Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and anti-sense RNA

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1 Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and anti-sense RNA Waterhouse et al Presented by Sean Montgomery and Jeff Chen

2 How are these things related?

3 Outline Background Paper Discussion (Waterhouse et al. 1998) PART 1: Viral gene silencing in tobacco Nicotiana tabaccum PART 2: Reporter gene silencing in rice PART 3: Proposed model for PTGS Current models and significance Conclusion Orzya sativa

4 Generating knock outs and knock downs At the genetic level? CRISPR/Cas9 site-directed mutagenesis At the post-translational level? enzyme inhibitors At the post-transcriptional level? sirnas shrnas What is the difference between a knock out and a knock down?

5 The polarity of genes Sense Same sequence as mrna Antisense Same sequence as template Complementary to mrna

6 Discovery of PTGS in petunias Napoli et al Introduced pigmentation gene but got less pigmentation Trait cosegregated with introduced gene Transcription of gene not sufficient to produce trait But how does this occur?

7 Hypotheses of PTGS in 1998 Increased copy number increases strength of PTGS Sense and antisense genes at the same locus on the genome is required for effective PTGS Mechanism involves degradation of transgene and target mrna Antisense RNAs and crnas bind to target mrna to induce degradation?

8 Big questions in Waterhouse et al. (1998) Generally, how does post-transcriptional gene silencing (PTGS) occur? What are the contributions of sense genes, antisense genes, or both genes in effective RNA interference (RNAi)? Does increased copy number of RNA-coding genes increase silencing? Do sense and antisense genes need to be at the same locus in the genome for effective RNAi?

9 Question and hypothesis Question: Are mrna transcripts capable of forming a duplex more or less effective in PTGS than either sense or antisense alone? Hypothesis: If dsrna is the trigger that induces silencing, then a stronger PTGS would be observed in duplex forming transcripts rather than single polarity RNA.

10 PART I Viral Gene Silencing

11 Viral gene silencing Transformation with plasmids containing single genes Pro = protease gene from potato virus [s] = sense [a/s] = antisense stop = transcription but no translation

12 Tobacco transformation

13 Methodology of viral gene silencing Transform into tobacco Infect with potato virus Score symptoms

14 Results Which construct should have the most effective silencing? Predictions? Fraction of Immune Plants Percentage of Immune Plants 5/57 8.8% 1/54 1.9% 10/ %

15 Rationalization of results? Why would we see highest silencing in the Pro[stop] gene construct? Why would we expect to see lowest silencing in Pro[a/s]? Generally, do these results agree with our hypotheses?

16 Role of copy number Immune plants all had 3 or more copies of the transgene Susceptible plants only had 13 copies of the transgene

17 Design the experiment How to introduce both sense and antisense constructs into a single plant? Double transformants? Crossing existing mutant lines?

18 Viral gene silencing Transformation with plasmids containing both sense and antisense genes

19 Methodology of viral gene silencing Transform into tobacco Infect with potato virus Score symptoms

20 Results Plants with sense and antisense genes were much more immune to the virus than any one gene on its own.

21 So how is PTGS inherited? What is the mode of inheritance? Does it follow Mendelian inheritance?

22 Selfing Pro[s]Pro[a/s] heterozygous plants Pro[S]Pro[A/S] Essentially, a single locus gene. What do we expect if we assume Mendelian inheritance?

23 Selfing Pro[s]Pro[a/s] plants 3:1 15:1 Thus, the sense and antisense construct acts like a single locus gene that is inherited in a Mendelian way. Is this strong evidence? Why are the ratios not perfect?

24 Selfing Pro[s]Pro[a/s] plants 3:1 15:1 Furthermore, how can we explain the 2:18 ratios?

25 Crossing susceptible Pro[s] and Pro[s] lines ] Pro[S X Pro[S ] Expectations?

26 Crossing susceptible Pro[a/s] and Pro[a/s] lines /S Pro[A ] X Pro[A /S] Expectations?

27 Crossing susceptible Pro[s] and Pro[a/s] lines Pro[S ] X Pro[A /S] Expectations?

28 Genetic cross of Pro[s] and Pro[a/s] lines P1: Pro[s] x Pro[s] P1: Pro[a/s] x Pro[a/s] P1: Pro[s] x Pro[a/s] F1: Pro[s], Null F1: Pro[a/s], Null F1: Pro[s]Pro[a/s], Pro[s], Pro[a/s], Null

29 Previous model disproved Previous hypotheses postulated that antisenserna hybridized to the target mrna Thus, coexpression of senserna would only compete with the target to hybridize to antisenserna and decrease PTGS However, this is disproved by the crossing results.

30 Genetic cross of Pro[s] and Pro[a/s] lines Conclusions -Expression of sense and antisense strands together confers immunity -Sense and antisense genes are not required to be located at the same locus

31 Genotyping immune and susceptible progeny Linking phenotype and genotype All immune plants had both sense and antisense genes, suggesting that both are required for effective PTGS.

32 PART II Reporter Gene Silencing

33 Endogenous reporter gene silencing Insertion of crippled GUS transgene to silence endogenous GUS gene Control Which construct are controls? Control

34 Endogenous reporter gene silencing Insertion of GUS inverted repeat to form a duplex panhandle structure

35 Results Minimal interference Minimal interference High interference (90%)

36 PART III Proposed model for PTGS 5 main steps

37 Step 1 Sense and antisense RNA are transcribed and form a duplex. These are exported to the cytoplasm.

38 Step 2 RNA polymerase transcribes the dsrna into crna, which is attached to RNA endonucleases

39 Step 3 The crna (with the endonucleases attached) hybridizes to the target mrna or viral RNA Step 4 The endonuclease cleaves the target RNA at the ends of the crna

40 Step 5 The crna releases from the cleaved target RNA and is free to bind to more mrnas for cleavage, ie. recycled.

41 Current model of RNAi

42 Two main types of RNAi mirna must be post-transcriptionally modified in plants, almost perfect base pair complementarity in animals, imperfect base pair complementarity sirna derived from unique long double stranded RNA

43 Summary of findings from Waterhouse et al. (1998) Copy number not main source of PTGS Sense/antisense pairing mediates, not interferes with, silencing Panhandle structure of RNA operates similarly to two separate molecules

44 Post-paper events 1998, February Fire et al. is published 1998, November Waterhouse et al. is published 2001 Discovery of Dicer endonuclease 2006 Nobel Prize awarded to Mello and Fire for RNAi discoveries in C. elegans 2013 First in human trials of sirnas blocking angiogenesis inducing genes in tumors.

45 RNAi in medicine RNAi has a very wide range of applications in human medicine sirnas have been tested as a cancer therapeutic sirna drugs were used in trials to treat ebola

46 How are these things related?

47 Future directions/questions Potential applications? Unanswered questions? Flaws in the paper?