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”
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
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
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
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
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?
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
Plant immunity likely spread through plasmodesmata
• Plasmodesmata directly connect cytoplasm of adjacent cells
– Animal cells use gap junctions for communication
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
RNA silencing- viruses fight back• Viral protein p25is a movement protein that prevents cellular
RdRp activity
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
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:
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