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Molecular and functional characterization of an Arabidopsis protein that interacts with coat protein of turnip crinkle virus
The interaction between plants and their pathogens has been of great research interest for most of the past century. In this thesis work, I have used Arabidopsis thaliana and its viral pathogen, Tunip crinkle virus, as a model system to elucidate mechanisms of plant-virus interaction, especially the mechanism of race-specific resistance. One Arabidopsis protein was found to interact specifically with the capsid protein (CP) of TCV through yeast two-hybrid screening of an Arabidopsis cDNA library. This protein, designated TIP (for T&barbelow;CV-i&barbelow;nteracting p&barbelow;rotein), is a member of NAC family of proteins and the interaction between TIP and TCV CP was verified by in vitro binding assay. TIP is 451 amino acids (AA) in length, and its C-terminal 100 AA region is sufficient for interacting with TCV CP. TIP is capable of transcriptional activation and this function was mapped to the N-terminal 268 AA region. Fine mapping of TCV CP reveals that its N-terminal 25 AA region is responsible for interaction with TIP. Site-directed mutagenesis within this region revealed that loss of the TIP-CP interaction in the yeast two-hybrid assay correlated with loss of the ability of TCV to induce a hypersensitive response and resistance in the TCV-resistant Arabidopsis ecotype Dijon 17. These data suggest that TIP is an essential component in the TCV resistance response pathway. ^ Further experiments confirmed that TIP is a transcription factor. The evidence includes (1) TIP activates the transcription of reporter genes in yeast; (2) TIP binds DNA in vitro, albeit nonspecifically; (3) TIP contains a putative nuclear localization sequence (NLS), and GFP fused TIP localizes to nuclei in Nicotiana benthamiana leaves. Significantly, co-expression of TCV CP and GFP-TIP prevents nuclear localization of GFP-TIP, leading to the formation of inclusion-like bodies within the cytoplasm. This demonstrates that CP does interact with TIP in plants cells, resulting in the disruption of the normal cellular localization and function of TIP. My research results support the hypothesis that TIP plays a critical mediating role in the signal transduction pathway leading to Arabidopsis resistance to TCV, consistent with the role of “Guardee” in the “Guard Hypothesis”. ^
Biology, Molecular|Biology, Genetics|Agriculture, Plant Pathology
Ren, Tao, "Molecular and functional characterization of an Arabidopsis protein that interacts with coat protein of turnip crinkle virus" (2003). ETD collection for University of Nebraska - Lincoln. AAI3092586.