Characterization of cDNAs from Nicotiana benthamiana that encode proteins which interact with tomato golden mosaic virus AL2 protein in the yeast two-hybrid system

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dc.contributor.advisor Fred J. Fuller, Committee Member en_US
dc.contributor.advisor Eric S.Miller, Committee Member en_US
dc.contributor.advisor Ian T.D. Petty, Committee Chair en_US Yu, Ming en_US 2010-04-02T18:07:22Z 2010-04-02T18:07:22Z 2003-07-23 en_US
dc.identifier.other etd-07182003-150714 en_US
dc.description.abstract The AL2 protein of tomato golden mosaic virus (TGMV) is multifunctional. It is required for derepression of the TGMV AR1 gene in phloem tissue, and for trans-activating the AR1 and BR1 genes in mesophyll cells. It also enhances virus susceptibility when expressed in transgenic plants. It is thought that TGMV AL2 protein accomplishes these functions by interactions with unknown host factors. In this study, cDNAs from Nicotiana benthamiana plants that encode proteins which interact with TGMV AL2 were characterized. A yeast two-hybrid assay identified two cDNA clones, Nb#51 and Nb#62, that specifically interacted with TGMV AL2, but not with negative control proteins. Sequences of these two cDNA clones were determined by primer walking, which revealed that Nb#51 appears to be a 3'-coterminal truncated version of Nb#62. Inspection of the amino acid sequences encoded by Nb#62 found the presence of both ankyrin-repeats and tetratricopeptide repeats (TPR). Deletion analysis showed that the TPR motif, together with its flanking regions, was sufficient to confer on Nb#62 the ability to interact with TGMV AL2, whereas the ankyrin-repeats were not required for this interaction. Nb#62-specific mRNAs were detected in N. benthamiana plants by northern hybridization in potato virus X (PVX) infection experiments, but not in heat-shock experiments. Virus-induced gene silencing (VIGS) assays were used to investigate the possible function(s) of the Nb#62-encoded protein in normal plants and in the context of a TGMV infection. When PVX carrying a 618-bp fragment from the 5'-end of the Nb#62 cDNA was used as a silencing trigger in N. benthamiana plants, VIGS effectively targeted the transcripts which contained sequence similarity with the trigger fragment. However, the infected plants didn't have difference in the phenotype when compared to the PVX vector infection. When TGMV carrying a 93-bp fragment from the 5'-end of the Nb#62 cDNA infected plants, viral DNA accumulation in the upper leaves was reduced, when compared to wild-type TGMV or a control TGMV construct containing a 95-bp fragment from the tobacco Sulfur gene. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject Nicotiana benthamiana en_US
dc.subject yeast two-hybrid system en_US
dc.subject tomato golden mosaic virus AL2 protein en_US
dc.title Characterization of cDNAs from Nicotiana benthamiana that encode proteins which interact with tomato golden mosaic virus AL2 protein in the yeast two-hybrid system en_US MS en_US thesis en_US Microbiology en_US

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