Nuova piattaforma tecnologica per lo studio dell'interazione tripide-tospovirus: sviluppo di un sistema a genetica inversa per Tospovirus e uso della tecnologia siRNA per l'analisi genomica funzionale dei tripidi

Responsabili di progetto

Massimo Turina, Renato De Oliveira Resende

Accordo

BRASILE - CNPq - Conselho Nacional Desenvolvimento Cientifico y Tecnologico

Bando

CNR/CNPq 2012-2013

Dipartimento

Agroalimentare

Area tematica

Scienze bio-agroalimentari

Stato del progetto

Nuovo

Proposta di ricerca

Introduction
The genus Tospovirus is a limiting factor for the economic feasibility of a number of horticultural crops, causing significant yield losses in crops such as tomato, pepper, tobacco, potato, onions and in ornamental crops. This genus belongs to the family Bunyaviridae, which includes four other genera, able to infect animals and  humans. All Tospoviruses are characterized by three genomic segments (L, M and S) of single strand RNA (ssRNA), of negative (L) or ambisense polarity (M and S). Viruses are transmitted in a persistent and propagative manner exclusively by thrips (Thysanoptera:Thripidae). Currently thirteen species have been identified as vectors of tospoviruses. The first instar-larvae acquire the virus from infected plants upon ingestion, virions travel into the midgut where virus replicates, spreads and infects muscle cells and salivary glands and adults transmit the virus from plant to plant by the injection of viruliferous saliva in plant tissues. The project will explore the feasibility of new technological platforms to study tospovirus-thrips interactions using as model system Tomato spotted wilt virus (TSWV), still the most detrimental tospovirus worldwide, and Frankliniella occidentalis, its main insect vector species. A better knowledge of the virus-vector relationship and acquisition/transmission determinants is of strategic importance in order to implement new control strategies against thrips and tospoviruses.
Establishing a proof of concept reverse genetic system for Tospoviruses    The main obstacle to a thorough study of Tospovirus molecular biology is the lack of a reverse genetic system for this viral genus, although such system is available for other members of the Bunyaviridae infecting humans and animals. A critical step in the development of a reverse-genetic system for TSWV is the possibility to express in planta viral-like genomic RNAs, i.e. without the 5’GMet-cap and the polyA-tail, which are instead general characteristics of plant mRNAs. We will use two different strategies for “in cell” viral-like RNA production:
i)   First, we will use a recombinant plasmid carrying the cDNA of interest between the S35 promoter from CaMV and Nos terminator. The cDNA sequence will be flanked by two ribozyme sequences at the 5’ and 3’ borders for generation of viral-like RNAs with the exact ends by auto-catalysis.
ii)   An alternative approach, already used for animal Bunyaviruses, is to generate a plasmid expressing viral RNA with the phage T7 promoter for RNA transcription by T7 phage polymerase. The T7 phage polymerase will be expressed transiently through agroinfiltration or transgenically.
Once one of such constructs is obtained, it can be agro-infiltrated in Nicotiana benthamiana. At the same time, on the same leaves, we will inoculate wild type TSWV. In order to facilitate the trans-replication of the genomic RNA derived from cDNA, we will use a selection method so far never implemented: we will clone cDNA of the S or M segment from RB strains (carrying the determinants for the RB phenotype, respectively from TSWV infecting resistant pepper or resistant tomato) and we will express such RNA in N. benthamiana transiently; then we will mechanically inoculate sap from locally infected and agroinfiltrated N. benthamiana on resistant pepper or tomato.  In this way we will have a TSWV wild type infection in cells where the S (or M) segment of an RB strain of TSWV is expressed and derived from cDNA. We then will mechanically inoculate sap from N. benthamiana tissue to resistant pepper in order to select the viral population which has a reassorted genome: the S (or M) segment will be from the RB strains and will be provided from cDNA, the other segments will be provided from the wild type viral strain. Once such reverse genetic system is established, chimeric cDNA and site directed mutagenized cDNA of the S (or M) segment will be engineered and each construct will be evaluated for its ability to systemically infect resistant pepper and for its relationship with the insect vector.  The result of this preliminary work will allow us to develop a novel approach for a “pilot” reverse genetic system for tospoviruses.
Use of RNAi technology for functional genomic application in the thrips vector   A great contribution to the study of genes involved in the tospovirus-vector relationship could derive from the development of the RNA-interference (RNAi) technology for the Thripidae family, particularly taking into account that a cDNA library and RNAseq data are available and the F. occidentalis genome is likely to be sequenced in the next future.  RNAi has been widely used in several insect species, including vector of plant diseases, for functional analysis of target genes.  