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Use of Green Fluorescent Protein GFP in the Study of Plant-Colletotrichum spp. Interactions

GFP (Green Fluorescent Protein). Fig. Jellyfish (Aequorea victory). 1962: Isolation from Jellyfish 1992: Cloning 1994: First use as a reporter gene ~ Present: Extended to all organisms. 238 amino-acid, 27kDa protein Absorption: 395nm~475nm Emission: at a maximum 508nm light Under UV or bl

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Use of Green Fluorescent Protein GFP in the Study of Plant-Colletotrichum spp. Interactions

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    1. Use of Green Fluorescent Protein (GFP) in the Study of Plant-Colletotrichum spp. Interactions Sep. 15, 2005 Horticultural Crops Breeding Laboratory Sanghoon Kim ??????? ???????? ??2??? ??????. ?? ?? ??? ??? ???? ?????? colletotrichum? ????? ?? ??? ??? ?? ??? ?? GFP???? ??? ??? ??? ????? ?????.??????? ???????? ??2??? ??????. ?? ?? ??? ??? ???? ?????? colletotrichum? ????? ?? ??? ??? ?? ??? ?? GFP???? ??? ??? ??? ????? ?????.

    2. ? GFP???? 1962?? ??? ????? ???? ??? ????, 1992?? ????? ??????. ?? 1994?? ???? ????? ?? reporter gene???? ??? ???? ?? ????? ??? ??? ??????, ?? ???? GFP? ??? ?? ???? ??? ?????. ? ??? 238?? ?????? ???? ???, ??? ?? ?????? ??? ??? ?? ???? ?? ???? ???? ?????. (??????? GFP???? 1962?? ??? ????? ???? ??? ????, 1992?? ????? ??????. ?? 1994?? ???? ????? ?? reporter gene???? ??? ???? ?? ????? ??? ??? ??????, ?? ???? GFP? ??? ?? ???? ??? ?????. ? ??? 238?? ?????? ???? ???, ??? ?? ?????? ??? ??? ?? ???? ?? ???? ???? ?????. (??????

    3. Advantages of GFP No other co-factors Species-independent Without cell lysis or tissue distortion Stable under diverse conditions ( up to 65?, pH 3-12, inorganic solvents) Overcome of inaccessible information, spatially, temporally GFP? ??? ?? ??? ??? ????. ?? ?? Reporter genes? ???? ?? ??? ??? ???? ??? ?? ???? ??? ?????, ?? ?? ??? ?? ???? ??? ???. ?? bacteria, fungi, plant? ???? ?? ???? ??? ????, ??? ??? ??? ???? ??? ???? ??? ?????. ?? ??? ???? ??? ?????, ????? ?? ? ??? ???, ????? ????? ???? ???? ??? ?? ?? ?? ???? ????. ?, ?? ???? ??, ???, ???? ??? ???? ??? ? ?? ?????. (inorganic solvents: SDS, urea, formaldehyde?, ?? reporter gene? ??? ??? ????) GFP? ??? ?? ??? ??? ????. ?? ?? Reporter genes? ???? ?? ??? ??? ???? ??? ?? ???? ??? ?????, ?? ?? ??? ?? ???? ??? ???. ?? bacteria, fungi, plant? ???? ?? ???? ??? ????, ??? ??? ??? ???? ??? ???? ??? ?????. ?? ??? ???? ??? ?????, ????? ?? ? ??? ???, ????? ????? ???? ???? ??? ?? ?? ?? ???? ????. ?, ?? ???? ??, ???, ???? ??? ???? ??? ? ?? ?????. (inorganic solvents: SDS, urea, formaldehyde?, ?? reporter gene? ??? ??? ????)

