Plant virus control employing RNA-based vaccines: A novel non-transgenic strategy FA 0806 Start date: 30/03/2009 End date: 29/03/2013 Year: 2 nd Andreas Voloudakis Chair of the Action Agricultural University of Athens / HELLAS
Scientific context and objectives (1/2) • Background / Problem statement Develop suitable, efficient and cost-effective methods for reactive and proactive response to viral diseases of plants for a sustainable Agriculture. • FA 0806 Mo. U objectives: 1. Co-ordinate frontier European research on plant virus control through gene silencing approaches. 2. Develop novel non-transgenic control strategies for managing plant viral diseases in Europe. 3. Optimize protocols for high-throughput production and delivery of suitable resistance inducer molecules. 4. Transfer innovations to end-user groups (i. e. SMEs, plant protection organizations, growers’groups). 5. Disseminate results via publication of all meeting proceedings, developing a web site with bibliography and announcements for long-term networking. 2
Scientific context and objectives (2/2) • Research directions: Approach and methodology a) To design & produce different molecules (nucleic acid forms) to be used for the induction of virus resistance in plants Monitor the outcome of the application of the produced material in small scale (laboratory) trials b) To establish a high throughput production method of the selected molecules (nucleic acid forms) Evaluate the efficacy of the produced material in large scale (greenhouse and field) trials Innovative work within the Action a) Produce efficient, cost-effective inducer of the plant resistance nucleic acid forms b) Improve the delivery machinery of the selected nucleic acid forms 3
Working groups 1. Working group (WG 1) Development of novel non transgenic strategies for plant virus control NA R ds phi 6 2. Working group (WG 2) Application (small & large scale) of novel non transgenic strategies for plant virus control 3. Working group (WG 3) Socio-economic aspects of the novel application methods 4
Action Parties Grant Holder: GH Institution - AUA GH Representative Dr G. Papadakis (Vice Rector) GH Country - HELLAS 5
Action participants 6
Use of COST Instruments Activity (No. ) Year 1 Year 2 MC/WG Meetings 2 2/2* STSMs 4 5/7/10** Training Schools 2 1/1*** 0/0 12/59 3/46 Workshops or Conferences Joint Publications Year 3 Year 4 * Other meeting (Steering committee or Core group meeting) ** done/applications/estimated *** Trans COST activity 7
Results vs. Objectives • Co-ordinate frontier European research on plant virus control through gene silencing approaches (Objective 1) • Various ds. RNA molecules targeting the genomes of selected plant pathogenic viruses are under development (Objective 2) • Different production methods (3 in vitro and 1 in bacteria) have been used for the production of ds. RNA molecules (Objective 2) • Updating of the FA 0806 web site, announcement of STSM calls, development of Action’s poster (Objective 5) • 1 training schools (T. S. ) +1 trans-COST T. S. completed (Objective 5) • EU proposals with participants from FA 0806 were submitted: in FP 6 and FP 7. – Not successful until now … Other national applications were made in 2009 (Objective 5) 8
Results vs. Objectives • An improved device for biolistic introduction (Sikorskaite et al. , 2010) • A simplified and low-cost method for biolistic transfection of herbaceous experimental plants • Genetic diversity and evolution of the main viral pathogens - of sweet potato in East Africa - of Plum pox virus, Watermelon mosaic virus, Zucchini yellow mosaic virus and Wheat streak mosaic virus • A microarray-based method for the fast analysis of secondary structures of large RNA molecules • Short RNA profile in the Arabidopsis-Oilseed rape mosaic virus • Artificial mi. RNAs employed to mediate resistance against Potato spindle tuber viroid • Expression of entire metabolic pathways with the IL-60 system • Suppressor of PTGS was identified in the Grapevine leafroll associated virus 3 9
Innovative knowledge in FA 0806 • New composite RNA-based vaccines were designed and produced against Cauliflower mosaic paparetrovirus (Ca. MV) • RNA-based vaccines derived from selected viral sequences were developed against the EU quarantine virus: Citrus Tristeza virus (CTV) • An in vivo (in specialized bacterial cells) ds. RNA production system was optimized • Preparation of small RNA libraries from tobacco plants after the application of a Cucumber mosaic virus (CMV)-derived RNA-based vaccine • Standardization of RNA extraction protocols to obtain good quality RNA and analysis of virus-derived small RNAs from banana plants infected with Banana streak virus • Characterization of the Sweet potato chlorotic stunt virus (SPCSV) RNase 3 • Molecular characterization of vsi. RNAs to elucidate the function of the vsi. RNAs in the N. benthamiana-CMV pathosystem 10
