Inside or outside? A new collection of Gateway vectors allowing plant protein subcellular localization or over-expression.

Transient expression of proteins based on agro-infiltration techniques has proven very efficient and straightforward to study the intrinsic properties of proteins. The level of protein expression has been enhanced by the use of vector plasmids containing virus-derived sequences and the cloning step has been facilitated by recombination technologies. The pEAQ-HT-DEST series of vectors fulfilling these improvements are vectors of choice. However, they lack the possibility to directly and easily fuse the protein of interest to a fluorescent tag or to address it to the secretion pathway. In the present work we describe the production of 15 pEAQ-HT-DEST1-based plasmids designed to use the Gateway® cloning technology and to generate high levels of fluorescent fusion protein by agro-infiltration, in planta. This collection of plasmids includes binary vectors allowing N-terminal or C-terminal fusion to the bright tags EGFP or TagRFP for cytoplasmic accumulation or secretion and represents therefore a valuable tool for subcellular localization or biochemical studies. A viral protein, the blue fluorescent protein TagBFP, the green fluorescent protein variant T-Sapphire and an Arabidopsis protein were transiently expressed in N. benthamiana to demonstrate the potential of these vectors.

Nanobody-mediated resistance to Grapevine fanleaf virus in plants.

Since their discovery, single-domain antigen-binding fragments of camelid-derived heavy-chain-only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode-transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell-to-cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.

Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus-like particles.

Virus-like particles (VLPs) derived from nonenveloped viruses result from the self-assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self-assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N- and C-terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant-based production of nucleic acid-free VLPs. Remarkably, expression of N- or C-terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.

The 50 distal amino acids of the 2AHP homing protein of Grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis.

Avirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2AHP , particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2AHP fused to an enhanced green fluorescent protein (EGFP) tag in N. occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N. occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression.