| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 98 === Whitefly-transmitted geminiviruses, members of the genus Begomovirus, can infect a variety of dicotyledonous plants and inflict different types of symptoms, causing severe economical losses. Previous studies in our laboratory revealed that Nicotiana benthamiana plants infected by Ageratum Yellow Vein Virus (AYVV) display severe upward leaf curling symptoms. In contrast, the leaves of those infected by Tomato Leaf Curl Virus (TLCV) or Squash Leaf Curl Virus (SqLCV) showed distinct downward curling. Further studies using recombinant viruses have identified C4 protein coding region as the possible determinant of the directions of leaf curling. However, the host factors involved in leaf curling symptoms remain elusive. Therefore, the objectives of this study are to identify the host factors and underlying mechanisms controlling the directions of leaf curling, using the C4 proteins of AYVV, TLCV and SqLCV as materials. The C4 gene of AYVV, TLCV, and SqLCV were amplified and cloned in a pET21d vector. The respective C4 proteins were over-expressed in Escherichia coli, purified, and used as the antigens to raise specific sera against C4 proteins. The bacterially expressed C4 proteins were used as baits to identify the interacting host proteins of N. benthamiana using two dimensional- polyacrylamide gel electrophoresis (2D-PAGE), far-western blot analyses, and co-immunopricipitation assays. At least four host factors interacting with various C4 proteins were identified as membrane-bound proteins: chloroplast photosynthetic oxygen-evolving protein, 24K germin-like protein, plastidic aldolase, and photosystem I light-harvesting chlorophyll a/b-binding protein. Six additional host factors were identified as soluble proteins: Formin-like protein 19, Luminal-binding protein, NADH-ubiquinone oxidoreductase, 26S protease regulatory subunit 6A homolog B, MAR-binding filament-like protein 1-1 and Putative disease resistance protein RPP8-like protein. The biological functions of individual C4 proteins were further assayed in N. benthamiana by cloning the C4 genes of different begomoviruses in the transient expression vector, pBin35S, Bamboo mosaic virus satellite RNA expression vector, pCass-satBaMV, and the Bamboo mosaic virus expression vector, pCass-BaMV, followed by subsequent inoculations. Although C4 mRNA could be clearly detected, neither C4 proteins nor abnormal symptoms were observed in the inoculated plants, confirming that C4 gene functions at the protein level instead of the RNA level. Taken together, this study has identified several host factors involved in energy metabolism or cell proliferation which can interact with C4 proteins of different geminiviruses. It is expected that through the understanding of the underlying mechanisms, more insights could be provided into the development and differentiation of plant cells, and effective measures may be developed for the “curing” of diseased caused by geminiviruses.
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