Purification Of An Extracellular B-1, 3-Glucanase Secreted By Gaeumannomyces Graminis Var. Tritici And Its Immunolocalization In Infected Wheat Root

Take-all is one of the destructive diseases on wheat which widely distributed on the world. Because of it is a kind of soilborne disease which difficult to control by chemicals and the poor understand of the pathogenesis of the pathogen, as well as the absence of resistant varieties, the disease still seriously affects the production and quality of the wheat. Analysis and understand the pathogenesis of take-all pathogen is the key and foundation for make up the control method. In this work, the pathogenesis of the take-all pathogen was investigated chemically, cytologically and cytochemically.β-1,3-Glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt), which incite the take-all disease of wheat, was purified and characterized. And the role this enzyme may play was explored during infection and colonization of Ggt on wheat.The results are as follows:1. The carbon and nitrogen sources in the MS medium for Ggt produce extracellularβ-1,3-glucanase were screened, the result showed us the optimal combination were using wheat leaves as the carbon source and beef extract as the nitrogen source, the enzyme activity in the culture filtrate 7 d after inoculation were 0.072 U/mL, keep up to the level which using wheat bran as the carbon source and beef extract as the nitrogen source 21 d after inoculation (0.074 U/mL).2. Ammonium sulfate precipitation (60% saturation), hydrophobic chramotography (Phenyl-Sepharose), anion exchange chramotography (DEAE-sepharose) and gel filtration chramotograpy (Superdex200 10/300 GL) were adopted to purify the extracellularβ-1,3-glucanase in the culture filtrate of Ggt to homogenesis, and designated as GluGgt.3. The extracellularβ-1,3-glucanases secreted by Ggt has two isoforms, which was discovered by Native PAGE and further confirmed by SDS-PAGE, designated as Ia and Ib. GluGgt consists of two subunits, the molecular weights of them, determined by SDS-PAGE, were 66.2 KD and 56.0 KD, respectively. The pI of Ia and Ib were determined to be 6.3 and 6.4, respectively, by IEF. Analysis with Native PAGE and periodic acid/Schiff reaction, Ia and Ib appeared to be glycosylated in Native PAGE.4. The study of properties showed the optimal temperature and pH of GluGgt were about 50℃and pH 5.0, this enzyme was stable at pH 4.0-7.0 and temperature below 60℃. The activity of the enzyme was strongly enabled by Mn²⁺, was strongly inhibited by Co²⁺, Hg²⁺ and KMnO4, was slightly inhibited by EDTA. Other metal ions, Fe3+, Mg²⁺, Zn²⁺, Ba²⁺, Ca²⁺ and Cu²⁺ have little effect on the enzyme activity. The Km and Vmax of the enzyme are 4.91 mg/mL, 0.11 mg/mL/min for the laminarin, respectively.5. The products of laminarin hydrolyzed by GluGgt in different time was analysed by TLC, the result showed us that glucose was the sole product, indicating that GluGgt was an exo-β-1,3-glucanase. The enzyme could hydrolyze salicin and glucans containing 1,3-β- linkages, but not glucans containingα/β-1,4-or 1,6-linkages.6. The antiserum was prepared by immunize rabbit using GluGgt as the antigen and the specific antibody was purified, designated as Anti-GluGgt. Analysis with double immunodiffusion, Elisa and Western blotting, Anti-GluGgt showed high specificity to GluGgt, and have high titer.7. Immunogold labelling and Transmission electron microscopy, as well as Anti-GluGgt were used to investigate GluGgt during Ggt infection of wheat. The result showed that GluGgt were distributed on the cell wall and membranous structure of the axenic culture mycelia. During the infection and colonization process the pathogen secreted exo-β-1, 3-glucanase, the antigen sites were found in the plant cell wall and the lignituber which contacted the infection hyphae besides the infection hyphae cell wall.This study for the first time report the purification and characterization of the extracellularβ-1,3-glucanase secreted by take all disease of wheat, G. graminis var. tritici. The distribution of the enzyme in the pathogen and the infected host tissue was investigated, the result shows us this enzyme was involved in degradingβ-1,3-glucan in the host cell wall and lignitubers. This paper demonstrate partly the role the pathogen originβ-1,3-glucanase played during Ggt infection of wheat cytochemically.