Regulation Mechanism Of E3Liagase SIE3for Receptor Kinase SymRK In Lotus Japonicus

Symbiotic nitrogen fixation system between Legumes plants and rhizobia or mycorrhizal fungi is the most important nitrogen fixation way in nature and has a critical role in agriculture, because of its efficiency and economy. The research on two model legume species, Lotus japonicus and Medicago truncatula, has been done during the past decade and established a model of early symbiotic signaling pathways. The symbiosis receptor kinase, SymRK, which is located in transmembrane, is required for perception of microbial signal molecules. Its mutant results in rhizobia cannot enter the plants. Based on the importance of SymRK in symbiosis system, we identified a SymRK-interacting E3ligase (SIE3), using SymRK-EC as a bait to screen a Lotus japonicus AD-cDNA library. SIE3ubiquitination modified the SymRK and participated in the symbiotic nitrogen fixation. In current work, we have obtained these results as follows:1. According to the ubiquitination substrate protein database UbiProt, we forecast SymRK ubiquitination sites. The results show that the ubiquitnation may be modified at SymRK198,332,408,417,556,567Lys amino acid sequences, and a SUMO ubiquitination site is located at296amino acid residue.2. Based on the forecast of SymRK, a series of truncated protein of SymRK were constructed to pSCYNE, pVYNE and pSCYCE(R) plant expression vectors and expressed in Nicotiana tabacum. In protein degradation experiments between SIE3and SymRK in vitro, which showed that only part of SymRK can be degraded, and the degradation initiation site may be located in417Lys.3. In yeast two-hybrid, data showed that the self-interaction site of SIE3is the266Cys amino acid. Then we mutant the Cys to Ser, their interaction disappeared. The SIE3C266S were constructed to vectors pSCYNE and pSCYCE(R) and expressed in N. benthamiana. According to the protein degradation experiments, SIE3no longer has the degradation function.4. Overexpression technologies were used to study the symbiotic function of SIE3and SIE3C266S in L. japonicas. And the results showed that overexpression of SIE3C266S didn’t promote nodulation in transgenic hairy roots.