1. 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”

2. 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

3. 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

4. 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

5. 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

6. 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?

7. 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

8. Plant immunity likely spread through plasmodesmata Plasmodesmata directly connect cytoplasm of adjacent cells –Animal cells use gap junctions for communication

9. 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

10. RNA silencing- viruses fight back Viral protein p25is a movement protein that prevents cellular RdRp activity

11. RNA silencing Viruses come in DNA or RNA forms 35S promoter nitrate reductase Nitrate reductase NO 2 - NH 4 = yellow leaves Transgene Palauqui and Balzergue Curr. Biol. 9:59-66, 1999

12. 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 reductase or Conclusion: Is DNA causing PTGS?? Observe:

13. 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

14. 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