    4. ?? GFP? ?????? ???? ??? ??????? ??? ??? ?? ????. ?? ?? ??? ??? ???? GFP? ?????? ??? ??? ? ???? ??? ??? ?? ????? ?? ??? ?????. ??? GFP? ??? ??? ????? ?? ? ?? ?? ?? ??? ???? ????? ? ? ?????. (??: ???? ???? ??? ????? ?? ??, ????? ?? ???? ??? ?? ??: ??? ????? Genek????????? Zebra fish) ?? GFP? ?????? ???? ??? ??????? ??? ??? ?? ????. ?? ?? ??? ??? ???? GFP? ?????? ??? ??? ? ???? ??? ??? ?? ????? ?? ??? ?????. ??? GFP? ??? ??? ????? ?? ? ?? ?? ?? ??? ???? ????? ? ? ?????. (??: ???? ???? ??? ????? ?? ??, ????? ?? ???? ??? ?? ??: ??? ????? Genek????????? Zebra fish)

    5. 1. Use as a reporter gene 2. Visualization of fungus 3. Quantification of fungus 4. Investigation cell dynamics ???? ???? ?????? GFP? ??? ?? ??? ?? 4??? ??? ? ?????. ??, ???? ?????? ??? ???? ?? Reporter gene??? ??? ? ? ???, ????, ???? ??? ??? ????? ??????? ?? ?? ??? ?? ????. ???? ???? ??? ????? ?? ??? ??? ??? ???? ?? ???, ???? ??? ?? ???? ??? ????? ???? ????.???? ???? ?????? GFP? ??? ?? ??? ?? 4??? ??? ? ?????. ??, ???? ?????? ??? ???? ?? Reporter gene??? ??? ? ? ???, ????, ???? ??? ??? ????? ??????? ?? ?? ??? ?? ????. ???? ???? ??? ????? ?? ??? ??? ??? ???? ?? ???, ???? ??? ?? ???? ??? ????? ???? ????.

    6. ??? GFP? ??? ???? ????? ??? ???? ??? ?? GFP???? ???? ???, GFP????? ???? ??? ?? agrobacterium? ???? ??? ???? ??? ?? electroporation??? ???? ????. ? ? agrobacterium? ???? ??? hygromycin ??? ???, gpd????, trpC?????, GFP, T-DNA? ??? ?? vector? ?? ??, ??? ????? ???? ??? ?? ??? ???????? ???? ???. ?? ????? ???????? ???? ????? ???????? ????? ?????? ????? ?? ????? ???? ?? ???. ??? GFP? ??? ???? ????? ??? ???? ??? ?? GFP???? ???? ???, GFP????? ???? ??? ?? agrobacterium? ???? ??? ???? ??? ?? electroporation??? ???? ????. ? ? agrobacterium? ???? ??? hygromycin ??? ???, gpd????, trpC?????, GFP, T-DNA? ??? ?? vector? ?? ??, ??? ????? ???? ??? ?? ??? ???????? ???? ???. ?? ????? ???????? ???? ????? ???????? ????? ?????? ????? ?? ????? ???? ?? ???.

    7. ?? reporter gene???? ??? ???????. ?????? ?? ?????? ???? endopolygalacturonase? ???? ??? ???? ?? ??? ?????? ???? ?? ??? homogalacturonan? ???? ???? ???? ???? ??? ??? ?? ???? ??? ????. ? ??? ??? ???? ??? endopolygalacturonase? GFP? fusion?? ???? ????? ? ?? ???? ??? ?? ???.?? reporter gene???? ??? ???????. ?????? ?? ?????? ???? endopolygalacturonase? ???? ??? ???? ?? ??? ?????? ???? ?? ??? homogalacturonan? ???? ???? ???? ???? ??? ??? ?? ???? ??? ????. ? ??? ??? ???? ??? endopolygalacturonase? GFP? fusion?? ???? ????? ? ?? ???? ??? ?? ???.

    8. ???? in vitro, in vivo?? ???? GFP? ????? ????? ?? ???? ??? ??, ???? appressorium ????? appressoruim? ????? ??? ?? ??? ? ????, ?? endopolygalacturonase? ? ??? ???? ?????. ? ???? ???? ???? ???? ??? ?????? ?? ??? endopolygacturonase? ????? ?????? ?? ???? ???? ???? appressorium?????? appressorium?? ????? ????? ? ? ?????.???? in vitro, in vivo?? ???? GFP? ????? ????? ?? ???? ??? ??, ???? appressorium ????? appressoruim? ????? ??? ?? ??? ? ????, ?? endopolygalacturonase? ? ??? ???? ?????. ? ???? ???? ???? ???? ??? ?????? ?? ??? endopolygacturonase? ????? ?????? ?? ???? ???? ???? appressorium?????? appressorium?? ????? ????? ? ? ?????.

