Functions And Regulation Mechanisms Of Small GTPase ROP6in Nodulation Signaling In Lotus Japonicus

Production of Nod factor molecule is activated by the release of plant predominantly flavonoids, Nod factors are a novel general class of signal molecules produced by rhizo-bia, which play a key function in the initial steps of nodulation. Both NFR1and NFR5are predicted to participate in the reception and transduction of Nod factor signal, and trans-mit it to the downstream genes. Then, there are a series of physiological and biochemical changes in host plant cells. Although NFR5is known to be essential in the Nod factor signaling in Lotus japonicus, the biochemical mechanism leading to Nod factor-induced transcriptional activation is obscure. In this report we describe a small GTPase ROP6in-teracted with Nod factor receptor NFR5and our experimental data demonstrate a role of ROP6as a positive regulator of infection thread formation and nodulation in L. japonicus. The specific research results are as follows:1. We use the atypical kinase domain (NFR5-PK) as bait to screen a Lotus cDNA li-brary constructed in the prey vector pGADT7-Rec and identify a small GTPase ROP6. Protein pull-down assay in vitro and Co-IP assay in planta show that the full-length NFR5could interact with ROP6. On the other hand, we construct ROP6-CA (constitutively ac-tive) and ROP6-DN (dominant negative) mutants. Using protein pull down assay in vitro and BiFC assay in planta, we find that NFR5is able to interacte with ROP6-CA and ROP6-DN respectively.2. ROP6is localized to the plasma membrane and cytoplasm in onion epidermal cells through particle bombardment. Similar localization results are also observed in Lo-tus hairy roots. We perform bimolecular fluorescence complementation (BiFC) to assess the interaction between NFR5and ROP6in tobacco leaves. ROP6and NFR5are fused to the split cyan fluorescent protein N terminus (SCN) and C terminus (SCC), respectively. When SCC:NFR5with ROP6:SCN are coexpressed in the leaf cells, strong cyan fluores-cent signals are detected at the plasma membrane. BiFC assay suggests that ROP6inter-acted with NFR5at the plasma membrane of living plant cells.3. Purified ROP6is incubated with [a-32P]GTP, and the reaction product [a-32P]GDP is detected by autoradiography on thin-layer chromatography plates (TLC). ROP6is able to hydrolyze GTP to GDP. Only wild-type ROP6has intrinsic GTPase activity without the requirement for NFR1and/or NFR5proteins in vitro.4. Real-time PCR approach showes that ROP6transcripts are highly expressed in the roots of L. japonicus after inoculation with M. loti. To investigate the spatial pattern of ROP6expression at the cellular level, the ROP6gene promoter is used to drive the ex-pression of the GUS reporter in transgenic hairy roots. Histochemical staining showes that the gene expression is dramatically increased in root vascular bundles, root tips and root hairs after inoculation with M. loti. The activity is concentrated in the cortex of develop-ing nodules and is down-regulated drastically in mature and senescent nodules. These observations are consistent with the results of real-time PCR and confirm that the expres-sion of ROP6gene is indeed dependent on rhizobial infection. These results indicate that ROP6involves in the process of rhizobial infection and nodule development in L. ja-ponicus.5. Down-regulation of ROP6gene expression by RNA interference (RNAi) signifi-cantly impairs nodulation by inhibiting infection thread formation and nodule initiation. The two nodulation-related marker genes NIN and ENOD40-2transcripts decrease mark-edly in ROP6RNAi hairy roots. These results suggest that ROP6is required for the NF induced expression of early nodulin genes. Thus, ROP6may act as a positive regulator in the process of symbiosis in L. japonicus.6. Overexpression of ROP6, ROP6-CA and ROP6-DN in L. japonicus lead to differ-ent root hair deformation phenotypes after inoculation with M. loti. Overexpression of ROP6and ROP6-CA increase the root hair deformation, while overexpression of ROP6-DN significantly inhibits the root hair deformation. These results suggest that dif-ferent activation state of ROP6can affect the Nod factor perception of root hairs. Over-expression of ROP6and ROP6-CA can enhance the Nod factor perception, while overex-pression of ROP6-DN greatly reduces the Nod factor perception.