*Post-trascriptional gene silencing (PTGS) is an ubiquitary mechanism of adaptative defence against viruses and mobile genetic elements

Transcription

1 The research activities of the present FIRB project involving the use of PTGS* are: 1) PTGS to improve plant resistance against viruses 2) PTGS for studying the function of genes involved in fruit set and growth (work in progress) *Post-trascriptional gene silencing (PTGS) is an ubiquitary mechanism of adaptative defence against viruses and mobile genetic elements PTGS is triggered by double-stranded RNA homologous to target RNA. Target RNA is specifically degraded

2 PTGS for Systemic disease resistance to Plum Pox Virus

3 Sharka disease is caused by Plum Pox Virus PPV is a Potyvirus. Its RNA genome consists of approximately bases PPV genome is expressed as a poliprotein of 350 kda, that is processed in smaller functional peptides Some fruit (Prunus) species affected by Sharka are Prunus amygdalus, P. persica, P. domestica, P. salicina, P.armenica, P.avium, P. cerasus.

4 Virus infection via Aphids The virus enters in the epidermal cells. From the infected epidermal cell PPV moves to mesophyll cells, and then it reaches the cells of the vascular system. From cells of the vascular system (i.e. companion cells), the virus moves to the phloem, and via the phloem PPV spreads to the whole plant.

5 From local to systemic infection: a model (hypothetical) of virus systemic spreading

6 Can we confer resistance to Sharka by Genetic Engineering? Yes So far, gene constructs have used a constitutive promoter (CAMV 35 S) to drive expression of a PPV genome fragment coding for virus proteins (Coat protein, Helicase, Replicaseprotease) E.g.: Scorza et al., 2001; Wittner et al., 1998; Guo et al., 1998 However, virus resistance appears not to be caused by the expression of the viral protein(s) but by Post-Transcriptional Gene Silencing (PTGS)

7 The molecule which triggers PTGS is double-stranded RNA (RNAds) HOW?

8 dsrna is processed by the Ribonuclease activity of DICER to small interfering RNAs (sirna). sirna interacts with the RISC complex. sirna confers specificity to the RISC complex. Target RNA is degraded dsrna DICER 5 P 3 3 P 5 sirna RISC

9 sirnas are short RNA molecules confering specificity to the RISC protein complex by pairing to homologous sequences of the target RNA. Target RNA is degraded in the cytoplasm sirnas are involved in the amplification of PTGS. Process that involves RNAdependent-RNApolymerase and produces secondary sirnas In plants, PTGS is transmitted from cell to cell via plasmodesmata. Long distance transmission takes place via phloem.

10 General Aim Enhance the endogenous plant defence system against PPV Specific Aim Avoid PPV systemic disease without affecting local infection (N.B. PPV damages are caused by systemic disease while local symptoms might be useful to monitor the presence of PPV)

11 Nicotiana benthamiana is a model plant for PPV studies wt Most plants infected with PPV do not survive (figure at the left) Very few plants do survive (figure in the middle). Such plants, however, appear very sick. These plants do contain sirnas homologous to PPV genome (Figure at the right; northern blot)

12 promoter sense intron antisense Termination seq rolc PPV PPV rolc ihprolc-pp197 A gene to produce in phloem cells dsrna homologous to PPV genome. RolC promoter is not expressed in epidermal and mesophyll cells. hairpinrna produced by intron containing constructs are very efficient in triggering PTGS. Reference:Smith et al.,2000

13 sirnas are present in plants transgenic for the ihprolc-pp197 gene construct C sirna homologous to PPV genome

14 sirnas are present in the progeny of plants GM with the ihprolc-pp197 gene construct C sirna PPV specific

15 Inoculation of Nicotiana benthamiana with PPV: Local Symptoms: chlorosis Systemic Symptoms: leaf curling and wilting

16 GM Plants Resistant to PPV Systemic infection GM plants do not show symptoms of systemic infection. Only in few cases, light symptoms (mottling) are visible GM Wild type

17 Test DAS-Elisa Apical leaf (not inoculated) ELISA 1 and 6 weeks after inoculation of the basal leaves Basal leaves (inoculated) ELISA 1 week after inoculation

18 PPV detection in independent GM lines Elisa on inoculated basal leaves (1 week after inoculum) Threeshold level Linee Plant , WT

19 ELISA on not-inoculated apical leaves (1 and 6 weeks after inoculation of the basal leaves) Threeshold value Linee Lines Plant 1 week 6 weeks 1 week 6 weeks 1 week 6 weeks 1 week 6 weeks 1 week 6 weeks * * * * * * WT * transgenic plants showing leaf mottling (mild symptoms of PPV infection)

20 Conclusions the gene construct ihprolc-pp197 (containing 197 bases homologous to the PPV genome) confers systemic resistance to PPV Virus resistance takes place by PTGS Virus infection and local multiplication are not significantly affected

21 References Guo HS, Cervera MT and Garcia JA: Plum pox potyvirus resistance associated to transgene silencing that can be stabilized after different number of plant generations. Gene 1998, 206: Pandolfini T, Molesini B, Avesani L, Spena A and Polverari A: Expression of selfcomplementary hairpin RNA under the control of the rolc promoter confers systemic disease resistance to plum pox virus without preventing local infection. BMC Biotechnology 2003, 3:7 Scorza R, Callahan A, Levy L, Damsteegt V, Webb K and Ravelonandro M: Posttranscriptional gene silencing in plant pox virus resistant transgenic European plum containing the plant pox potyvirus coat protein gene. Transgenic Research 2001, 10: Smith NA, Singh SP, Wong MB, Stoutjesdijk PA, Green AG and Waterhouse PM: Total silencing by intron-spliced hairpin RNAs. Natue 2000, 407: Wittner A, Palkovics L and Balazs E: Nicotiana benthamiana plants transformed with the plant pox virus helicase gene are resistant to virus infection. Virus Research 1998, 53:97-103