Study On Mechanism Of Inducing Disease Resistance In Tomato Of Protein Elicitor BcSpl1 From Botrytis Cinerea

BcSpll is a protein elicitor isolated from the Botrytis cinerea, it belongs to a member of the cerato-platanin family and it is able to cause a strong necrosis in infiltrated tomato and tobacco leaves. In order to elucidate the molecular mechanism that BcSpll elicits tomato immune response, we expressed the recombinant proteins by Pichia pastoris and Escherichia coli., respectively. Both recombinant proteins showed necrosis activity and induced local acquired resistance and systemic acquired resistance. Recombinant protein BcSpll could up-regulated expression of resistance-related genes and protein kinase genes in tomato, caused the variation of phosphorylated protein expression profile. The construction features of the phosphorylated proteomics and some specific protein kinases and resistance proteins induced by BcSpll were obtained by bioinformatics analysis. The major results are as follows:1. The recombinant protein BcSpll was produced by P. pastoris and E. coli. expression system respectively, and the two recombinant proteins were identified as a same protein by mass spectrometry. Two recombinant proteins from two different expression systems all could cause a rapid necrosis in tomato leaves, and induce tomato disease resistance. After infiltrating in tomato leaves, BcSpll protein induced observable necrosis 1 h post-infiltration and an apparent necrotic zone in the infiltration area at 24 h post-infiltration. The combinant protein could induce the tomato resistance to B. cinerea, BcSpll expressed by P. pastoris and E. coli conferred the resistance in tomato against B. cinerea, and the disease infection degree is 22.7% and 21.8% lower than the control, respectively.. Above bioassay of wo recombinant proteins expressed by two different expression system showed same biological activity, and E. coli. expression system could instead of P. pastoris expression system for recombinant protein BcSpll.2. Protein BcSpll triggered elevation of transcript levels of some defence-related genes PR-la, Prosystemin, PI Ⅰ, PI Ⅱ, and protein kinase genes SIPK, WIPK, TPK1b. Realtime quantitative PCR results showed that SAR markers PR-1a up-regulated 200-fold at 12 h post-infiltration, and the up-regulated folds still remained at a high level 24 h post-infiltration. Prosystemin, PI I and PIⅡ genes were up-regulated 2.4,9 and 100 folds, respectively 6 h post-infiltration, and then increased to 7,400 and 900 folds. In addition, BcSpll up-regulated protein kinase TPK1b, SIPK and WIPK expression. SIPK and WIPK expresssion were increased significantly after 1h and increased by 5 and 3 folds respectively after 6 h. TPK1b up-regulated 10 folds at 6 h post-infiltration, and the up-regulated folds increased to 17 24 h post-infiltration. It suggested that the up-regulations of resistance-related genes and protein kinases were an important mechanism of BcSpll-induced disease resistance.3. BcSpll induced the variation of specific phosphorylated proteins in tomato. By using IMAC technology, we analyzed the difference phosphorylation proteomics in tomato leaves between treatment group (tomato leaves treated by BcSpll) and control group (tomato leaves treated by buffer), and in total 871 phosphorylated peptides from 483 proteins were identified, and 573 phosphorylated peptides from 332 proteins were quantifiable. We identified 6 phosphorylation sites in 5 phosphorylated proteins were up-regulated and 68 phosphorylation sites in 49 phosphorylated proteins were down-regulated. Through the analysis of Motif-X software, we got 5 conseved motifs, that is, S(ph)P, R**S(ph), S(ph)D*E, S(ph)D*D and T(ph)P. A GO functional classification showed that the quantifiable differentially expressed phosphorylated proteins play a very important role in cell metabolism process and the response to stimuli. In addition, the results showed that 19 phosphorylated zinc finger proteins, containing RING-H2、C2H2 or other domains, were located in nucleus, it indicated that they may participate in BcSpll induced signal transduction and transcriptional regulation. Meanwhile, in total 36 protein kinases were identified,9 of which were located in plasma membrane and 28 of which contained Serine/threonine/tyrosine -protein kinase catalytic domain. In those 9 plasma membrane-located protein kinases,4 of which contained LRR domain, belonging to receptor-like protein kinase, and 2 of which contained LysM domain.