Molecular Mechanisms Of CHC2 In Symbiotic Signaling Pathway In Lotus Japonicus

Legume and rhizobia establish the symbiotic nitrogen-fixation, which are recognized mutually signal molecules of both sides. Rhizobia recognizes the plant predominantly flavonoids of legume released, activates the nod genes. Then nod factors are produced and released to surroundings. After legume recognizing nod factor, signal transduction net is gated, and the symbiotic genes are transcripted & expressed, the nodules are formed at last. The signal of nod factor is recognized by two Lys M RLK—NFR1/NFR5 in Lotus japonicus, and then is transmitted to downstream genes. Although we know that SYMRK is the first downstream protein, the mechanism of signal transduction is not clear. We know from the document published that ROP6 interacted with NFR5 and regulated positively nodulating by influencing development of infect thread. In this report, we screened the AD-c DNA library of Lotus japonicus using ROP6. We get the following results:1. We use the ROP6 as the bait to screen the AD-c DNA library of Lotus japonicus through yeast two hybrid, and identify a clathrin heavy chain protein named CHC2. Protein pull-down assay in vivo and Bi Fc assay in plant demonstrate they can interact. The other hand, we analyze the structure of CHC2 using SMART web, and divide CHC2 into 9 segments of different length. By yeast two hybrid, the shortest interaction domain is identified.2. Blast the Lotus data, we find the other CHC gene named CHC6, and test the interaction between CHC2 and ROP6 by yeast two hybrid. The result shows they don’t interact. We select 4 small GTPase genes from Lotus data on the other hand. They interact respectively with CHC2 in yeast. The result show RAC1—homologue protein of ROP6 can interact with CHC2. Therefore CHC2 interacts specially with ROP6 each other.3. We clone the CLCs from c DNA, and test the interactions with CHC2 in yeast. The result shows they interact both with CHC2, but the interaction is stronger CLC1 than CLC2 with CHC2. In vivo, CLC1 compete the interaction between CHC2 and ROP6.4. The spatial & temporal expression is analyzed via real-time PCR. The result demonstrate that CHC2 is expressed in all tissues. After inoculating rhizobium, the transcript of CHC2 increase first and decrease then, the transcript is highest at seventh day.5. By the transfection of agrobacterium-mediated, We get transgenic plants which express CHC2:GFP and CHC2:RFP/NFR5:GFP. Before inoculating rhizobium, CHC2 is localized to cytoplasm & cell membrane in root hairs. After inoculating rhizobium, CHC2 is localized to vesicles & cell membrane in the early phase, then move to top of root hairs, is localized to infection threads & infection thread initiation site in the phase of infection thread. On the other hand, before inoculating rhizobium, CHC2/NFR5 are colocalized to cell membrane & cytoplasm in root hairs. After inoculating rhizobium, CHC2/NFR5 are colocalized to vesicles and cell membrane.6. The transcript level of CHC2 is regulated down in the RNAi plants. And nodule number is reduced significantly which inoculate rhizobium after 3 weeks.