Comprehensive analysis of infection-specific proteins in Betula platyphylla var. japonica plantlets infected with a birch canker pathogen, Inonotus obliquus

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In the present study, infection-specific proteins were detected in the crude protein preparations from Betula platyphylla var.japonica No.8 plantlets infected with a birch canker pathogen, Inonotus obliquus IO-U1 or IO-B2 strain, followed by estimation and identification of the detected proteins with using data base search. The infected plantlets with I.obliquus IO-U1 or IO-B2 strain, control 1 plantlets (no infection and no injury), and control 2 plantlets (injured) were collected everyday after the inoculation for one week. These plantlets were deep-frozen with liquid N_2, powdered, and then subjected to extraction of proteins with buffer. The obtained protein solutions were concentrated, desalted, and then used for two-dimensional electrophoresis (2DE). After the electrophoresis, the gels were stained with silver, and the images were inco**rated into personal computer with scanner, followed by image analysis of the gels. Corresponding proteins to infection-specific ones dete** by im … More age analysis were searched on the data base, based on their molecular weight and isoelectric point. As the results, carbonic anhydrase, NADH-ubiquinone oxidoreductase, cytochrome C oxidase, glutathione S-transferase (GST), L-ascorbate peroxidase, and glutathione peroxidase were estimated to be produced as the proteins involved in oxidative burst. In addition, the following proteins were also presumed to be formed : PR10-1, caffeoyl-CoA O-methyltransferase, chalcone-flavonone isomerase, heat shock 70kDa protein, glyceraldehyde 3-phosphate dehydrogenase, 20S proteasome, and calreticulin. In the case of IO-U1 infection, the proteins estimated to be involved in oxidative burst were detected in the most of culture days. IO-B2 infection also caused production of the proteins estimated to be involved in oxidative burst. Hence, it is assumed that in both the infection with IO-U1 and IO-B2 oxidative burst occurred as a main protective reaction in birch plantlets within one week after infection. While in the case of IO-U1 infection many infection-specific proteins were detected 2 days after infection, protein preparation was obtained from the infected plantlets. The sample was subjected to 2DE, and the gel was stained with silver by the modified method for MS analysis. Twelve infection-specific protein spots were cut off from the gel, they were in-gel-digested, and then analyzed with MALDI-TOF-MS. Using the obtained peptide maps, Mascot data base search was performed. As the results, GST and 60kDa chaperonin were identified as infection-specific proteins. From these results, it is clarified that oxidative burst occurred as a protective mechanism and protective genes were actively expressed in the birch No.8 plantlets infected with IO-U1. Less