    9. ???? ????? ??????? ???? ?? GFP? ????? ????? GFP? ???? ?????? ???? ???? ??? ??, ? ????? ??? ?????. ?? ????? ?????? ?? ? ??? ???? ?? ? 15??? ??? ?? ??? ??? ????, 20??? ???? Appressorium? ?????. ?? ? 3??? epimermal cell??? ??? ????, ?? ? 4??? ??? ???? colony? ??? ??, ?? ? 14??? mesophyll ?? ??? ?? ??? ? ????.???? ????? ??????? ???? ?? GFP? ????? ????? GFP? ???? ?????? ???? ???? ??? ??, ? ????? ??? ?????. ?? ????? ?????? ?? ? ??? ???? ?? ? 15??? ??? ?? ??? ??? ????, 20??? ???? Appressorium? ?????. ?? ? 3??? epimermal cell??? ??? ????, ?? ? 4??? ??? ???? colony? ??? ??, ?? ? 14??? mesophyll ?? ??? ?? ??? ? ????.

    10. ?????? ?????? ?? ? 6?? ??????? ?? ??? ????? ??? ??, 15???? ????? ???? ?? ??? germ tube? ???? ? ???? ??? ? ??? ???? ?? ??? ? ????.?? ? 20??? ??? ???? Appressorium? ???? ?? ? 5??? ???? trichome? ?? ??? ??? ?? ??? ? ????. ???? ?????? ??? ??????? ?? ???? ??? ???? ?????, ??? ??? ???? ??? ??? ????, ??? ???? ????. ?? ???? ???? apoplast?? ???? ???? ???? ??? ??? ??, ????? ???? ???. ?????? ?????? ?? ? 6?? ??????? ?? ??? ????? ??? ??, 15???? ????? ???? ?? ??? germ tube? ???? ? ???? ??? ? ??? ???? ?? ??? ? ????.?? ? 20??? ??? ???? Appressorium? ???? ?? ? 5??? ???? trichome? ?? ??? ??? ?? ??? ? ????. ???? ?????? ??? ??????? ?? ???? ??? ???? ?????, ??? ??? ???? ??? ??? ????, ??? ???? ????. ?? ???? ???? apoplast?? ???? ???? ???? ??? ??? ??, ????? ???? ???.

    11. ???? ??? ????? ??? ??? ??? ???? ????? ???? ???? ??? Fluorescence microplate reader? ???? GFP? ?? ???? ?? ??? ???? ???? ???? ???. ??? ??? GFP? ??? Fluorescence microplate reader? ??? ?? ?? ????? ???? ????? ??? ??? ?? ?? ????? ??? ??? ? ????.???? ??? ????? ??? ??? ??? ???? ????? ???? ???? ??? Fluorescence microplate reader? ???? GFP? ?? ???? ?? ??? ???? ???? ???? ???. ??? ??? GFP? ??? Fluorescence microplate reader? ??? ?? ?? ????? ???? ????? ??? ??? ?? ?? ????? ??? ??? ? ????.

