Grapevine Red Blotch Disease was first observed in 2008 and is caused by Grapevine Red Blotch-associated Virus (GRBaV) which has been detected in numerous cultivars across the United States and alters fruit chemistry, flavor, and yield. The objective of the study was to evaluate GRBaV transcription by northern blot analysis which involves large-scale nucleic acid extractions from grapevine leaf tissue, RNA hybridization using chemiluminescence, and determining the 5' termini of transcripts. Specifically designed probes detected viral RNA transcripts and RACE PCR followed by cloning and sequencing can identify the 5'-ends of transcripts to further the understanding of Geminivirus transcription and genetics.

1. Elucidating the Transcriptome: Grapevine Red Blotch-associated Virus
Antonio Cerullo, Dr. Keith Perry, Dr. Jeremy Thompson, Jose Asencio Vargas
Perry Laboratory
Department of Plant Pathology and Plant-Microbe Biology
Grapevine Red Blotch Disease was first observed in 2008 and the causal agent named
Grapevine Red Blotch-associated Virus (GRBaV). This virus has been detected in
numerous cultivars across the United States and is found to alter fruit chemistry, flavor,
and yield. Currently, very little is known about this virus’s replication. My primary
objective was to evaluate GRBaV transcription by northern blot analysis. To address this
we did 1. large-scale nucleic acid extractions (>mg quantities) of grape leaf tissue, 2.
RNA hybridization utilizing chemiluminescence, and 3. 5’ termini determination of
transcripts. Nucleic acid extraction followed by DNase treatment results in the isolation
of mRNA molecules present in grapevine tissue. Following the agarose gel
electrophoresis of RNA and transfer to a nylon membrane, hybridization using
chemiluminescent methods identifies the number and size of RNAs synthesized from
each targeted open reading frame. Specifically designed hybridization probes detected
viral RNA transcripts. Rapid Amplification of cDNA Ends (RACE) PCR of reverse-
transcribed cDNA can be used to identify the 5’-ends of transcripts. Amplicons were
cloned into Escherichia coli using the pGEM®-T vector and sequenced. Continuation of
this study will further understanding of Geminivirus transcription and genetics.