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Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato
Authors:Takenaka Shigehito  Yamaguchi Keishi  Masunaka Akira  Hase Syu  Inoue Tsuyoshi  Takahashi Hideki
Affiliation:a Memuro Research Station, National Agricultural Research Center for Hokkaido Region, National Agriculture and Food Research Organization (NARO), Hokkaido 082-0081, Japan
b Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan
c Faculty of Agriculture, Yamagata University, Yamagata 997-8555, Japan
d Department of Life Science, Graduate School of Agricultural Science, Tohoku University, Miyagi 981-8555, Japan
Abstract:The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.
Keywords:CBEL, Cellulose-binding elicitor lectin   CWP, Cell wall protein fraction   D1, Dimer of POD-1   D2, Dimer of POD-2   D(1+2), Heterodimer of POD-1 and POD-2   DTT, Dithiothreitol   ET, Ethylene   JA, Jasmonic acid   LeCAS, β-Cyanoalanine synthase   MALDI-TOF MS, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry   M1, Monomer of POD-1   M2, Monomer of POD-2   MAMP, Microbe-associated molecular pattern   ME, 2-Mercaptoethanol   PAMP, Pathogen-associated molecular pattern   PR-2b, Intracellular basic β-1 3-glucanase   PR-6, Proteinase inhibitor II   PTI, Pattern-triggered immunity   SA, Salicylic acid   SEC, Size exclusion chromatography   TGase, Transglutaminase
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