Significant scientific breakthroughs • Extend the application methodology in other plant-virus interactions & to suppress gene expression of endogenous plant genes (i. e. GFP transgene) • ESR training: Training schools (1+1) & open call STSMs • Trans. COST activity (FA 0806, TD 0801, FA 0604, Triticeae. Genome) – Training school “Applied Bioinformatics in Plant Sciences” • Interaction with other EU consortia: Shar. CO, Triticeae. Genome, SIROCCO • Dr V. Pantaleo (MC member of FA 0806) 1 st prize ESF-EMBO Symposium 11
Socio-economic impacts Achieved • The application of the novel method is communicated within the scientific community (WG meetings, interaction with other consortia, etc) • Contacts with stakeholders Expected • Dialogue workshop “Workshop on Value Communication in the Field of Novel Biotechnological Methods”. A COST Trans-Domain activity • National publications will be attempted in the next Grant system year • The Economics of the novel methodology are under investigation 12
Spin-off proposals/projects • Research work in FA 0806 members’ laboratories is supported by several national and international funding agencies - EU proposals with participants from FA 0806 were submitted in FP 7 • CNRS-AUA-Syngenta “Application of ds. RNA vaccination for non-transgenic RNA silencing in crops” • Bilateral (Slovenia-Poland) collaboration 13
Inter-disciplinary networking • FA 0806 members were supported by the Action to attend international meetings in order to bring new interdisciplinary information to the Action - The International BARD Workshop, Eilat (IL) (March 14 -17, 2010) “Plasmodesmata 2010”, Sydney (AU) (21 -26 March 2010) ESF-EMBO, Sant Felieu (ES) (30 May-4 June 2010) EMBO Workshop, Fenestrelle (IT) (12 -16 June 2010) 11 th International Plant Virus Epidemiology Symp. , Ithaca (USA) (20 -24 June, 2010) • Trans-COST training school (FA 0806, TD 0801, FA 0604, Triticeae. Genome) “Applied Bioinformatics in Plant Sciences” (December 2010) • Trans-COST Domain Workshop (3 -4 November 2011) entitled “Workshop on Value Communication in the Field of Novel Biotechnological Methods” • Trans-COST Domain training school (May 2012) in Bioinformatics • Several FA 0806 members are members of other COST Actions and EU consortia • New members, non. COST participants & Contacts with stakeholders 14
New networking • New MC members & MC substitutes, new WG members • New non. COST participants • Training schools (1+1) - “small RNA cloning” jointly w/ SCIROCCO - “Applied Bioinformatics in Plant Sciences” Trans. COST activity (FA 0806, TD 0801, FA 0604, Triticeae. Genome) • Contacts with stakeholders 15
“Workshop on Value Communication in the Field of novel Biotechnologies” - A trans-COST activity • Why is this needed? Develop communication strategies for novel biotechnological methods § New biotech methods are perceived as “loaded” with risks and controversially discussed by the public Public perception § Consumer psychology, perception of risks, consumer trust, agricultural ethics Market research § Marketing analysis, corporate and marketing communication Discourse strategies § Risk communication, planning of large scale social interventions, application of social psychological know-how in the fields of consumption and new technologies • Interested parties COST FA 0806 (www. aua. gr/COSTFA 0806) - Application of protective molecules in Agriculture ( COST FA 0804 (molfarm. ueb. cas. cz) - Molecular Farming ( COST FP 0905 (www. cost-action-fp 0905. eu) - Biosafety of forest transgenic trees ( • Expectations: Length of T. S. : 2 days # Participants: 25 -30 # Invited speakers: 6 -7 place: Munich (DE) time: 3 -4 November 2011 16
Training School in “Applied Bioinformatics …” A trans-COST activity • Why is this needed? New Generation Sequencing technologies § 454 -Pyrosequencing, Solexa-Ilumina, SOLID, HELICOS, Pacific Biosystems Handling of high throughput sequencing data § Quality Analysis, Mapping vs Reference Genome, Clustering and Assembly Statistical analysis of high throughput sequencing data § Peak Detection, Noise Reduction, Cross Validation with other Experiments Comparative Genomics • Interested parties COST FA 0806 (www. aua. gr/COSTFA 0806) - Deep sequencing data, small RNA profiling ( COST TD 0801 (ww. statseq. eu) - Statistical analysis and integration of sequence data ( COST BM 1006 (ww. seqahead. eu) - Next Generation Sequencing Data Analysis Network ww. seqahead. eu • Expectations: Length of T. S. : 5 days # Trainees: 25 -30 # Trainers: 4 -5 place of T. S. : Upsala (SE) time of T. S. : mid May 2012 The course will take place in connection to a more advanced workshop. 17
Self evaluation, Strengths and Weaknesses Strengths • FA 0806 has fulfilled all millstones set in Mo. U for the respective period • New MC members & MC substitutes, new WG members • Interaction between participants was maximized (joint activities) • FA 0806 poster was presented at International conferences • ESR training: Training schools (1+1) & open call STSMs Weaknesses – GRANT HOLDER system • Some delays in administrative implementation of COST rules (e. g. payments, reporting) • Extra paperwork required by the Grant Holder • Extra work load for the Action Chair 18
Grazie Thank you Dank u Takk Blagodarya Kiitos Aitäh Dakujem Hvala Köszönöm Gracias Obrigado Dzieki Merci Tak Efcharisto Tack Dankë Merci Dankë Toda
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