| The root nodule symbiosis (RNS) between legume plants and rhizobia is the most efficient and productive source of nitrogen fixation, and has critical importance in agriculture and mesology. Nodule symbiosis formation is a tightly regulated process that coordinates the activation of developmental mechanisms to synchronize bacterial infection and organ development and is accompanied by a series of signal transduction. Nod factor signaling is an essential element in the establishment of the symbiosis. During the past decade, a lot of genes have been identified from various symbiotic mutants of the two model legumes:Lotus japonicus and Medicago truncatula. A signaling pathway necessary for Nod factor signal transduction was revealed by genetic dissection of nodulation. The symbiosis receptor kinase, SymRK, is indispensable for rhizobial signaling cascade in legumes and may function at the decision rhizobial enter the plants whether or not. However, little is known about how SymRK receive and transduce the NF signal to downstream components. In this study, using SymRK-EC as a bait to screen a Lotus japonicus AD-cDNA library, we identified a SymRK-interacting E3ligase (SIE3). SIE3represents a novel protein family of containing the CTLH, RanBPM-CRA and RING domains, which are conserved in higher plants with no known biological function till now. The exploratory study of SIE3was mainly focus on the following several aspects in this study.1. According to the two EST clones Lj-G0027263and Lj-G0032470, and the peptide sequence homology alignment analysis of SIE3, the reconstituted full-length coding region of SIE3was identified. Then we designed primers according to the reconstituted full-length coding region and amplified its cDNA using RNA isolated from L. japonicus roots inoculated with M. loti. The1,161-bp coding region encodes387amino acids. The peptide sequence analysis showed that SIE3containing a CTLH (C-terminal to LisH) motif, a RanBPM-CRA domain and a RING (Really Interesting New Gene) finger domain. The RING finger in SIE3is a degenerated type of C3HC4RING finger, and it belongs to the RING-S/T variant. SIE3exists as a reverse parallel homodimer and the dimerization of SIE3could be detected when expressed in yeast cells. Deletion analysis demonstrated that both SymRK-EC and SymRK-PK can interact with SIE3in yeast, and the RING domain is nessary for the interaction between SIE3and SymRK. BIFC and Co-IP assays showed that SIE3can interact with SymRK in plant and the interaction happens in the plasma membrane.2. The study of the ubquitin ligase activity of SIE3showed that in the presence of wheat (Triticum aestivum) E1activating enzyme, human E2(UBCh5b) conjugating enzyme and Arabidopsis ubiquitin. SIE3was converted to a mixture of high-molecular weight ubiquitinated protein products, suggesting that SIE3has great ubquitin ligase activity. The assay of detecting the SIE3ligase activity in tobacco cells by co-expression in tobacco leaf cells via agroinfiltration showed that the intensity of the SymRK poly-ubiquitinated products were much stronger in the presence of SIE3. These results suggested that SIE3could mediate the ubiquitination of SymRK in vivo. In vivo and in semi-in-vivo assays demonstrated that SIE3can not promote the ubquitin-dependant degradation of SymRK. It means that there exist a nonproteolytic functions of SIE3-mediated ubiquitination of SymRK, while the detail regulation mechanism need to be further studied.3. SIP1(SymRK interacting protein1) is a ARID transcription factor, can bind specifically to the nodule inception (NIN) promoter. SIP1protein can be easily degradated, and the degradation of SIP1mainly takes place via the ubiquitination pathway rather than another pathway in N. benthamiana. SIE3can interact with SIP1in yeast, and the deletion analysis showed that both SIE3-N and SIE3-C are nessary for the interaction between SIE3and SIP1. Further more, BIFC assay showed that SIE3can interact with SIP1in plant and the interaction happens in the plasma membrane and nuclei. In vivo and in semi-in-vivo assays demonstrated that SIE3can promote the ubquitin-dependant degradation of SIP1, and the homodimer form of the SIE3-GFP protein was increased with SIP1degradation in SIP1protein semi-in-vivo degradation assay. So SIE3homodimer may be nessary for promoting SIP1degradation and SIP1may be another ubquitination substrate of SIE3except SymRK.4. Real-time RT-PCR results showed that the SIE3mRNA was present in all tested tissues of L. japonicus plants, Rhizobium-inoculated roots with developing nodules were found to have the highest expression level of SIE3mRNA. The lowest expression was found in mature nodules. During the nodulation period, the expression of SIE3was elevated6days after rhizobial infection and stay at high levels10days post inoculation. These observations demonstrated that the expression of SIE3is induced by Nod factor in the early state of nodulation. SIE3-GFP was expressed in transgenic onion (Allium cepa) epidermal cells by agroinfiltration, and its subcellular localization was found in both the nuclei and plasma membrane, less in cytoplasm. The same subcellular localization could be found in transgenic hairy roots of L. japonicas and transgenic N. benthamiana leaves by hairy root transformation and transient expression in tobacco leaves.5. Overexpression and RNA interference technologies were used to study the symbiotic function of SIE3ubquitin ligase in Lotus japonicas. And the results showed that Overexpression of SIE3promoted nodulationin transgenic hairy roots. Whereas down-regulation of SIE3transcripts by RNA interference (RNAi) inhibited infection thread development and nodule organogenesis. Through further investigate the expression level of NIN, ENOD40-2and Lb mRNAs in SIE3-RNAi transgenic hairy roots, we found that the expression levels of these genes except Lb were reduced drastically as compared with those in the control hairy roots. These results demonstrate a role of SIE3in the early events of nodulation. |
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