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Posttranscriptional gene silencing Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS) • Promoters active • Promoters silenced • Genes hypermethylated in • Gene hypermethylated in coding region promoter region • Purpose Viral • Purpose - Viral immunity? immunity? This has recently been termed “RNAi” S. Grant Cell 96:303, 1999 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 • Has been used effectively in some animal models S. Grant Cell 96:303, 1999 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 21-23 nt fragments dsRNA dsRNase RNA degrading enzyme RNA fragment renders specificity to the RNA degrading enzyme Carrington,Nature 408:150, 2000 RNA silencing• Model 2 RNA-directed RNA polymerases (RdRps) produces “aberrant” RNA 2 Aberrant RNA produced RdRp viral ssRNA 1 RdRp viral ssRNA RdRp plant mRNA 3 dsRNase Carrington, Nature 408:150, 2000 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 Age of plant Protected from subsequent infection Infect with blackring nepovirus No virus 1st infection 2ndinfection 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 NO2Nitrate reductase 35S promoter nitrate reductase = yellow leaves NH4 Transgene Palauqui and Balzergue Curr. Biol. 9:59-66, 1999 RNA silencing Observe: 35S promoter nitrate reductase or nitrate reductase 35S promoter or nitrate reductase Into plant leaves via DNA-coated tungsten Silencing of endogenous transgene Conclusion: Is DNA causing PTGS?? Palauqui and Balzergue Curr. Biol. 9:59-66, 1999 RNA silencing by ectopic pairing Three phases suggested: Introduced DNA pairs with homologous gene Transcriptional interference- aberrant RNA produced Aberrant RNA spreads to adjacent cells through plasmodesmata RNA silencing Alternative models for PTGS • 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