Investigating the Gene Expression Strategy of Grapevine Red Blotch Virus
Grapevine red blotch virus (GRBV), a geminivirus, is the recently identified causative agent of the emerging grapevine disease known as red blotch. The monopartite circular ssDNA GRBV genome consists of genome-sense open reading frames (ORFs) (V1, V2 and V3), and complementary sense ORFs (C1, C2, and C3). Based on sequence comparisons to related viruses, C1 is hypothesized to be spliced with C2 to give rise to a C1-2 mRNA transcript. Recent unpublished data provides mRNA evidence of a possible seventh ORF referred to as V0 as well as evidence of V0 splicing with V2 to form a V0-2 mRNA transcript. So far, none of the predicted protein products have been detected, and gene expression mechanisms are primarily deduced from comparative sequence analyses. Thus, one aim of this study was to investigate the protein expression profile of GRBV in part by analyzing infected grapevine protein extracts with tandem mass spectrometry. Current GRBV diagnostic resources all depend on enzymatic amplification of viral DNA sequences, though false-negative results have been problematic. Another aim of the study was thus to determine the relative translation levels of the ORFs by expressing them in Nicotiana benthamiana and analyzing protein products by western blot in order to identify potential diagnostic protein biomarkers. Additionally, expressing the ORFs in N. benthamiana generated mRNA transcripts that were analyzed to test the hypothesized C1-2 and V0-2 splicing mechanisms. C3 and V2 proteins were detected by tandem mass-spectrometry in infected grapevine. Furthermore, C3 was found to have the highest translation level, followed by V2, and then by C1-2; other proteins were not translated at levels high enough for detection. Considering several factors, V2 appears to be the best candidate for a diagnostic biomarker against which antibodies can be developed. Lastly, PCR analysis of mRNA transcripts provides evidence for the hypothesized C1-2 and V0-2 splicing mechanisms.
Grapevine Red Blotch Virus; Gene Expression; Splicing; Translation
dissertation or thesis