| The wheat sharp eyespot, caused by Rhizoctonia cerealis Vander Hoeven, is one of the major wheat diseases in our country. Despite the widespread and economic impact of this necrotrophic pathogen, very little is known about how it causes disease and little is appreciated about the significance in pathogenesis of the protease which is produced during infection. In order to further reveal the important role of cell wall-degrading enzymes in plant-microbe interactions and provide new theory basis for controlling the disease, one of the extracellular protease which produced by Rhizoctonia cerealis and the function which it served in virulence have been studied. The main results and conclusions are as follows:1. 19 isolates of the pathogen from sharp eyespot lesions of wheat in Shandong province of China were obtained and identified with Giemsa staining as Rhizoctonia cerealis Vander Hoeven. 2. Rhizoctonia recealis when grown in liquid medium by using wheat cell walls as the sole carbon and nitrogen source secretes several extracellular protease according to the result of the gelatin-coated X-ray film blotting. The maximal protease activity was obtained at 48h after the fungus were cultured in inducing medium. A kind of protease, named RCP1, was purified after ammonium sulfate precipitation, SP-Sepharose Fast Flow chromatography, Phenyl-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography. It appeared as a single band corresponding to molecular weight(MW)of approximately 40kD on SDS-PAGE with silver staining. 3. The optimum temperature for RCP1 activities is 37℃ and optimum pH is 8.0. 90% of the protease activities were kept 60 minutes after treatment below 25℃. When temperature is higher than 35℃, the protease activity lost rapidly with the increasing of it. Its activity level remained nearly half at 35℃ for 60 minutes after trentment, but it was completely lost beyond 45℃. It was stable after 1h incubation at a broad pH range of 6 to 10 in the temperature condition of 4℃ . The enzyme activity was unaffected by the metal ions Na+, and Zn2+, partly inhibited by Mn2+, Mg2+, and heavily inactivated by Cu2+, Fe2+, Pb2+ at a range of 2.6 to 10 mM, however it was stimulated by calcium ions, optimally at 2.6 mM. 4. In order to ascertain the nature of the protease concerned, pure RCP1 was assayed against a range of nitroanilide substrates to assess substrate specificity. It showed no activity against the chymotrypsin substrate Suc-Ala-Ala-Pro-Phe-NA or the subtilisin substrate CBZ-Gly-Gly-Leu-NA. Whereas its high or proper activities were revealed against the trypsin substrate Benz-Phe-Val-Arg-NA, and also the substrate D-Val-Leu-Arg-NA,Benz-Pro-Phe-Arg-NA and D-Val-Phe-Lys-NA. It means that RCP1 has the ability to cleave at the carboxyl side of both Arg and Lys, and is a trypsin-like protease. The pure protease was classified with the use of standard inhibitors. It was strongly inhibited by aprotinin,leupeptin,soybean trypsin inhibitor and partly inhibited by PMSF, manifested the characteristics of trypsin-like protease. The cysteine and aspartic inhibitors iodoacetamide and pepstatin were ineffective against the protease.5. To investigate the possible importance of RCP1 in the infection processes of the pathogen to the host, the change of protein content in extracted cell wall and hydroxyproline content released from it after treatment with protease were tested. The result show that incubation of wheat cell wall with the RCP1 depleted the protein content by about 34.58%, resulted in an approximately 3.5-fold increase in release of hydroxyproline-containing protein from cell wall compared with that of containing trypsin protease inhibitor aprotinin. This suggested that this protease has the ability to degrade structural proteins of the wheat cell wall.6. The impact of this protease on wheat tissue could be observed directly by transmission electron microscopy. A new method was adopted in this investigation, which could ensure not only the protease entering into plant tissue...
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