Establishment of functional genomics in Vitis ssp. is still challenging, due to the lack of reliable high-throughput tools for grapevine transformation. Virus-induced gene silencing (VIGS) makes use of a plant virus-based vector carrying a sequence of an endogenous plant gene. By delivery of the vector and replication of the recombinant virus within the plant, the plant defence mechanism known as post-transcriptional gene silencing (PTGS) is activated against the virus resulting in silencing of the plant gene. This system has been recently applied on Vitis vinifera plants with vectors based on the Grapevine Virus A (GVA) and Grapevine Leafroll-Associated Virus-2 (GLRaV-2) to silence endogenous reporter genes. We developed a new VIGS system using a vector based on Grapevine Algerian Latent Virus (GALV), a member of the genus Tombusvirus, first isolated from an Algerian vine without any relevant symptom. Preliminary infections revealed that GALV infective transcripts could replicate and spread systemically in grapevine. The ability of the GALV-based VIGS vector to replicate and induce silencing against target genes was first confirmed in N. benthamiana introducing a portion of the phytoene desaturase (PDS) coding region into the viral vector. Preliminary results of Agrobacterium-mediated GALV infections in grapevine have been obtained. In particular, the presence of a ribozyme sequence immediately downstream of the viral sequence was crucial for viral replication. The GALV-based VIGS vector could be precious for grapevine functional genomics as it is a systemic and latent grapevine virus with the positive features of tombusviruses. Indeed they are one the most extensively studied messenger-sensed RNA plant viruses, showing a broad experimental host range and a rapid and strong viral replication and expression. These characteristic have contributed to the successful use of different Tombusvirus vectors for developing plant VIGS systems.
Development of a new VIGS vector for grapevine based on Grapevine Algerian Latent Virus.
LOVATO, Arianna;PEZZOTTI, Mario;POLVERARI, Annalisa
2013-01-01
Abstract
Establishment of functional genomics in Vitis ssp. is still challenging, due to the lack of reliable high-throughput tools for grapevine transformation. Virus-induced gene silencing (VIGS) makes use of a plant virus-based vector carrying a sequence of an endogenous plant gene. By delivery of the vector and replication of the recombinant virus within the plant, the plant defence mechanism known as post-transcriptional gene silencing (PTGS) is activated against the virus resulting in silencing of the plant gene. This system has been recently applied on Vitis vinifera plants with vectors based on the Grapevine Virus A (GVA) and Grapevine Leafroll-Associated Virus-2 (GLRaV-2) to silence endogenous reporter genes. We developed a new VIGS system using a vector based on Grapevine Algerian Latent Virus (GALV), a member of the genus Tombusvirus, first isolated from an Algerian vine without any relevant symptom. Preliminary infections revealed that GALV infective transcripts could replicate and spread systemically in grapevine. The ability of the GALV-based VIGS vector to replicate and induce silencing against target genes was first confirmed in N. benthamiana introducing a portion of the phytoene desaturase (PDS) coding region into the viral vector. Preliminary results of Agrobacterium-mediated GALV infections in grapevine have been obtained. In particular, the presence of a ribozyme sequence immediately downstream of the viral sequence was crucial for viral replication. The GALV-based VIGS vector could be precious for grapevine functional genomics as it is a systemic and latent grapevine virus with the positive features of tombusviruses. Indeed they are one the most extensively studied messenger-sensed RNA plant viruses, showing a broad experimental host range and a rapid and strong viral replication and expression. These characteristic have contributed to the successful use of different Tombusvirus vectors for developing plant VIGS systems.
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