| Ubiquitin is a highly conserved micromolecule protein which is composed of 76 amino acids, and is named for it's aboard existence in all eukaryotic cells. Ubiquitination is one of the post-translational modification processes. Similar to the signal transduction role of protein phosphorylation, ubiquitination plays an important role in many processes of cell life, such as endocytosis, histone activity, DNA repairation. Ubiquitylated proteins are degraded through 26S proteasome. Ubiquitin-26S proteasome pathway as a highly efficient and specific protein degradation regulation mechanism, is involved in almost all activities in plant life. It have been found that ubiquitination is closely related with regulation of human disease, but the documents on its role in plant disease is very limited. In this study, the role of two ubiquitin -26S proteasome pathway genes in regulation of resistance was analyzed employing virus induced gene silencing (VIGS) technique. In addition, proteomic analysis on non-host resistance to Xanthomonas oryzae pv. oryzae (Xoo) was analyzed. Ubiquitylated proteins in Xoo-infiltrated Nicotiana plants were identified. The main results were as follows:(1) Role of the ubiquitin fusion degradation protein (18-8) gene in regulation of plant disease and stress resistance was analyzed by VIGS technique. Compared to control plants, those were VIGS-treated for 18-8, showed less severe Cf-4/Avr4- and Cf-9/Avr9-dependent and Xoo-induced hypersensitive response (HR) symptom. VIGS-treated 18-8 plants showed delayed and less severe disease symptoms after inoculated with Pseudomonas syringae pv. tomato (Pst) and Sclerotinia sclerotiorum (Ss).Plants were VIGS-treated for 18-8, showed enhanced degradation of chlorophyll resulted from high concentration of salt stress. Taken together, these results revealed that 18-8 was a pivotal regulator of plant disease and stress resistance.18-8 gene might be involved in positive regulation of Xoo-induced HR and salt tolerance, and in contrast, negative regulation of the resistance to Pst and. Ss.(2) Function of ubiquitin carboxyl-terminal hydrolase (18-5) gene was studied by VIGS technique. Compared with CK, plants silenced for 18-5 gene displayed weaker Xoo-induced HR, earlier and more severe wilt symptom caused by drought treatment, while,shown similar disease severity after inoculated with Ss to CK. VIGS treated 18-8 plants showed enhanced degradation of chlorophyll resulted from high concentration of salt stress. These results demonstrated that 18-5 may be involved in regulation of plant non-host resistance to Xoo, drought tolerance and salt tolerance.(3) Proteomic analysis for the non-host resistance of N. benthamiana to Xoo was conducted. Proteomic profiles for leaf tissue of tobacco treated with MG132 and both treated with MG132 and infiltrated with Xoo (MG132+Xoo) were compared.45 spots differently expressed in the MG132-treated and (MG132+Xoo)-treated leaf tissue. These 45 protein spots showed over 2-fold difference of in protein abundance in two treatments. Among them,36 proteins were identified successfully through MALDI-TOF-TOF-MS analysis.4 spots among them were down-regulated, while the remains were up-regulated in (MG132+Xoo)-treated leaf tissue. These differentially expressed proteins involved regulation of metabolism (25%), Photosynthesis (25%), defense (12%), biosynthesis (amino acid biosynthesis 10%,protein synthesis 3% and thiamin biosynthesis3%),protein degradation (5%), rcell skeleton (2%), anscription (2%), signal transduction (5%), cell motility (2%), nucleocytoplasmic transportation (2%)and post-transcriptional gene expression regulator (2%). There were 2% proteins with unknown function.(4) The ubiquitylated proteins in (MG132+Xoo)-treated N. benthamiana leaf tissue were indentified by immunoaffinity chromatography and LC-MS techniques. Affinity chromatography using ubiquitin antibody column were employed to purify the ubiquitylated proteins, which were then subjected to liquid chromatography and nano mass spectrometry analysis to identify the ubiquitylated proteins.10 ubiquitylated proteins with confidence percentage of over 90% were successfully identified in the initial analysis for one partition of the total sample, which included an OsAGO1-like protein, an aldehydes-keto reductase family protein, an ATP synthase and two predicted proteins with unkown function. Among them,6 proteins were agreed with the two-dimensional electrophoresis results, demonstrating the high reliability of the results. |
PDF Full Text Request