Presentation 5: Analysis of Trigger and Suppression of Antiviral Defences by Grapevine Pinot Gris Virus Flashcards
What is the taxonomy of Grapevine Pinot Gris Virus?
Family: Flexiviridae
Genus: Trichovirus
Species: Grapevine Pinot Gris Virus
What are the symptoms of GPGV?
Mottling, deformation, short internodes, shunted shoots, yield loss, also may be symptomatic
How is GPGV transmitted?
Contact with infected equipment
What is the GPGV genome like?
\+ssRNA ORF1 encodes replicase ORF2 encodes movement proteins ORF3 encodes viral coat proteins 5' UTR and 3' UTR
How post transcriptional gene silence activated as a viral defence?
RNA silencing recognizes dsRNA to target related ssRNAs in the cytoplasm
Complementary siRNA duplexes produced through dsRNA cleavage via Dicer
Strand of siRNA duplex associates with AGO in RISC
RISC uses siRNA as a template to recognize mRNA
Programmed RISC degraded targeted mRNA RNA-dependent RNA polymerase activity amplifies siRNA
PTGS is responsible for symptom recovery and fighting viral RNA
How does viral suppression of post-transcriptional gene silencing activity?
Majority of viruses encode viral suppressors of RNA silencing
VSRs are diverse, and are able to target all effectors of RNA-silencing
–> VRS can mimic important structures involved in PTGS
–> VSRs can target and suppress the amplification of silencing effects
—> VSRs can inhibit translation of essential proteins to alleviate antiviral functions
–> VSRs can sequester siRNAs
VSR are essential to proliferation of viral infection
What is the hypothesis of this paper?
Ability of certain viruses to overcome the RNA-based defence is the main factor by which symptomatic phenotypes are restored
What are the objectives of this paper?
Investigate the RNA-silencing pathway that mediates the plant response to GPGV infection
Evaluate the putative ability of the virus to overcome antiviral defences by suppressing the RNA silencing mechanisms
What were the methods used in this paper?
GPGV infectious clones
How was the siRNA processing in the genome identified?
High through-put sequencing from 16 sRNA libraries
Mapping sequence allowed for detection of siRNA processing
siRNAs are distributed throughout the genome
How were predicted genes expressed in RNA silencing?
RNA was extracted and reverse transcribed
Real-time PCR was performed using cDNA of each sample
Expression of NbDCL4, NbAGO5, and NbRDR6 genes in pRI inoculated plants are higher than mock and healthy plants, NbAGO1 is inhibited in inoculated plants
How was construction of a viral plasmid for transformation performed?
Coding sequences from pRI were PCR amplified
Final plasmid vectors were created by recombining GPGV proteins and FLAG epitope into destination vector
Expression cassettes were transfected into Agrobacterium tumefaciens
How was GFP signal quantification done?
Leaf samples harvested, and real time PCR used to extract RNA and reverse transcribed into cDNA
No fluorescence signals seen except for samples of p35s GFP inoculated with pP19 and PGPGV
How was GFP mRNA quantified?
Helped to identify CP protein abundance it is may be necessary to PTGS silencing
How was GFP siRNA quantified?
Stem loop PCR revealed that there was overall less action of siRNAs in presence of CP