    12. ??? GFP???? ??? ??? ?? ???? ?????, GFP? ??? ??? RFU?? ?? ??? ??? ?? ??? 72??? 1? ??? ???? 96??? 2???? ??? ?? ??? ? ????. ?, 96??? ???? ???? ???? ???? ???? Biotrophic ???? ?? ???? ???? ???? Necrotrophic??? ???? ?? ??? ? ???, ? ??? RFU?? ??? ???? ?? ??? ? ??.??? ?? 48??? Multi vesicle? ???? ??? ? ??? Biotrophic ???? Necrotrophic??? ???? ???? ????, ???? RFU?? ??? ???? ?? ??? ? ????, ? ???? RFU? ???? ????? ????? ??? ? ???, Biotrophic ???? Necrotrophic ??? ???? ??? ?? ??? ???? ??? GFP? ??? ??? RFU?? ??? ??? ??? ??? ? ??? ? ? ?????.??? GFP???? ??? ??? ?? ???? ?????, GFP? ??? ??? RFU?? ?? ??? ??? ?? ??? 72??? 1? ??? ???? 96??? 2???? ??? ?? ??? ? ????. ?, 96??? ???? ???? ???? ???? ???? Biotrophic ???? ?? ???? ???? ???? Necrotrophic??? ???? ?? ??? ? ???, ? ??? RFU?? ??? ???? ?? ??? ? ??.??? ?? 48??? Multi vesicle? ???? ??? ? ??? Biotrophic ???? Necrotrophic??? ???? ???? ????, ???? RFU?? ??? ???? ?? ??? ? ????, ? ???? RFU? ???? ????? ????? ??? ? ???, Biotrophic ???? Necrotrophic ??? ???? ??? ?? ??? ???? ??? GFP? ??? ??? RFU?? ??? ??? ??? ??? ? ??? ? ? ?????.

    13. ? ???? ???? ??? ??? ??? ??? ??? ?? ???? GST? ??? ??? ??? ????, ??? ??? RFU?? ?? ???? ????. ??? insensitive type? Tetr1? ???? wild type?? ????? ??? ?? ????? RFU? ??? ???? ???, GST? ?? ?? GST? VIGS? ??? ??? ???? ?? ?? ????? ????? RFU? ??? ???? ????. ????? ? ? ??? ?? ??? ??? RFU? ?? ?? ???? ??? ? ????. ? ???? ???? ??? ??? ??? ??? ??? ?? ???? GST? ??? ??? ??? ????, ??? ??? RFU?? ?? ???? ????. ??? insensitive type? Tetr1? ???? wild type?? ????? ??? ?? ????? RFU? ??? ???? ???, GST? ?? ?? GST? VIGS? ??? ??? ???? ?? ?? ????? ????? RFU? ??? ???? ????. ????? ? ? ??? ?? ??? ??? RFU? ?? ?? ???? ??? ? ????.

    14. ????? ??? ?? ???? ??? GFP? ???? ??? ????, ????? ??? ??? ???? NLS sequence? GFP? fusion?? ???? ????? ? ?? ??? ?? ? ??? ?? ??? ???? ???. ?? ???? ???? ???? ?? ????? ???? ??? ???? ?? ??? ?? ??? ???? ??? ??? 24??? ???? ????? Appressrium ??? ????. 48???? ?? ??????? ???? ???? ?? ???? ??? Necrotrophic ?? ????? ?? ? ?? ???? ????. ????? ??? ?? ???? ??? GFP? ???? ??? ????, ????? ??? ??? ???? NLS sequence? GFP? fusion?? ???? ????? ? ?? ??? ?? ? ??? ?? ??? ???? ???. ?? ???? ???? ???? ?? ????? ???? ??? ???? ?? ??? ?? ??? ???? ??? ??? 24??? ???? ????? Appressrium ??? ????. 48???? ?? ??????? ???? ???? ?? ???? ??? Necrotrophic ?? ????? ?? ? ?? ???? ????.

    15. ??? ???? ???? ?? ???? ?? ?? ??? ??? ??? ????, ?? ? 24??? ???? ???? ???? ?? ????, 48???? ?? ?? ??? ???? ???. ??? ???? ???? ???? ??? ?? ??? ?? ??? ??? ?? ???, ? ????? ?? ??? ??? ???? ??? ???? ??? ?????? ???? ???? ?????? ??? ??, ???? ??? ?? ?? ????? ??? ???? ???? ???? ? ??? ????? ??? ????, ?????? ??? ???? ???? ??? ?? ? ??? ??? ?? ??? ????? ???? ????. ??? ???? ???? ?? ???? ?? ?? ??? ??? ??? ????, ?? ? 24??? ???? ???? ???? ?? ????, 48???? ?? ?? ??? ???? ???. ??? ???? ???? ???? ??? ?? ??? ?? ??? ??? ?? ???, ? ????? ?? ??? ??? ???? ??? ???? ??? ?????? ???? ???? ?????? ??? ??, ???? ??? ?? ?? ????? ??? ???? ???? ???? ? ??? ????? ??? ????, ?????? ??? ???? ???? ??? ?? ? ??? ??? ?? ??? ????? ???? ????.

