13. VL Corinna siRNA/RNAi

• plant response against invasive nucleic acids

• Occurs after the infection of susceptible plants (no R gene), plants are able to recover and are then resistant to this and similar pathogens

plant response against invasive nucleic acids

If a susceptible plant is inoculated with a mild strain of a virus, it develops immunity against related viral strains that would otherwise cause severe symptoms.

No further information than:

Mild virus has a deletion of 2b protein, which usually inhibits AGO in the RISC and prevents gene silencing. —> PTGS (posttranscriptional gene silencing) protects plant

PTGS might be triggered by preinfection with a virus lacking a PTGS suppressor (2b) —> leads to immunity against viral strain (already activated PTGS can’t be silenced anymore)

• resistant leaves contain more siRNA before secondary infection because the RNA of the coat protein (CP) from the first viral infection is cleaved

• the additional siRNA then leads to faster silencing of the viral RNA in the secondary infection

viral synergism: mixed infection of a susceptible plant by 2 or more viruses leads to increased susceptibility to at least 1 of the viruses.

Example: PVX (aggressive virus) but PTGS protects plant + PVY (mild virus) with suppressors of PTGS —> single infection no symptoms

simultaneous infection with PVX+PVY allow suppressors of PTGS from PVY replication of agressive PVX

In SAR the plant has an R-gene, recognising the pathogen and initiating HR. Upon second infection, the salicylic acid levels are higher and the plant reaction is faster, resulting in smaller lesions by HR.

In recovery, the plant is initially succeptible (no R-genes!). si-RNA travel from lower infected leaves to higher leaves. These new leaves can not be infected because they can prevent the expression of viral RNA by matching defense siRNA. (RNAi)

• nuclear run-on assay identifies RNAs while they are generated

Experimental design:

• In native state, only one gene e.g. Y is transcribed —> cell are frozen to arrest transcription

• nucleus isolation + incubation with nucleotides including 32P-GTP (radioacitvity marked) and heparin —> in presence of heparin, RNA Pol finishes transcription but doesn’t bind to promoter again —> transcription of genes whose transcription had already (e.g. Y) started is finished

• isolation of RNA

• Dot blot assay —> signal if radioactivity is detected which means that the gene of interest was transcribed

1. virus infects plant with plus strand (=viral genomic RNA)

2. plus strand is translated by plant host ribosome —> plus strand acts as mRNA

3. mRNA is translated into polyprotein (=RNA-dependent RNA polymerase (RdRP), movement protein, coat protein, protease

4. plus strand is replicated by RdRP

Plant RNAi response: viral dsRNA is processed to siRNA —> siRNA target viral plus mRNA strands

• Mechanism: RNAi

• viral infection leads to occurence of viral dsRNA species (with viral-origin RNA-dependent RNA Polymerase (RdRP))

  1. dsRNA is recognized by Dicer and cleaved into smaller parts of dsRNA (=dicing) —> further processed into siRNAs by other proteins

  2. siRNAs are loaded onto RNA Induced Silencing Complex (RISC) by ARGONAUTE (AGO) (part of the RISC) and transported to matching viral target mRNA

  3. either cleavage of mRNA target (=slicing) OR translation inhibition of mRNA target

• plant RNA-dependent RNA Polymerase (RDR)

• siRNA serves as primers for RDR —> more dsRNA

• This dsRNA is then recognized by Dicer, and more siRNAs are produced

AGO is part of the RISC (RNA induced silencing complex)

A sRNA antisense strand gets taken into the RISC and guides it to the complementary RNA sequence, which then gets sliced by AGO

Or AGO binds and represses translation of proteins

Dicing is the cleavage of dsRNA by Dicer (or DicerLike) proteins into smaller compartments

Slicing is the degradation of single strand (ss)RNA (target mRNA), marked by small (s)RNA by the RISC (RNA induced silencing complex), more specifically by ARGONAUTE

• Dicer (targeted by P14, p38)

• AGO (targeted by P19, 2b)

• Translational inhibition via AGO (targeted by P1)

• RDRs (V2, 2b)

dsRNA might be generated though transcription of adjacent genes

miRNA duplex during miRNA/sRNA formation for gene silencing

antisense sRNA in RISC annealing to complimentary marker sequence

Plant RNA-dependent RNA polymerase (RDR) generates more dsRNA

• PAMP flg22 induces expression of miRNA in arabidopsis

• the resulting siRNA targets the mRNA of the auxin receptor TIR1 (auxin increases susceptibility of plants towards bacteria —> repression of auxin signaling reduces suscptibility

• VIGS is a virus vector technology that exploits RNA defense

2 T-DNA plasmids encoding TRV genome (one encodes TRV RNA1, the other TRV RNA2 that carries inserted target sequence) —> propagation in Agrobacterium + infiltration of tobacco plant —> when plant is inoculated with agrobacterium containing this plasmid, DNA between LB and RB are transfered into plant cells and viral RNA sequences (including inserted target sequence) are transcribed under the control of the 35S promoter —> RdRP (=RNA dependent RNA polymerase) is translated from transcript which in turn replicates transcript into infectious viral RNA

Nicht sicher ob richtig:

• a typical t-dNA plasmid for expression of hairpin RNAs in plants. Plasmid can be transiently introduced in plants by bombardement or more commonly stably inserted into plant’s genome by Agrobacterium-mediated transformation (hence presence of selectable marker)