| The success of plant viruses as pathogens depends on their ability to recruit host factors to support their propagation whilst defeating host defense mechanisms early in the infection process. While the virus reprograms plant physiological processes during this critical early period, many of the events and interactions taking place remain unknown. In this thesis, I utilized Nicotiana benthamiana (Nb, a model host for most plant viruses) and Red clover necrotic mosaic virus (RCNMV, a typical plus strand RNA plant virus) to elucidate these early events in the host-virus interaction. The Nb unigene collection, a custom microarray representing 13,413 host genes (~38% of the Nb transcriptome) was created for this study along with a corresponding functional annotation resource. The performance of the Nb array demonstrated its potential usage as a genomic tool in a genome wide study.;This thesis is the first study to examine the host transcriptome at the earliest stages of the virus infection process of 2, 6, 12 and 24 hours post-inoculation (hpi). The 1,654 host genes exhibited differential expression at an FDR cutoff of 0.01. This global snapshot of gene expression revealed that host genes are significantly down-regulated at 2, 6 and 24 hpi and significantly up-regulated at 12 hpi. This suggests that (i) host gene expression was suppressed as early as 2 hpi, and (ii) one infection cycle within the primary infected cell takes 12-24 hours. Gene set enrichment analysis (GSEA) revealed that RCNMV affected the following key host functions/pathways: defense, translation, photosynthesis and chloroplastrelated functions, carbon fixation (Calvin cycle), metabolism, multidrug and toxin extrusion (MATE) transporter, cell wall-associated functions and protein kinases.;Four host genes representing various plant biological functions were transiently silenced or over-expressed to further functionally analyze their impact(s) on the RCNMV infection process. The genes chosen were: 1) NbSAR (systemic acquired resistance), the only host gene in the microarray study that was significantly regulated across all four time points (down regulated in the first 6 hours followed by up regulation), 2) NbWRKY (putative transcription factor) which was up-regulated (10 fold) at 24 hpi, 3) NbBTF3 (basal transcription factor 3) which was up-regulated (1.5 fold) at 6 hpi and 4) NbSI (C-8, 7- sterol isomerase) which was up-regulated (1.2 fold) at 12 hpi.;Pre-silencing of NbSAR decreased the replication levels of RCNMV RNAs suggesting that a certain NbSAR level may be required for RCNMV replication. NbWRKY is a novel WRKY gene that was first identified and characterized in this thesis. The putative NbWRKY polypeptide (325 amino acids in length) is most similar to potato StWRKY6, possessing a single WRKY DNA binding domain, four beta-stranded sheet structure and a nuclear localization signal which was confirmed by cellular localization studies with a GFP fusion. NbWRKY may possess dual functionality in regulating the defense response: shifting its function between both a positive and negative regulator of disease resistance depending on the duration of the RCNMV infection. NbBTF3 was predominantly localized to the nucleus suggestive of its transcription regulator function. NbSI was first isolated and characterized in this thesis. It is an integral membrane protein containing five alpha-helical transmembrane domains and predominantly localizes along cellular, nuclear and endoplasmic reticulum membranes. Functional assays suggested a potential transition of function from promoting membrane proliferation (RCNMV replication) to acting as a precursor of brassinosteroid (modulating host defense).;The genome-wide Nb screen in this thesis reveals the changes in early host gene expression patterns in response to RCNMV. This work forms the foundation for future studies into significant host factors/pathways that determine the fate of virus survival in an inhospitable cellular environment. |
