Small GTPase ROP6 Interacts With Clathrin Heavy Chain In Lotus Japonicus

Nodulation (Nod) factor signaling molecules are essential for rhizobia to initiate the symbiotic nitrogen-fixing interaction with legumes in the early stages of process. The LysM-RLK is required for the recognition of Nod factor in the symbiotic nitrogen-fixing interaction, the receptor kinases of Nod factor are called NFR1 and NFR5 in model plant Lotus japonicus, they may form heterodimer to recognize and receive the common nodulation factor to start the symbiosis signal transduction pathway. For further understanding the mechanism of the symbiosis process between the Rhizobium and the Legume, we screened the AD-cDNA library of Lotus japonicus by yeast two-hybrid using the small GTPase ROP6 as the bait, and identified the small GTPase interacted proteins. This work was base on the fact that this small GTPase is identified by yeast two-hybrid system using NFR5(NFR5-PK) as the bait. We get the following main results:1. The prey plasmids was isolated from the positive clones, PCR amplification and restriction endonuclease were used to identify the insertion fragment of the prey plasmids. By sequence BLAST in NCBI databanks, the insertion fragment encoded two motifs of the clathrin heavy chain, called CHCR in this work.2. The interaction was checked between CHCR proteins and ROP6, CA (constitutively activated mutant of ROP6), DN(dominant negative mutant of ROP6) by yeast two hybrid systems. Results indicated that proteins could interact with each other.3. The CBD-CHCR and His-ROP6 fusion protein was expressed and purified in E. coli. The interaction between CHCR and ROP6 was confirmed by in vitro pull-down. At the same time, the result of Bimolecular Fluorescence Complementation(BiFC) indicated CHC interacted with ROP6 in Nicotiana tabacum.4. The interaction between different deletion versions of CHCR and ROP6 was studied. The result showed that CHCR containing a single motif can not interact with ROP6 unless both of the two motifs existed.5. Using CHCR as a bait in the yeast two-hybrid screening, the clathrin light chain and the RING domain of C3HC4-type E3 ubiquitin-protein ligase were identified.