L10 - RNA silencing and artificial resistance Flashcards
What layers of resistance do plants have against viruses
- Barriers blocking virus vector
- ETI (from virus encoded effector proteins)
- RNA silencing system
- No PTI as viruses never exposed to outer membrane! (delivered by vector)
Briefly outline how the RNA silencing system helps to protect against viruses
1) Viral ssRNA forms viral dsRNA via 3 potential mechanisms (discussed later)
2) dsRNA recognised by plant and chopped into small interfering RNAs (siRNAs)
3) siRNAs loaded onto protein complex + used to find similar viral RNA from complementary base attraction
4) Once bound, slicer (part of Argonaute protein in protein complex) cleaves the RNA
5) Translation and replication prevented, normally limiting virus accumulation
Why is viral RNA targeted by this RNA silencing mechanism but the plant’s own RNA is not?
- Recongnises dsRNA, not ssRNA
- dsRNA uncommon in uninfected cells
dsRNA formed in infected cells from:
1) Viral replication intermediate
2) Fold back regions of viral genomes
3) RNA-dependent RNA polymerase (RdRP) uses viral ssRNA as a substrate
Can the RNA suppressing response be spread throughout the plant, if so how?
Why is this such a good defence system?
- Yes, siRNA can move through plasmodesmata and the phloem
- Suppresses spread of virus
PROS of RNA silencing:
- sequence specific (no problem if virus mutates)
- amplifiable
- mobile
- active against all viruses
Quick aside, why can RNA silencing cause problems with transgenes?
- Transgene RNAs recognised as if viral
- Often silenced
How do viruses counter RNA silencing?
Describe an example of the evidence for this mechanism
- Viruses encode suppressors of RNA silencing
- Combination of RNA silencing by host and RNA silencing suppression determine steady state virus accumulation level
- E.g. p19 from Carnation Italian Ringspot Virus
Example:
- Plants expressing transgene GFP = green under UV
- Plant without GFP = red under UV from auto-fluorescence
- Spontaneous silencing of GFP can = loss of green fluorescence
- Infection w/ virus restores green signal via silencing suppressors
Describe the evolution of RNA silencing
How has RNA silencing been coopted beyond its defence role?
Describe the similarities and the differences between the RNA silencing regulators used in these different roles
- Present in many eukaryotes
- Likely present in common ancestor of plants + animals
Also used for regulation of endogenous RNA:
- micro RNAs (miRNAs) used as RNA silencing regulators
- help fine tune TF level
Similarities between miRNA and siRNA:
- about 21nt long
- produced by Dicer-like proteins
- Use AGO proteins as effectors
Differences between miRNA and siRNA:
- miRNA derived from foldback RNAs, only one miRNA per precursor
- siRNAs can have perfectly ds precursors, multiple overlapping siRNAs from each precursor
How can miRNAs influence defence against pathogens? How can this be utilised to increase immunity?
Give an example
- miRNAs may help negatively regulate defence mRNAs
- Costs and benefits of disease resistance tuned by miRNAs
- E.g. miRNAs in tomato target NLR mRNAs
- Knockdown of specific miRNAs increases immunity against pathogens targeted by NLRs
- Immunity balance can be increased in crops
How else can RNA silencing be exploited generally to increase immunity?
1) Transgene inserted w/ small amount of virus
- Triggers silencing response, pre-preparing plant
2) miRNAs engineered to target viral RNAs
3) Host induced gene silencing - HIGS (see more below)
How can RNA silencing be used to increase immunity against nematodes?
Form of HIGS:
- Plant parasitic nematodes take up RNA from infected plant cells
- Specific RNA could be expressed in plant
- RNA taken up by nematode silences key genes for nematode parasitism = resistance
- E.g. targeting HYP effectors of plant parasitic nematodes
How can RNA silencing be used to increase immunity against fungi?
Form of HIGS:
- Fungi use sRNA as effectors to suppress defence mRNAs triggered by PAMP
- Transfer of silencing RNA from plant to fungus via transgene or virus could silence fungal effectors
How else can RNA silencing be used for immunity outside of introducing transgenes?
Name two CONs of this method
- “Spray Induced Gene Silencing” - SIGS
- RNA sprayed on plants
- E.g. protects against Colorado potato beetle, virus + more
CONs:
- RNA unstable under UV so potential issues with degradation of foliar application
- Depends on efficiency of pathogen RNA uptake
