Grapevine Leafroll Disease Flashcards
What are the symptoms of Grapevine Leafroll Disease?
Leaf margin downward rolling
Interveinal discolouration
Chlorosis
What is the taxonomy of GLRaV-3?
Family: Closteroviridae
Genus: Ampelovirus, Closterovirus, Crinivirus, Velarivirus
Explain the GLRaV-3 virus
Phloem-limited, positive single stranded RNA
Conserved feature: filamentous, no polyA tail, HSP70, semi-persistent transmission by Hemiptera
Explain the GLRaV-3 genome
Monopartite, 18.4 - 18.6 kb, 13 ORFs
Replication gene block: genome replication, transcription: ORF1a, ORF1b (genomic RNA)
Quintuple gene block: assembly and movement: ORF3- ORF7 (sub genomic RNA)
Unique gene block: unique to each virus: In GLRaV3; p208: RNA silencing suppressor: silence the viral RNA
Explain mRNA-Seq
1) Library preparation: Capture of mRNA: Olgo(dT) beads
2) Direct mRNA extraction
3) Double strand cDNA synthesis
4) Enzymatic fragmentation of cDNA: smaller the fragment, easier to sequence, but more error
5) End Repair and A addition
6) Addition of adapters with 96 unique barcodes
7) Amplification by PCR
8) 96 RNA-seq libraries
Explain cluster generation/amplification
Use bridge amplification
Explain how the sequencing works
1) Fluorescently labeled DNA bases
2) Sequencing cycles
3) Data is exported to an output file –> “Base calling”, the machine determines the base, contains all data
How is the RNA-sequencing data processed?
1) Trim the raw data
- -> Adaptor sequence and low quality reads: trim low Q score
2) Transcriptome assembly
- -> Where do the fragments come from in the genome? Millions of fragments read, so assemble reads to genome location/genome feature, reference genome, spliced transcripts alignment to a reference (STAR): cloud computer/super computer
- -> Very computational intensive
3) Count gene features
- -> Raw expressional data: cannot use this data as final results
4) Data normalization: account for factors that affect reads numbers
- -> Machine bias: not every read is the same, could have double sequenced in certain locations
5) DEG analysis: Higher expression on certain genes
6) Gene annotation
What is a Q score?
Q20: incorrect based called 1 in 100 times, accuracy 99
Q30: incorrect based called in 1000 times, accuracy 99.9
Higher Q score, means more accurate
What was seen in RNA sequencing for the Leaf for the primary metabolism (EL31 and 35)?
There was a down regulation of photosynthesis, chlorophyll biosynthesis and sugar assimilation
There was an up regulation of cell wall biosynthesis
What is the development stages of the leaf?
EL31: pea-sized berry
EL35: veraison
EL38: harvest
In the berry and the leaf
What was seen in RNA sequencing for the Berry for primary metabolism (EL31)?
Photosynthesis and sugar assimilation are up regulated
There was an up regulation in cell wall biosynthesis
What was seen in RNA sequencing for the Leaf in the mitochondria?
Structural component and mitochondrial activity was downregulated
What was seen in RNA sequencing for the Berry in the mitochondria?
Structural component and mitochondrial activity was down regulated
What was observed for RNA-sequencing and sugar translocation?
Hexose transporter 5 (HT5) was unregulated
Sucrose transporter 2 (SUT2) was downregulated
What was observed for RNA-sequencing and sugar translocation for the transporters?
HT2, HT5 was unregulated
HT3/7, SUT2, SUT 27 was downregulated
What does the up regulation in HT5 show?
Increased hexose retrieval to leaf cells
What are SUT2 and SUT27?
They are paralogs
Explain SUT2 and SUT27
They are localized to the plasma-membrane in the companion cell, and are downregulated
There is increased sucrose in the apoplast, but have repressed activity, so less sucrose seen in the cytoplasm
What is seen in RNA sequencing for the flavonoids?
The flavonoid biosynthetic pathway (flavanol, flavan-3-ol/tannins, anthocyanin) are unregulated
What was seen with the defence response with RNA sequencing?
The pathogenesis related protein (PR-2, PR-4, PR-5, PR-10) , NB- LRR R protein family, RNA-induced gene silencing mechanisms (E-L 35 berry, RdRp1, RdRp6, Dicer 2, Dicer 4, AGO5, AGO4A, RDM3) , HSP and HSP co-chaperones are all up-regulated
What is the PR-2?
Beta - 1,3- gluconase which blocks PD to inhibit viral transport by depositing Callaces in the PD to block transport
What is the working model?
Sucrose transport is driven by ATP, but damaged mitochondrial lacks ATP, and as such sucrose cannot be transported, so the mitochondria produces ATP, so if damaged, cannot produce ATP
There is increased sugar in the cytoplasm and increased osmotic pressure and cell wall fortification