Overall, these studies provided the basis for possible practical applications by developing RNAi-based strategies for defence of plants against insect pests. RNAi was first applied to insects through injection of dsRNA of a selected number of genes which provided proof of concept studies demonstrating gene down regulation and its potential insecticidal use. Oral delivery of dsRNA molecules through artificial diet also resulted in specific down regulation of genes, but mostly limited to epithelial cells in the gut lumen: in facts, some authors showed that siRNA technology through oral delivery seems to be confined locally in the insect, likely because homologous of RdRp are absent from a number of sequenced insect species; nevertheless, possible alternative siRNA amplification-transport systems have been hypothesized. A further step for oral delivery of molecules with the potential to stimulate siRNA response is the production in planta of dsRNAs or hairpin RNAs, often through production of transgenic plants or through transient expression through local agroinfiltration. Another technique recently applied to stimulate specific RNAi in insect is the use of VIGS (viral induced gene silencing): plant virus vectors are engineered to express fragments of specific insect genes; accumulation of dsRNA during the replication cycle in plants will stimulate production of siRNA specific to the insect gene; acquisition of the dsRNA or the siRNA through feeding on the part of the insect will induce specific knock down of insect genes at least in the gut epidermis; effects of such knock down on insect fitness were demonstrated in a number of species. This last technique has the advantage of a systemic infection of the plant, allowing at the same time its use for high throughput screen necessary in functional genomic analysis.
So far RNAi technique for thrips is not available. Given the extremely small size of the thrips species involved in vectoring plant viruses, lack of a transgenic system for this insect order, and the fact that thrips do not complete their cycle on artificial diet, delivery of dsRNA molecules can only be envisioned through direct feeding on plants (or leaf discs) expressing such RNAs. Our previous experience with viral vectors, will allow the set up of a pilot system using TMV and PVX vectors.
General References for Thrips and Tospoviruses
Goldbach, R. and D. Peters (1996). Molecular and Biological Aspects of Tospoviruses. The Bunyaviridae. R. M. Elliot. New York, Plenum Press: 129-157.
Hogenhout, S. A., E. D. Ammar, et al. (2008). "Insect vector interactions with persistently transmitted viruses." Annual Review of Phytopathology46: 327-359.
Jones, D. R. (2005). "Plant viruses transmitted by thrips." European Journal of Plant Pathology113(2): 119-157.
Moritz, G., S. Kumm, et al. (2004). "Tospovirus transmission depends on thrips ontogeny." Virus Research100(1): 143-149.
Mound, L. A. and D. C. Morris (2007). "The insect order Tysanoptera: Classification versus systematics."Zootaxa(1668): 395-411.
Pappu, H. R., R. A. C. Jones, et al. (2009). "Global status of tospovirus epidemics in diverse cropping systems: Successes achieved and challenges ahead." Virus Research141(2): 219-236.
Rotenberg, D. and A. E. Whitfield (2010). "Analysis of expressed sequence tags for Frankliniella occidentalis, the western flower thrips." Insect Molecular Biology19(4): 537-551.
Whitfield, A. E., D. E. Ullman, et al. (2005). "Tospovirus-thrips interactions." Annual Review of Phytopathology43: 459-489.
 References for reverse genetic systems in Bunyaviridae
Brennan, B., S. R. Welch, et al. (2011). "Creation of a Recombinant Rift Valley Fever Virus with a Two-Segmented Genome." Journal of Virology85(19):10310-10318.
Flick, R. and R. F. Pettersson (2001). "Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs." Journal of Virology75(4):1643-1655.
Habjan, M., N. Penski, et al. (2008). "T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus." Journal of General Virology89:2157-2166.
Kohl, A., T. J. Hart, et al. (2004). "A Bunyamwera virus minireplicon system in mosquito cells." Journal of Virology78(11):5679-5685.
Lowen, A. C., C. Noonan, et al. (2004). "Efficient bunyavirus rescue from cloned cDNA." Virology330(2): 493-500.
Margaria, P., M. Ciuffo, et al. (2007). "Evidence that the nonstructural protein of Tomato spotted wilt virus is the avirulence determinant in the interaction with resistant pepper carrying the Tsw gene." Molecular Plant-Microbe Interactions20(5): 547-558.
Neumann, G., M. A. Whitt, et al. (2002). "A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned?" Journal of General Virology83:2635-2662.
Nguyen, H. T., S. Leelavathi, et al. (2004). "Bacteriophage T7 RNA polymerase-directed, inducible and tissue-specific over-expression of foreign genes in transgenic plants." Plant Biotechnology Journal2(4): 301-310.
  References for RNAi in insects
Baum, J. A., T. Bogaert, et al. (2007). "Control of coleopteran insect pests through RNA interference." Nature Biotechnology25(11): 1322-1326.
Bettencourt, R., O. Terenius, et al. (2002). "Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos." Insect Molecular Biology11(3): 267-271.
Gordon, K. H. J. and P. M. Waterhouse (2007). "RNAi for insect-proof plants." Nature Biotechnology25(11): 1231-1232.
Huvenne, H. and G. Smagghe (2010). "Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review." Journal of Insect Physiology56(3): 227-235.
Jaubert-Possamai, S., G. Le Trionnaire, et al. (2007). "Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum." Bmc Biotechnology7.
Lindbo, J. A. (2007). "High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors." Bmc Biotechnology7.
Mutti, N. S., Y. Park, et al. (2006). "RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum." Journal of Insect Science6.
Price, D. R. G. and J. A. Gatehouse (2008). "RNAi-mediated crop protection against insects." Trends in Biotechnology26(7): 393-400.

