Characterization Of The Binding Site For The Protein Elicitor Peat1 On Tobacco Plasma Membrane

Presently, elicitor is one of the most popular biomolecule in controlling plant diseases. Elicitors are inducers of plant defense genes and the chemical natures of them are different including proteins, glycoproteins, lipids, and oligosaccharides. The discovery of elicitors to induce responses has led to interest in manipulating the inducible responses in plants for crop protection. Some elicitors have also been reported to provide stress tolerance to the crops against certain abiotic factors. Generally, mode of action for elicitors has been categorized into signal recognition, signal transduction and PR gene expression. The present study is aimed at the characterizations of the binding site for the protein elicitor, PeaT1 on tobacco plasma membrane. The transcription level of PR -1a gene and induction of acid PR protein was found in the tobacco plant treatment with PeaT1 even at extremely low concentration. These results indicated that there was mechanism which triggered the plant defense. In order to find out the mode of action for the PeaT1 different studies were carried out, which are summarized as below: 1. The FITC-labeled PeaT1 was incubated with tobacco suspension cells and it was found that PeaT1 could bind with cell surface when observed under laser confocal microscope. The binding site was specific, when compared with control. 2. The tobacco protoplasts were prepared by enzymolysis, according to the method of Immunofluorescence, and it was found that PeaT1 could bind with tobacco cell membrane when observed under laser confocal microscope. 3. For quantitative characterizations of binding sites for PeaT1 on tobacco cell, the membrane vesicle of tobacco cell was prepared, and then purified it by hydrophilic-double phase-system. The incubation of labeled PeaT1 with 125I revealed that the binding sites were specific, saturable and reversible. It was also found that binding sites were decreased significantly when different fragments of PeaT1 were used. It indicated that integrity of PeaT1 was important to activate the mechanism. 4. The 125I-labelled PeaT1 was incubated with tobacco cell membrane and reacted with covalent cross-linking regent. The SDS-PAGE and radioautography analysis revealed that PeaT1 could bind with two kinds of molecules on the tobacco plasma membrane. Similarly, it was found that PeaT1 could not bind with the heat or proteinase treated tobacco plasma membrane. It had indicated that that these two binding molecular were proteinaceous in nature. 5. Further analysis of two interacting molecules with two dimensional electrophoresis, Far Western Blot and MS suggested that a 22 kDa transmembrane peptide may be a potential receptor of PeaT1 for activation of defense responses in plant.