-localized with rhizobia. Knockdown of the AsOPR1 gene decreased the nodule number to 34.6% that of the control roots, and significantly reduced the JA level in both transgenic roots and nodules, while overexpression of the AsOPR1 gene resulted in enlarged nodule meristem, but caused no changes in nodule number. Taken together, these results indicate that AsOPR1 may participate in the regulation of nodule formation and development, as well as affect endogenous JA metabolism.

In a previous study, we identified the novel opa22 (DQ199621) gene in Mesorhizobium huakuii 7653R, which is required for nodulation. In this study, we determined that opa22-homologous genes exist in the multiple species of bacteria in Rhizobiales. A conserved domain analysis revealed that the Opa22 homologues are a group of β-barrel outer membrane proteins, belonging to the surface antigen superfamily. A phylogenetic tree was constructed based on the Opa22 homologues and showed that Opa22 supports the validity of the 16S rRNA gene for phylogenic classification. Isogenic mutants were made for each homologue of Sinorhizobium medicae USDA1037, Rhizobium leguminosarum bv. trifolii LPR5045, and Bradyrhizobium japonicum USDA2110 via double-cross replacement. Plant experiments showed that the mutants had lost nodulation ability with their original host plants, and that this symbiotic phenotype could be restored by functional complementation. Interestingly, opa22 from M. huakuii 7653R partially restored nodulation ability of S. medicae isogenic mutant, but not that of B. japonicum isogenic mutant. Taken together, these results indicate that Opa22 is phylogenetically conserved in rhizobial families and is essential for root nodule formation in some species.

The AsPPD1 gene from Astragalus sinicus encodes a purple acid phosphatase. To address the functions of AsPPD1 in legume-rhizobium symbiosis, its expression patterns, enzyme activity, subcellular localization, and phenotypes associated with its over-expression and RNA interference (RNAi) were investigated. The expression of AsPPD1 was up-regulated in roots and nodules after inoculation with rhizobia. Phosphate starvation reduced the levels of AsPPD1 transcripts in roots while increased those levels in nodules. We confirmed the acid phosphatase and phosphodiesterase activities of recombinant AsPPD1 purified from Pichia pastoris, and demonstrated its ability to hydrolyze ADP and ATP in vitro. Subcellular localization showed that AsPPD1 located on the plasma membranes in hairy roots and on the symbiosomes membranes in root nodules. Over-expression of AsPPD1 in hairy roots inhibited nodulation, while its silencing resulted in nodules early senescence and significantly decreased nitrogenase activity. Furthermore, HPLC measurement showed that AsPPD1 overexpression affects the ADP levels in the infected roots and nodules, AsPPD1 silencing affects the ratio of ATP/ADP and the energy charge in nodules, and quantitative observation demonstrated the changes of AsPPD1 transcripts level affected nodule primordia formation. Taken together, it is speculated that AsPPD1 contributes to symbiotic ADP levels and energy charge control, and this is required for effective nodule organogenesis and nitrogen fixation.