Grapevine Leafroll Disease

Question 1

What are the symptoms of Grapevine Leafroll Disease?

Answer 1

Leaf margin downward rolling
Interveinal discolouration
Chlorosis

Question 2

What is the taxonomy of GLRaV-3?

Answer 2

Family: Closteroviridae
Genus: Ampelovirus, Closterovirus, Crinivirus, Velarivirus

Question 3

Explain the GLRaV-3 virus

Answer 3

Phloem-limited, positive single stranded RNA

Conserved feature: filamentous, no polyA tail, HSP70, semi-persistent transmission by Hemiptera

Question 4

Explain the GLRaV-3 genome

Answer 4

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

Question 5

Explain mRNA-Seq

Answer 5

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

Question 6

Explain cluster generation/amplification

Answer 6

Use bridge amplification

Question 7

Explain how the sequencing works

Answer 7

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

Question 8

How is the RNA-sequencing data processed?

Answer 8

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

Question 9

What is a Q score?

Answer 9

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

Question 10

What was seen in RNA sequencing for the Leaf for the primary metabolism (EL31 and 35)?

Answer 10

There was a down regulation of photosynthesis, chlorophyll biosynthesis and sugar assimilation
There was an up regulation of cell wall biosynthesis

Question 11

What is the development stages of the leaf?

Answer 11

EL31: pea-sized berry
EL35: veraison
EL38: harvest
In the berry and the leaf

Question 12

What was seen in RNA sequencing for the Berry for primary metabolism (EL31)?

Answer 12

Photosynthesis and sugar assimilation are up regulated

There was an up regulation in cell wall biosynthesis

Question 13

What was seen in RNA sequencing for the Leaf in the mitochondria?

Answer 13

Structural component and mitochondrial activity was downregulated

Question 14

What was seen in RNA sequencing for the Berry in the mitochondria?

Answer 14

Structural component and mitochondrial activity was down regulated

Question 15

What was observed for RNA-sequencing and sugar translocation?

Answer 15

Hexose transporter 5 (HT5) was unregulated

Sucrose transporter 2 (SUT2) was downregulated

Question 16

What was observed for RNA-sequencing and sugar translocation for the transporters?

Answer 16

HT2, HT5 was unregulated

HT3/7, SUT2, SUT 27 was downregulated

Question 17

What does the up regulation in HT5 show?

Answer 17

Increased hexose retrieval to leaf cells

Question 18

What are SUT2 and SUT27?

Answer 18

They are paralogs

Question 19

Explain SUT2 and SUT27

Answer 19

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

Question 20

What is seen in RNA sequencing for the flavonoids?

Answer 20

The flavonoid biosynthetic pathway (flavanol, flavan-3-ol/tannins, anthocyanin) are unregulated

Question 21

What was seen with the defence response with RNA sequencing?

Answer 21

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

Question 22

What is the PR-2?

Answer 22

Beta - 1,3- gluconase which blocks PD to inhibit viral transport by depositing Callaces in the PD to block transport

Question 23

What is the working model?

Answer 23

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