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Structural And Molecular Interaction Analysis Of Protein Elicitor PevD1from Verticillium Dahliae

Posted on:2013-01-21Degree:MasterType:Thesis
Country:ChinaCandidate:L HanFull Text:PDF
GTID:2230330374457018Subject:Biochemistry and Molecular Biology
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Fungal protein elicitor is a type of protein that derived from plant pathogens, which can induceplant defense responses and enhanced plant disease resistance. PevD1, a novel elicitor protein frompathogenic fungus Verticillium dahliae, can trigger hypersensitive response and cause the productionand accumulation of resistance substances in tobacco plants. In order to study the function and themolecular mechanism of PevD1protein, In this study, PevD1was expressed and crystallized. Afterslightly optimization, well-diffracting crystals were obtained and yielded a good diffraction pattern.The structure of PevD1consists of a compact ten-stranded antiparallel β-barrel connected by variableloops and is similar to the canon C2domain, which has the ability binding to lipids. The interactingproteins in the Arabidopsis thanliana were screened by Yeast two-hybrid. The interaction betweenPevD1and Receptor4was further tested by in vitro GST pull down. The transgenic strains wereobtained by plant overexpression vector, and used to analyse the preliminary subcellular localizationin the overexprssion lines.1The well-diffracting crystals were obtained. PevD1was expressed in E.coli and purified withdifferent columns. The homogeneous protein was obtained and used for crystals screening. Thewell-diffracting crystals were obtained from an optimized precipitant concentration of3.8Msodium formate at a protein concentration of2mg ml-1. The native and derivative datasets werecollected for structure determination.2The structure of PevD1consists of a compact ten-stranded antiparallel β-barrel connected byvariable loops which is similar to the canon C2domain. Two disulfide bonds were formed by Cys50and Cys64, Cys105and Cys115.which would be important for maintaining the overall structure ofPevD1protein.3The structure of PevD1was similar to the canon C2domain, which bound to lipid byCa2+-dependent and Ca2+-independent forms. The result of protein-lipid overlay assay identified theinteraction between PS,PI and PevD1. This interaction could associate with PevD1getting inside thecell.4The interacting proteins (Receptor1,Receptor2,Receptor3and Receptor4) in the Arabidopsisthanliana were screened by Yeast two-hybrid.77positive interactions were screened for furthertesting experiments, and then4real positive interactions were obtained. The binding assay betweenPevD1and Receptor4, which was soluble expression in E.coli, was further tested by in vitro GST pulldown. The results showed the binding interaction of Receptor4protein and PevD1could be observed.It provided a basis for studying how PevD1interferes with plant immunity system.5The subcellular localization of PevD1was in the plasma membrane. Incubation the roots ofArabidopsis thanliana with recombinant protein GFP-PevD1showed that PevD1could touch to thesurface of cells. The transgenic Arabidopsis thanliana strains were obtained by transformationmediated by Agrobacterium tumefaciens. And the localization of PevD1on the overexpression lines showed in the plasma membrane.
Keywords/Search Tags:Protein Elicitor, X-ray crystallography, Protein crystal, Yeast two-hybrid, Interactingproteins
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