Poster: Plant Pathogen/Symbiont Interactions

Abs # 736: Analysis of the relationship between Tobacco BY-2 cells and Alternaria alternata

Presenter:	Shinya, Tomonori , tomochan@cc.tuat.ac.jp
Authors	Shinya, Tomonori  (A)   Hanai, Kazunari  (A)   Kozone, Ikuko  (A)   Saito, Mikako  (A)   Matsuoka, Hideaki  (A)
Affiliations:	(A): Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology

Tobacco BY-2 chitinase (TBC) and lytic activities were induced by the addition of the pathogenic fungus Alternaria alternata culture medium. To investigate of the relationships between BY-2 cells and A. alternata, TBC was purified and the elicitor active compounds from A. alternata culture medium were isolated and analyzed. Three BY-2 chitinases (TBC-1, 2, 3) were induced at 3h after the addition of A. alternata culture medium. TBC-1, 2, 3 were purified and these molecular weights were 28.8, 33.3 and 28.5 kDa respectively. N-terminal amino acid sequences of TBC-1, 3 were analyzed. Database research analysis revealed that the sequences of TBC-1, 3 were indicated high homology with the sequences of class IV chitinases. The A. alternata culture medium was autoclaved and filtrated to remove the fungus body. 99% methanol-insoluble fraction of the filtrated solution had elicitor activity. The activity fraction was separated by removing protein, sodium hydroxide extraction and 75% methanol extraction. Finally, the elicitor was isolated by a gel filtration chromatography. The elicitor activity was assayed by the TBCs induction method. The isolated elicitor was composed of carbohydrates with molecular weight of >4000. TBCs were induced with the concentration of 5 mg/ml. Hydrolysates were converted the alditol acetates and analyzed with a gas chromatography. As the results, the carbohydrates elicitor from A. alternata was composed of mannose, glucose, and galactose.