Development of a new virus-based vector to control TSWV in tomato plants
- Universidade de Évora(líder)
- Universidade do Algarve(parceiro)
Tomato spotted wilt virus (TSWV) is a virus that causes a disease of major concern in tomato plants all over the world being responsible for extremely significant economic losses in this crop which constitutes 72% of the value of fresh vegetables produced worldwide. So far, virus control strategies rely only on preventive sanitary measures or genetic resistance as antiviral products are not available for field use, however its continuous resurgence and spread into new hosts make essential innovative and efficient means of control.
Virus induced gene silencing (VIGS) allows specific silencing of foreign genes that can be inserted in an optimized virus vector and then inoculated in plants. When a sequence of a viral suppressor gene is introduced in a VIGS vector, the plant infected with this vector will target viral RNA. This will give to the plant a significant advantage in its protection against a possible infection of that virus. The VIGS approach provides the generation of rapid phenotype, no need for plant transformation and can be used for plant protection purposes, at a relatively low cost. Several plant viruses have been used as VIGS vectors however, their large genomes, their difficult manipulation and the reduced number of hosts they infect restrain their use as vectors.
The Alphanecroviruses Olive mild mosaic virus(OMMV) and Olive latent virus 1 (OLV-1) have characteristics that place them as very promising vector tools. Their small genome makes them easy to manipulate, in addition, they are symptomless or cause only mild systemic symptoms in a wide range of crops, which will ease their manipulation into symptomless constructs, and allow their application to a high number of plants. To our knowledge no studies have been done concerning this matter using these small necroviruses.
Objectives, activities and expected/achieved results
O objetivo principal deste projeto é criar, otimizar e estabelecer para usos futuros, um vetor viral baseado em necrovirus, para ser usado como uma ferramenta de proteção de plantas. Como uma utilidade imediata, o vetor VIGS será usado para silenciar TSWV conferindo proteção às plantas sem o recurso à transformação genética.
The research plan will focus on:
1. Construction of OMMV and OLV-1 VIGS vector
OMMV and OLV-1 VIGS vector will be constructed from cDNA clones of viral RNA. Efficiency of the vector will be confirmed using reporter genes following agroinoculation of N. benthamiana 16C plants. The establishment of an OMMV and OLV-1 VIGS vectors will allow its general use in rapid functional analysis studies of a high number of plant species.
2. Tests on optimization and evaluation of vector genetic stability
Vectors will be tested for their ability to maintain the expression of reporter genes.
3. Development of a VIGS vector to induce TSWV silencing
Vectors will be manipulated to include TSWV silencing suppressor genes sequences to be further used as TSWV protection tools. Alternatively, the optimized Tobacco rattle virus vector will be used.
4. Optimization and stability tests of VIGS vector as a protective measure against TSWV
A protective measure against TSWV will be developed based on the silencing of TSWV suppressors. Field trials will also be performed.
In this work, it will be developed a virus based vector carrying TSWV sequences so that it can be used as a plant protection tool, protecting the plant against TSWV, a virus of major concern for tomato producers, through silencing, without the need of plant transformation. Based on this, tomato TSWV-protected plants will be produced.
This has a significant interest in that no antiviral products are available for plant disease management, leaving eradication or prevention through sanitary measures as the only immediate control strategies. Until date, the best control strategy for a viral disease is the introduction of genetic resistance in the plant host. However, the continuous increase in the number of varieties of tomato makes that goal almost impossible.
In addition, the virus construct designed here will be capable of replicating in several plants, being highly efficient in silencing endogens of a high number of plants and will be available for further uses as a VIGS vector, namely studies of silencing and reverse genetics.
We intend to protect the results obtained here by the registration of one European patent concerning the vector carrying TSWV sequences.