Protein Elicitor PeaT1 Stimulates Early Signaling Responses And Differential Protein Expression In Arabidopsis

A protein elicitor, PeaT1 was identified and purified from Alternaria tenuissima with the molecular weight of 36 kDa. Its encoding gene was cloned and expressed in E.coli. It was found that PeaT1 could enhance the growth in wheat and trigger the resistance against tobacco mosaic virus. In this study, we have studied the drought tolerance, salt tolerance and disease resistance against Botrytis cinerea, the signaling events, and the differentially expressed proteins induced by PeaT1 in Arabidopsis which initially explained to be the mode action for PeaT1.The results are summarized as follows:1. Expression and purification of PeaT1.The PeaT1 was expressed in E.coli and it was found that its apparent molecular weight was about 36 kDa in SDS-PAGE analysis. The expressed protein was detected by Western-Blotting analysis which revealed that expression was reliable. The PeaT1 was purified using the classical three-step purification procedure of affinity chromatography, anion-exchange chromatography and molecular sieve chromatography. The protein obtained with more than 90% of purity was used for further experiments.2. Analysis of the biological function of PeaT1. A series of assays were designed for the determination of the biological function using highly purified PeaT1.The results showed that PeaT1 could increase drought tolerance, salt tolerance and disease resistance against Botrytis cinerea in Arabidopsis thaliana. These results demonstrated that PeaT1 is a multi-functional protein elicitor.3. The signaling events in Arabidopsis thaliana induced by PeaT1.The signaling events in Arabidopsis thaliana induced by PeaT1 were the production of reactive oxygen species, PH variation, increase in calcium ion concentration, nitric oxide production, and activation of phenolic compounds. These results proved the basis for determination of mode of action for PeaT1.4 Differential expression of proteins in Arabidopsis thaliana induced by PeaT1. The Arabidopsis plants were labeled through the stable isotope labeling method, and verified for the saturated labeling. The results of mass spectrometry were blasted with the database, and quantified with MSQuant software. The function of these proteins was analyzed and these proteins were categorized into stress-related proteins, PR proteins, metabolism-related proteins and transport proteins. The result showed that these proteins could enhance the disease resistance, and stress tolerance in Arabidopsis thaliana.5. Verification of the differential expression of proteins in Arabidopsis thaliana induced by PeaT1 using RT-PCR.and Arabidopsis mutants.The two proteins, glutathione S-transferase 6 and peroxidase 52 which were significantly different from control were verified using RT-PCR and Arabidopsis mutants.the results showed that encoding genes of the two proteins were up-regulated in transcription leavel which reached the highest level at 24-hour in Arabidopsis after treated by PeaT1. The resistance in Arabidopsis induced by PeaT1 against Botrytis cinerea was decreased in Arabidopsis mutants which show that the resistance in Arabidopsis induced by PeaT1 against Botrytis cinerea was enhanced due to the induction of the two up-regulated proteins.In this study, the latest technology of proteomics was used to study the mode of action for PeaT1 which has established a basis for the mechanism of PeaT1. At the same time, a large number of differentially expressed proteins were obtained which has provided important information for the identification of key genes, related to disease resistance.