Association Analysis Of Fungal Protein Elicitor PeaT1and A Tobacco Plasma Membrane Protein

There are increasing studies about plant perception and recognition system, and scientists expect touncover the mechanism of plant immune system by studying the interaction between the plants andpathogenic microorganisms. Although new elicitor receptors are being identified every year, researchabout this field are immature, lacking systematicness. In this paper, a binding protein of elicitor PeaT1was isolated from tobacco plasama membrane via2D Gel Electrophoresis and Far Western Blot. Forfurther identification, the membrane protein was expressed in E. coli Transetta （DE3） and purified forbinding assays both in vitro and in vivo. Co-Immunoprecipitation, Far Western Blot, and BimolecularFluorescence Complementation （BiFC） indicated that PeaT1could associate with the membrane proteinnamed PeaT1-BP （binding protein of PeaT1）. Although there haven’t been enough evidences for thefunctional characterization of PeaT1-BP, in view of the binding activities, we speculate that PeaT1-BPmay be a potential receptor of PeaT1located on tobacco plasma membrane. The major results are asfollows:1. Isolation and functional analysis of a binding protein.2D Gel Electrophoresis and Far-Western Blotwere used to capture the binding proteins which could interact with protein elicitor PeaT1. The MSresult for the obtained protein indicated that it was a transmembrane protein from DREPP（developmental regulated plasma membrane protein） family, with the molecular weight of22KDa（GenBank: CAA65195.1）. The Blast result showed that the N-terminal sequence of the binding proteinwas homologus with that of Ca²⁺binding protein PCaPs，whose N-terminal sequence was reportedclosely related to early signal transduction events, such as Ca²⁺changes, phosphorylation,etc.2. Gene cloning of PeaT1binding protein. mRNA was extracted from tobacco leaves and reversetranscripted into cDNA.594bp CDS sequence, which could ecncode a22KDa protein with197aminoacids, was cloned using the specific primer designed according to the coding sequence for PeaT1binding protein.3. Membrane location of PeaT1binding protein in tobacco. The coding sequence for PeaT1bindingprotein was inserted into transient expression vector pGDG （with GFP tag） and pGDR （with RFP tag）.The recombinant constructs were transformed into Agrobacterium tumefaciens LBA4404and preparedfor agroinfiltation assay. Observation under laser scanning confocal microscope appeared a ubiquitousfluorescence on the surface of tobacco cell48h post treatment, which indicated that the cellular locationof the recombinant PeaT1-BP protein was on the membrane.4. Expression and purification of PeaT1binding protein. The coding sequence for PeaT1bindingprotein was inserted into pET-30His screened from various expression vectors, and transformed into E.coli Transetta. Soluable His-recombinant protein was induced to express by IPTG, and purified viaaffinity chromatography and anion exchange technology. This tobacco plasma membrane protein wasdesignated as PeaT1-BP （binding protein of PeaT1）.5. Association of protein PeaT1-BP with PeaT1in vitro binding assays. The Far-Western blot analysisshowed that the denatured protein PeaT1-BP could associate with the protein PeaT1, and Co-Immunoprecipitation showed that both of the expression protein could interact with each otherunder native condition.6. Association of protein PeaT1-BP with PeaT1in vivo binding assays. To further indentify theinteraction between protein PeaT1-BP and PeaT1, the CDS sequence of them were respectively insertedinto BiFC vector pSCYNE and pSCYCE. The recombinant constructs were transformed into A.tumefaciens LBA4404and prepared for agroinfiltation assay. Observation under fluorescencemicroscope appeared a cyan fluorescence on the surface of tobacco cell, which indicated that proteinPeaT1-BP and PeaT1could ineract with each other in living tobacco cells.