Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
Browse
2 results
Search Results
Item Characterization of the TMV replicase proteins: localization and interactions with Rab GDI proteins(2008-04-22) Kramer, Sabrina Renee; Culver, Jame; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco mosaic virus (TMV) is a model positive-strand RNA virus. TMV encodes two replicase proteins, both of which contain methyltransferase and helicase domains; the 183 kDa protein contains an additional RNA-dependent RNA polymerase domain. Using this virus, virus-host interactions important in the initial establishment of infection and formation of replicase complexes were investigated. Specifically, on the virus side, replicase proteins were examined for regions that may contribute to its localization to the endoplasmic reticulum (ER) during TMV infection. An ER localization domain was identified in a region between amino acids 599 and 701. Alanine substitutions were introduced into this region and examined for their effects on the virus. Several possible hypotheses are discussed as to how this domain may function during infection. Concerning the host, an interaction with a host protein, a Rab GDP Dissociation Inhibitor (Rab GDI), was examined. This interaction occurred with tomato and tobacco Rab GDIs as well as with the originally identified Arabidopsis thaliana Rab GDI (AtGDI2). Silencing of Rab GDI transcripts enhanced the number of infection sites in TMV:GFP-infected plants, but did not alter viral movement or overall accumulation, indicating a possible role in initial establishment of infection. Rab GDI-silenced Nicotiana benthamiana plants showed cellular morphologies similar to those of TMV-infected cells. Moreover, TMV infection results in Rab GDI proteins localizing to structures associated with viral replication. Taken together these data indicate a role for Rab GDI proteins in the initial establishment of infection. Two models of how Rab GDI proteins may contribute to TMV infection are discussed. These studies examine parts of the viral life cycle that are not very well understood, in particular the initiation and establishment of infection. Although vesicle trafficking has been shown to be important for several different pathogens, this is the first time that a Rab GDI protein has been identified as participating in viral replication. Understanding initiation of infection and susceptibility of a host to a pathogen are vital to elucidating pathogen-host interactions and developing disease resistance strategies.Item MOLECULAR CHARACTERIZATION OF INTERACTIONS BETWEEN TMV REPLICASE PROTEIN AND AUXIN RESPONSIVE PROTEINS: IMPLICATIONS IN DISEASE DEVELOPMENT(2006-11-25) Padmanabhan, Meenu Sreedevi; Culver, James; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco Mosaic Virus and Arabidopsis thaliana serve as ideal model systems to study the molecular aspects of virus - host interactions. Using this system, an interaction between the helicase domain within TMV replicase protein and an auxin responsive protein, IAA26 was identified. IAA26 is a member of the Aux/IAA family of transcription factors that function as repressors in signaling pathways controlled by the phytohormone auxin. Characterization of the interaction was carried out utilizing a helicase mutant defective in its interaction with IAA26 and by creating transgenic plants silenced for IAA26 expression. These studies suggest that the interaction was not essential for either viral replication or movement but promoted the development of disease symptoms. Cellular co-localization studies revealed that in TMV infected tissue, the nuclear localization and stability of IAA26 was compromised and the protein was relocalized to distinct cytoplasmic vesicles in association with the viral replicase. In keeping with its role as a transcription factor, the alterations in IAA26 function should lead to misregulation of downstream auxin responsive genes and this is supported by the fact that ~ 30% of the Arabidopsis genes displaying transcriptional alterations to TMV could be linked to the auxin signaling pathway. Aux/IAA family members share significant sequence and functional homology, and an additional interaction screen identified two more Arabidopsis Aux/IAA proteins, IAA27 and IAA18 and a putative tomato Aux/IAA protein, LeIAA26 that could interact with TMV helicase. The nuclear localization of these three proteins was disrupted by TMV and alterations in LeIAA26 levels induced virus infection-like symptoms in tomato. Additionally, transgenic plants over-expressing a proteolysis resistant mutant of IAA26 showed abnormal developmental phenotype, the severity of which was abrogated during TMV infection which blocked nuclear accumulation of the protein. Taken together, these findings suggest that TMV induced disease symptoms can partially be explained by the ability of the virus to disrupt the functioning of interacting Aux/IAA proteins within susceptible hosts. The significance of such interactions is yet to be determined but it appears that they may be advantageous to the virus while infecting tissues that are in a developmentally static stage.