Obiettivi della ricerca

 The project will be conducted by two research units in Italy, lead by the Plant Virology Institute (IVV) at CNR, combining complementary expertise on this specific topic such as the molecular know-how for implementing all the cloning steps and deal with the virological aspects of the project, and the traditional entomological expertise necessary for implementing the experiments dealing with the RNAi part of the project.The partners of the bilateral project in Brasil cover a very wide array of expertise, often complementing the ones in Italy: they have the tools and expertise for studying the cellular aspects of viral replication in the insect, and a very strong group dealing with expression of heterologous constructs in transgenic plants based on a general acceptance of the transgenic approach for plant defence supported by enabling legislation and possible market application in Brasil.
Specific objectives:
A)      Set up of a proof of concept reverse genetic system for Tospoviruses
i)    set up a pilot experiment with each of the two types of constructs to derive virus-like genomic RNA from cDNA “in planta” as described in detail in the other sections of the project
ii)   set up the “forced” reassortment through selection on resistant pepper and tomato varieties of mixed populations of genomic segments
iii)  locate specific RB mutations through site directed mutagenesis of the S or M genomic cDNA segments
 
B)      Set up of an RNAi system for functional genomics analysis of Thryps
i)    We will set up and RNAi system for thrips through expression of insect genes fragments through VIGS for an initial hi-throughput  screen of candidate “insecticidal” gene constructs.
ii)    Develop transgenic tomato and pepper plants with specific anti-thrips “insecticidal” properties
 

Ultimo aggiornamento: 03/05/2024