Mechanism Of Elicitor BcGs1 To Trigger Tomato Immunity Against Botrytis Cinerna

BcGs1 is an extracellular protein isolated from the metabolites of Botrytis cinerea which is a kind of cell wall degrading enzyme.Our previous research had proved that BcGs1 could activate immune defense response and improve the resistance in tomato and tobacco.In order to further clarify the regulatory mechanism that elicitor BcGs1 stimulated immune response in plant,the glycosidase activity of BcGs1and the bioassay of the optimum time for inducing tomato resistance to Botrytis cinerea was carried out.Furthermore,the elicitor activity of different functional domains of Bc Gs1 was identified by transient expression and prokaryotic expression method.The analysis of BcGs1 induced significantly differentially expressed proteins in tomato via i TRAQ was performed.Moreover,transcription profile of encoding genes for differentially expressed proteins,ROS accumulation,defense enzyme activity and histological observation were conducted,respectively.The major research results are as follows:1.Purified elicitor BcGs1 caused rapid cell necrosis in host plant leaves of tomato,tobacco,peas and cucumber after BcGs1 infiltraton.The resistance to Botrytis cinerea was significantly increased in tomato after BcGs1induction.Infected area caused by Botrytis cinerea was decreased by 23.3%at 72 h after the tomato treatment with the lowest induction concentration of 0.25?M BcGs1.2.BcGs1 has Glycosidase activity.BcGs1 could hydrolyze starch and Laminarin,but could not hydrolyze hydroxymethyl cellulose and microcrystalline cellulose.Both GH15 and CBM20 domains are required for Bc Gs1 full cell necrosis activity.Transient expression of BcGs1,GH15 and CBM20 was conducted in tobacco leaves.The results showed that BcGs1 induced a strong necrotic reaction,the necrotic activity of GH15 domain was significantly smaller than BcGs1 and CBM20 domain did not produce a necrosis reaction.The same results were shown after infiltrating leaves with the protein GH15expressed by Prokaryotic method.These results indicated that the synergy of domains GH15 and CBM20 was required for the activity of BcGs1.3.The mechanism of BcGs1 induced resistance to Botrytis cinerea in tomato was further analyzed by proteomics techniques.The total 109 proteins were obtained with ratio treatment/contol?1.3 or?0.77under P<0.05,which 71 proteins were up-regulated and 38 proteins were down-regulated.Total 66proteins were function-known based on genome information of Solanum lycopersicum and Arabidopsis thaliana.GO functional classification analysis of cellular component,molecular function and biological process and KEGG enrichment analysis of metabolic pathway showed that majority of functions of difference expression protein were focused on secondary metabolic synthesis,phenylpropanoid biosynthesis,phenylpropanoid metabolism,plant hormone signal transduction and Plant-pathogen interaction.4.QPCR was used to detect the transcriptional expression pattern of BcGs1 induced defense related genes in tomato.Encoding genes of PR proteins,peroxidase,chitinase,glucanidase,secondary metabolite-related proteins and phenylpropane synthesis-related proteins were verified by qPCR,according to the molecular function and biological process of differentially expressed proteins,GO analysis and KEGG enrichment analysis.The results showed the transcription level of above defensive-related protein coding gene were up-regulation with varying extent,the most with 600-700fold up-regulation.5.BcGs1 induced the accumulation of active oxygen and the phenylpropane metabolic pathway related defense substance in tomato.Reactive oxygen is not only an early signal of defense response,while oxygen metabolism is involved in the phenylpropane metabolic pathway and promotes the enhancement of plant cell walls.PAL and POD are the key enzymes and lignin is the end product of the phenylpropane metabolic pathway.In our paper,H₂O₂ accumulation,PAL and POD activity and lignin content were measured,and the cell wall structure was observed under electron microscope.The results showed that the H₂O₂ was rapidly accumulated,PAL and POD activity was significantly increased,the content of lignin was significantly increased and the cell wall was obviously thickened after BcGs1induction.These results indicated that the elicitor BcGs1 stimulated the base defense response and increased the resistance to Botrytis cinerea through the phenylpropane metabolic pathway.