Proteomic Analysis Of OSNPSN11-Transgenic Rice In Resistance To Rice Blast And Identification Of Interacting Proteins

Rice blast, caused by the filamentous ascomycete Mangnaporthe grisea (Hebert) Barr, is one of the most devastating diseases of rice, and also severely affect quantity and quality of rice. Elucidating the mechanism of pathogenesis and disease resistance to rice blast thus play an important role in the genetic improvement of rice breeding. The involvement of plant SNARE protein in resistance to pathogen was first discovered in the study of non-host resistance of Arabidopsis thaliana to the powdery mildew of barley. Previous study in our research showed that spores in the leaves of SNARE-type gene QsNPSN11-overexpressing transgenic rice and the blast-resistant cultivar had a delayed development, most spores stayed at the stage of appressorium, indicating that overexpression of OsNPSN11effectively prevented further invasion of blast pathogen, furthermore, the content of H2O2and activity of CAT, POD and SOD were significantly higher than the susceptible control. This thesis described the difference between OsNPSN11-overexpressing transgenic plant and wild-type Suyunuo from the proteomic level before and after inoculation with blast pathogen. In addition, the yeast two-hybrid technique was also used to screen the interacting proteins with OsNPSN11, the following results are here:PCR identification and semi-quantitative RT-PCR analysis showed that the expression level of target gene in two QsNPSN11-overexpressing transgenic lines,2-7and4-7, was much higher than the wild type and disease resistance of the two QsNPSN11-overexpressing transgenic lines to rice blast was also significantly improved after inoculation with rice blast. Investigation of the main Agronomic Traits showed no significant difference between wild type and transgenic lines, indicating the improvement of transgenic lines in disease resistance to rice blast had no effect on on rice yield, therefore, we focused on the2-7transgenic lines as our key point.Proteomic analysis of iTRAQ (isobaric tags for relative and absolute quantification) was applied to dentify the differently expressed proteins between OsNPSN11-overexpressing transgenic rice and wild-type Suyunuo before inoculation and24h after inoculation with blast pathogen, the results showed that45proteins had more than two-fold change of expression level among these four groups either up-regulation or down-regulation, of which5proteins, pathogenesis-related protein1, cytochrome c oxidase subunit, Expressed protein, BURP domain-containing protein16and Glycosyl hydrolase family14protein, showed more than two-fold up-regulated change in OsNPSN11-overexpressing transgenic rice before inoculation, and in wild type and OsNPSN11-overexpressing transgenic rice at24h when compared with wild type before inoculation, while3proteins, LTP family protein precursor, phenylalanine ammonia-lyase and expressed protein, showed more than two-fold down-regulated change, indicating that these8differently expressed proteins may act downstream of OsNPSNll and closely related with pathogenesis of rice blast. However,2proteins, LSM domain-protein and17.5kDa heat shock protein showed no significant difference or slightly down-regulated in OsNPSN11-overexpressing transgenic lines before and after inoculation, but more than two-fold up-regulated change in wild type at24h after inoculation, suggesting the expression of these2proteins were obviously inhibited in OsNPSNll-overexpressing transgenic lines, it may be inferred that the2proteins may act antagonisticly with the process of pathogenesis, after being inhibited, disease resistance of OsNPS11-overexpressing transgenic lines were improved.A cDNA library of membrane protein was constructed by using a mixture of samples untreated and treated with blast pathogen at8h,24h and48h after inoculation, with broad-spectrum and high blast-resistant rice cultivar, Heikezijing, as material, we constructed three bait vectors, pBT-3-N-OsNPSNll, pBT-3-N-OsNPSN12and pBT-3-N-OsNPSN13, respectively. Through the DUAL membrane System,We totally obtained14positive clones encoding7different proteins. Further analysis of identification showed that the positive interactors, inprll-1, inprll-5and inprl3-1can interact with OsNPSNll, OsNPSN12and OsNPSN13, so did the inprll-4, inpr13-3and inpr13-5, but with a lower affinity, sequencing analysis showed inpr12-4was only a cDNA sequence, no predicted gene or protein was reported at present. The full-length protein of inpr12-3and inprll-4encoded glutathione S-transferase and ankyrin repeat-domain protein28, previous study showed that these2proteins were related with disease resistance to pathogen, it may be inferred that OsNPSN11may be involved into the resistance to rice blast through interaction with the2proteins. In addition, the interaction type of OsNPSNll, OsNPSN12and OsNPSN13showed some difference, indicating that they may function differently in plant, hence further study is needed to dissect their individual role in signal transduction of plant.