2. Background1
• Grapevine red blotch disease was first described only in
2008.
• Red blotch disease is found to affect fruit chemistry,
flavor, and yield in afflicted cultivars.
• Previously thought to be grapevine leafroll disease, due
to leaf discoloration being a symptom of both.
• Diagnoses found that afflicted vines showed no sign of
known leafroll-causing agents.
• Large scale sequencing methods have indicated high
correlation between red blotch disease and the
presence of a novel ssDNA virus species.
3. The Virus
• The virus, aptly named Grapevine Red Blotch-
associated Virus (GRBaV), is not well understood.
• Its genome has been sequenced and putative Open
Reading Frames (ORFs) have been proposed.
• The function of 3 predicted ORFs (V0, V2, V3) are
unknown.
• Attempts to express the predicted capsid protein ORF
(V1) in bacteria have failed.
• Virus particles have not been observed.
• Sequence identity to other viruses suggests it is in the
family Geminivirdae.
4. Objectives
• To understand the strategies behind transcription of
the GRBaV genome & characterize its RNA transcripts.
– Determine size and number of transcripts generated from
each proposed ORF.
• To identify sequences of present mRNAs.
– Sequencing and identifying the ends of individual transcripts
will give greater understanding of transcription and the
translated protein.
– Transcripts can be compared to those of similar viruses.
5.
6. Methods: Extraction
• Conducted nucleic acid extractions from
plant tissue, both infected & healthy.
• PCR Screening confirms presence or
absence of the virus in respective samples.
7. Methods: RNA Hybridization
• Design & generate specific DIG-labeled DNA probes for
known sequences.
• Use gel electrophoresis to separate RNAs by size.
• Transfer RNAs to nylon membrane to be probed for desired
sequences.
• Washing with enzyme-linked anti-DIG antibodies and
addition of substrate allows chemiluminescent detection of
discrete mRNA transcripts.
9. Methods: 5’ RACE PCR*
• Reverse transcribe extracted mRNA to create cDNA.
• Modification of cDNA with poly-A tail.
• Rapid Amplification of cDNA Ends (RACE) PCRs
generates amplicons of decreasing size to increase
fidelity to 5’-termini.
• Amplicons were cloned into the pGEM-T vector to
transform Escherichia coli and DNA sequences were
extracted and sequenced.
*The RACE component was conducted primarily by Dr. Jeremy Thompson using materials from my extractions whereas
the subsequent cloning was completed in conjunction with my efforts.
12. Looking Ahead
• Further investigation into this area will provide
more information on the strategies geminiviruses
employ to transcribe and translate their genome.
• Continuation of this study can shed light on the
relationship between GRBaV and the
Geminiviridae family. Results will provide valuable
information on an emerging group of plant
pathogens.
13. References & Acknowledgements
1. Grapevine red blotch-associated virus Is Widespread in
the United States, B. Krenz, J. R. Thompson, H. L. McLane,
M. Fuchs, and K. L. Perry, Phytopathology 2014 104:11,
1232-1240
I would like to thank Keith Perry, Jeremy Thompson, and Jose
Asencio Vargas for their substantial efforts and advice in
helping me undertake this project, as well as Teresa
Pawlowska, Michael Milgroom, and all participants in the SIPS
Microbial Friends & Foes REU program for making this project
possible.
Editor's Notes
“Here is GRBaV. There are many shared elements between this virus, and members of the Geminiviridae family of viruses which points toward the notion that the two are related. Shown here are characteristics the Geminiviridae family, named such for the twin icosahedron capsids comprising the structure. Over here are genome schematics for several common geminiviruses. They are single-stranded DNA viruses and as such their genomes can be transcribed in either the Virion-sense or complementary sense orientation. You can see here that all of these viruses share the C-sense Rep ORF and the V-sense CP (capsid protein) spanning extremely similar regions of the genome. Not shown here is another commonality: the ori element is a highly conserved nine nucleotide sequence.”
“Here are some blots that we’ve done through probing the Capsid Protein or V1 ORF. After probing and visualizing, you can see the discrete bands formed by the detection of relevant RNA molecules. Shown here is detection of the v1 ORF, or the gene that encodes GRBaV’s capsid protein. After repeating the protocol using the same probes and different samples, we found a pattern of banding. The 3200 NT band is predicted to be the Genomic DNA, whereas the 1600 and 225 bands are proposed to be mRNA transcripts.
