Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component

Virus‐induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite‐like and single‐stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication‐associated protein open reading frame and the A‐rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene (sulphur, Su) or transgene (green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co‐agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co‐agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene‐silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes (NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants.     

A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development

Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.     

Efficient virus-induced gene silencing in Arabidopsis

Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.     

Delivery of macromolecules to plant parasitic nematodes using a tobacco rattle virus vector

Plant parasitic nematodes cause significant damage to crops on a worldwide scale. These nematodes are often soil dwelling but rely on plants for food and to sustain them during reproduction. Complex interactions occur between plants and nematodes during the nematode life cycle with plant roots developing specialized feeding structures through which nematodes withdraw nutrients. Here we describe a novel method for delivering macromolecules to feeding nematodes using a virus-based vector [tobacco rattle virus (TRV)]. We show that the parasitic nematode Heterodera schachtii will ingest fluorescent proteins transiently expressed in plant roots infected with a TRV construct carrying the appropriate protein sequence. A prerequisite for this delivery is the presence of replicating virus in root tips prior to the formation of nematode-induced syncytia. We show also that TRV vectors expressing nematode gene sequences can be used to induce RNAi in the feeding nematodes.     

Transient expression of human papillomavirus type 16 virus-like particles in tobacco and tomato using a tobacco rattle virus expression vector

The major capsid protein L1 of human papillomavirus type 16 (HPV16) was transiently expressed in tobacco (Nicotiana benthamiana and Nicotiana tabacum) and tomato (Lycopersicon esculentum) leaves using Agrobacterium tumefaciens. The expression vector pTV00 was derived from tobacco rattle virus (TRV). The highest L1 expression 15 μg g⁻¹(f.m.) was achieved when the coding sequence of L1 was optimized for expression in humans that caused an increase of the guanine and cytosine (GC) content from 38.2 % in wild type HPV16 to 64.1 % in optimized sequence. L1 monomers readily self-assembled into capsomeres and further into virus like particles (VLPs). Immunological characterization and electron microscopy showed that 89 % of L1 retained VLP structure also in extracts prepared from freeze-dried leaves. Plant expressed L1 in crude extracts was highly immunogenic without any additional adjuvant as vaccinated mice developed strong humoral and cellular immune response, comparable to that elicited by purified VLPs derived from insect cells. Further, the induced antibodies effectively neutralized infection of 293TT cells with pseudovirions. This finding demonstrates that the TRV expression system is comparable to other plant expression systems and due to the broad host range of TRV is particularly attractive when expression in plants with low content of toxic alkaloids is desired. Moreover, a monoclonal anti-L1 antibody E2 raised in the course of immunization with crude extract from freeze-dried leaves expressing L1 is specific preferentially against HPV VLPs and could be used in direct ELISA for monitoring of VLPs assembly and VLP purification protocols.     

Virus‐induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus‐based virus‐induced gene silencing (VIGS) approach, involving an Agrobacterium‐mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic‐mediated approach of inoculation of virus‐encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down‐regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45–90%), similar to results obtained via agro‐transformation. Thus, the biolistic‐based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium‐based approach, e.g. when difficulties arise with agro‐inoculations or when Agrobacterium‐free procedures are required to avoid plant defence responses.     

Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants

Virus-induced gene silencing (VIGS) is an effective tool for studying the functions of plant genes, but only a few VIGS vectors available for woody plants were reported so far. Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV) vectors. We first tested whether the TRV-vector can be directly applied to infect woody plant species, such as Vernicia fordii, Populus tomentosa Carr. and Camellia oleifera. The results revealed that TRV-mediated VIGS could be effectively elicited in V. fordii, weakly in P. tomentosa Carr., but not in C. oleifera. TRV-based VIGS vectors with heterologous phytoene desaturase (PDS) sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benthamiana and V. fordii. The photobleached leaf phenotype of silenced plants significantly correlated with the down-regulation of endogenous PDS as compared with controls. To further confirm the reliability of VIGS in V. fordii, we also isolated the cloroplastos alterados 1 gene from P. tomentosa Carr., and the silencing pheotypes of albino leaves were observed in V. fordii 2 weeks after inoculation using a heterologous TRV-based VIGS system. Taken together, we have successfully developed an Agrobacterium-mediated VIGS assay in V. fordii and demonstrated that V. fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.     

RNA 2 of tobacco rattle virus strain TCM encodes an unexpected gene.

The sequence of the 3'-terminal 1210 nucleotides of RNA 1 and the complete sequence of 3389 nucleotides of RNA 2 of tobacco rattle virus (TRV) strain TCM has been deduced. The sequence of the 3'-terminal 1099 nucleotides of RNAs 1 and 2 was found to be identical. Thus the genome of this TRV strain is partially diploid, encoding a 16K protein in both RNA 1 and RNA 2. The sequence that is unique to RNA 2 contains two open reading frames: the coat protein cistron and a cistron for a 29.1K protein, which shows no homology with the RNA 1 encoded 28.8K protein. cDNA probes corresponding to these two open reading frames cross-hybridized to pea early-browning virus RNA 2, but not to RNA 2 of five other tobraviruses tested.     

Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species

Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.     

Inheritance of insensitivity to tobacco rattle virus in potatoes

The sensitivity of 20 genotypes from each of 13 progenies of parental potato clones was assessed using recently developed glasshouse testing procedures. Significant heritable differences between the progenies with respect to their degree of sensitivity to TRV were observed. Significant general and specific combining abilities were also demonstrated. The results are discussed in relation to modes of inheritance of this trait. The results indicate that though a major gene for TRV insensitivity may be operating this does not explain all the observed variation and it is suggested that there is also a system inherited in a polygenic manner that can confer good levels of insensitivity to TRV.     

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus

Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.