    16. ???? ???? ??????? ????? ???? GFP? ??? ??? ???????. ?? ?? ??? ???? ? ??? ?? ????? ?????? ?? ??? ?? ???, ?????? ?? ???? ?? ?? ??????? ??? ??? ?? ?? ?? ???? ??? ?????, ?? ?? ????? ? ? ??? C. gloeosporioides? acutatum? ?? ??/?? ???? ?????/???? ???? ????? ?? ??? ??? ???, ?? ???? ?? ?????/???? ?? ?? ???? ??? ? ? ????. ???? ???? ??????? ????? ???? GFP? ??? ??? ???????. ?? ?? ??? ???? ? ??? ?? ????? ?????? ?? ??? ?? ???, ?????? ?? ???? ?? ?? ??????? ??? ??? ?? ?? ?? ???? ??? ?????, ?? ?? ????? ? ? ??? C. gloeosporioides? acutatum? ?? ??/?? ???? ?????/???? ???? ????? ?? ??? ??? ???, ?? ???? ?? ?????/???? ?? ?? ???? ??? ? ? ????.

    17. ?? ??? ?????? ??? ??? ?? ?? ????? ??? ?? ?? ???? ?? ?? ????? ?? ?? ???? ??? ??? ????. C. acutatum? ??? TSCa-1? ??? ???? C. annuum? ??? ????? ??? ?? ?? ??? ??? C. bacatum? ???? ?? ? ???? ?? ?? ???? ?? ?? ??? ? ????. ?? ??? ?????? ??? ??? ?? ?? ????? ??? ?? ?? ???? ?? ?? ????? ?? ?? ???? ??? ??? ????. C. acutatum? ??? TSCa-1? ??? ???? C. annuum? ??? ????? ??? ?? ?? ??? ??? C. bacatum? ???? ?? ? ???? ?? ?? ???? ?? ?? ??? ? ????.

    18. Application ?? ?? ??? ???? ???? ???, ?? GFP????? ???? ??? ??? ????? ????? ???? ???, ??? ??? ?? ??? ?? ????. ??????? ??? GFP????? ???? ??? ?? ????? ???? ?? ???? ??? ??? ?? ????? ???? ???? ??? GFP? ??? ??? ? ?? ??? ?????. ? ?? ??? ??? ??? ??? ?? ??? ??? ???? ??? ??? ??? ?? ??? ?? ??? ????? ????? ????? ?????. ???? ??? ??????.?? ?? ??? ???? ???? ???, ?? GFP????? ???? ??? ??? ????? ????? ???? ???, ??? ??? ?? ??? ?? ????. ??????? ??? GFP????? ???? ??? ?? ????? ???? ?? ???? ??? ??? ?? ????? ???? ???? ??? GFP? ??? ??? ? ?? ??? ?????. ? ?? ??? ??? ??? ??? ?? ??? ??? ???? ??? ??? ??? ?? ??? ?? ??? ????? ????? ????? ?????. ???? ??? ??????.

    19. Reference J. B. Yoon, 1999, MS Thesis. M. Robinson., 1999, Curr Genet, 36:98-104. B. Dumas et al., 1999, Appl Environ Microbiol, 65:1769-1771. J. M. Lorang et al., 2001, Appl Environ Microbiol, 67:1987-1994. S. Horowitz et al., 2002, Phytopathology, 92:743-749. D. Chen et al., 2003, Journal of Microbiological Methods, 53:113-122. N. Chen et al., 2003, Journal of Experimental Botany, 54(392):2449-2456. J. C. March et al., 2003, Appl Microbial Biotechnol, 62:303-315. S. Vorwerk et al.,2004, TRENDS in Plant Science, 9(4), 203-209. X. C. Shan et al., 2004, J. Phytopathology, 152:454-460. J. D. Dean et al., 2005, Journal of Experimental Botany, 56(416):1525-1533. S. K. Park, 2005, MS Thesis. http://www.ccrcuga.edu/~mao/intro/online.htm

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