The GLRaV-2-derived gene expression and VIGS vector described here has the potential to revolutionize grapevine biotechnology along three avenues of research and application. The first avenue is the investigation of virus-host interactions empowered by the ability to track virus infection. Our first glance at a pathway of virus transport already yielded surprises, including the virus invasion of the roots and berries and exit from the phloem to the mesocarp (). Because GLRaV-2 is strictly limited to phloem in other plant organs, this observation suggests the distinct nature of the plasmodesmata that interconnect berry phloem and mesocarp cells and are involved in pumping sugars. Therefore, the virus follows the entire pathway of sugar transport from leaves to roots to fruit, providing a tool for investigating this pathway via recombinant protein expression or RNAi.
The ability to follow virus transmission will shed new light on the regulation of virus-host interactions in a woody plant and facilitate the identification of the virus genes involved in these interactions. Our recent work already revealed novel roles for proteases that affect GLRaV-2 invasiveness in grapevine (33
) and suggested such roles for the AlkB RNA repair enzyme encoded by many viruses of woody plants, including Grapevine leafroll-associated virus-3
The second biotechnology avenue opened up by viral vectors is the functional genomics of grapevine using VIGS. The last decade has witnessed an explosion in the use of VIGS (2
). This facile technology is particularly important for plants with long life cycles, such as trees and vines. However, VIGS in woody plants is in its infancy and is trying to find its path through the thicket of technical problems, including the scarcity of known tree-infecting viruses and the vagaries of reintroducing vectors to these well-protected hosts. The VIGS demonstrated here for grapevine () opens immense opportunities for the functional mapping of the grapevine genome (28
). Some of the important research areas include (i) the investigation of the sugar transport (30
); (ii) metabolomics aimed at improving nutritional, medicinal, and wine-making qualities (50
); and (iii) mapping disease resistance and susceptibility genes to control pathogens (18
A third avenue of vector utilization is disease protection. The RNAi-susceptible pathogens, such as viruses, fungi, and invertebrate pests, are potential targets for VIGS. Previously, transgene-triggered RNAi was used to control plant-parasitic insects (3
), nematodes (27
), and viruses (24
). These approaches, although promising, involve labor-intensive GMO technology that is often faced with vocal opposition. In contrast, we have shown here that the RNA virus vector does not integrate into the grape genome, which is in full accord with the absence of any RNA virus sequences in the grapevine chromosomes despite the prolonged exposure of this plant to virus infections (6
Perhaps the closest analogy to RNA vector technology applied to disease resistance is provided by vaccination against human diseases such as poliomyelitis (32
). In both cases the genetically modified, live, attenuated RNA viruses are used to protect the host. Indeed, the insertion of an expression cassette into GLRaV-2 resulted in attenuated symptoms (D) of the already mild disease present and often tolerated throughout grapevine-growing areas. On the other hand, the poliovirus vaccine can protect only against poliomyelitis, whereas VIGS vectors can be used against a broad range of plant pathogens and pests.
The GLRaV-2 vector has several important advantages over other plant virus vectors. Whereas most of these vectors tolerate relatively small inserts (19
), our vector can accommodate inserts at least ~2 kb in size (A). This genetic capacity enables the simultaneous targeting of several pathogens by engineering VIGS cassettes harboring multiple RNAi triggers. The GLRaV-2 vector is also durable for more than a year versus days or weeks, which is typical of other vectors. The only vector comparable to GLRaV-2 in this respect is the Citrus tristeza
virus vector that is derived from a related closterovirus (11
). However, this promising vector could raise biosafety concerns due to potential escape via aphid transmission. In contrast, GLRaV-2 is transmissible only by grafting, thus minimizing the risks of inadvertent escape.
Why closterovirus-derived vectors exhibit a remarkably higher genetic stability compared to their competition from other lineages of plant RNA viruses is an intriguing evolutionary question. GLRaV-2 replication is prone to point mutations or deletions (this work), whereas the recombination-mediated occurrence of defective RNAs and chimeric isolates is well documented in diverse closteroviruses (47
). It is possible, however, that (yet uncharacterized) mutation or recombination rates in closteroviruses are lower than in other RNA viruses. This notion is supported by evidence for the proofreading ability of RNA polymerases in coronaviruses, a family of animal viruses whose large RNA genome evolution shows some analogies to closteroviruses (13
). A second distinct possibility is that the Hsp70 chaperone-aided assembly of the filamentous closteroviruses (1
) alleviates selection for shorter genomes that is typical for spherical or rod-shaped viruses. Finally, as proposed earlier (13
), the acquisition of strong RNAi suppressors (34
) and leader proteases (33
) that counteract distinct host defense mechanisms preconditioned closteroviruses for evolving larger genomes and increased their tolerance to accommodating larger genetic loads.
In conclusion, the GLRaV-2 vector provides a platform for multiple applications in grapevine functional genomics, biotechnology, and pathogen control. It also paves the way for generating analogous vectors for other woody plants used in agriculture, forestry, or the biofuel industry.