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Posttranscriptional gene silencing
• Promoters silenced• Genes hypermethylated in
promoter region • Purpose - Viral
immunity?
• Promoters active• Gene hypermethylated in
coding region• Purpose - Viral
immunity?
S. Grant Cell 96:303, 1999
Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS)
This has recently been termed “RNAi”
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Posttranscriptional gene silencing• Degrades all RNA
transcripts that are homologous, including unlinked genes
• Observed in filamentous fungi, ciliates, and animals – Injection of dsRNA into
worms or fruit fly inhibits endogenous gene expression
S. Grant Cell 96:303, 1999
• Has been used effectively in some animal models
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RNA silencing-
• plant “immunity” to viral infection involves spread of signals across membranes
• silencing triggered by dsRNA, transgenes, viruses
• If virus carries a plant gene, that plant gene is also silenced
• “Recovery”- when virus degrades the virus entirley, and is now protected against infection by that virus
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RNA silencing-
• Model 1
dsRNase
dsRNA
21-23 nt fragments
RNA fragment renders specificity to the RNA degrading enzyme
RNA degrading enzyme
Carrington,Nature 408:150, 2000
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RNA silencing-
• Model 2
viral ssRNA
Carrington, Nature 408:150, 2000
RNA-directed RNA polymerases (RdRps) produces “aberrant” RNA
RdRp
dsRNase
3
1
Aberrant RNA produced
viral ssRNA
plant mRNA
2
RdRp
RdRp
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Problem with models -
• Promoterless DNA can induce PTGS
• Not all genes undergo PTGS
• How can PTGS spread through an entire plant??
• What does an “aberrant” RNA look like?
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Recovery from plant infection and immunity
No virus
Infect with blackring nepovirus
1st infection 2ndinfection
Protected from subsequent infection
Age of plant
Ratcliff, Science 276:1558, 1997
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Plant immunity likely spread through plasmodesmata
• Plasmodesmata directly connect cytoplasm of adjacent cells
– Animal cells use gap junctions for communication
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RNA silencing- viruses fight back
• Virus spreads rapidly through plant via opening channels in the cell wall – e.g. Potato virus X (an RNA virus) has three movement proteins- p25,
p12 and p8
• Viral protein p25 is a movement protein that also prevents cellular RdRp activity
• Thus, virus spreads by two proposed mechanisms:– opening channels to adjacent to cells
– inhibiting RNA silencing • Note- viral RdRp does not result in silencing
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RNA silencing- viruses fight back• Viral protein p25is a movement protein that prevents cellular
RdRp activity
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RNA silencing
• Viruses come in DNA or RNA forms
35S promoter nitrate reductase
Nitrate reductase
NO2-
NH4
= yellow leaves
Transgene
Palauqui and Balzergue Curr. Biol. 9:59-66, 1999
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RNA silencing
Palauqui and Balzergue Curr. Biol. 9:59-66, 1999
Silencing of endogenous transgene
Into plant leaves via DNA-coated tungsten
35S promoter nitrate reductase
35S promoter
nitrate reductase
or
nitrate reductaseor
Conclusion:Is DNA causing PTGS??
Observe:
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RNA silencing by ectopic pairingThree phases suggested:
Introduced DNA pairs with homologous gene
Transcriptional interference- aberrant RNA produced
Aberrant RNA spreads to adjacent cells through plasmodesmata
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Alternative models for PTGS
RNA silencing
• DNA pairing activates RNA degradation
• dsDNA “looks like “ aberrant dsRNA and so activates RNA degradation
• hypermethylation leaves “mark” on transgene for formation of aberrant transcripts