An Aphid-Transmitted Virus Reduces the Host Plant Response to Its Vector to Promote Its Transmission.

The success of virus transmission by vectors relies on intricate trophic interactions between three partners, the host plant, the virus, and the vector. Despite numerous studies that showed the capacity of plant viruses to manipulate their host plant to their benefit, and potentially of their transmission, the molecular mechanisms sustaining this phenomenon has not yet been extensively analyzed at the molecular level. In this study, we focused on the deregulations induced in Arabidopsis thaliana by an aphid vector that were alleviated when the plants were infected with turnip yellows virus (TuYV), a polerovirus strictly transmitted by aphids in a circulative and nonpropagative mode. By setting up an experimental design mimicking the natural conditions of virus transmission, we analyzed the deregulations in plants infected with TuYV and infested with aphids by a dual transcriptomic and metabolomic approach. We observed that the virus infection alleviated most of the gene deregulations induced by the aphids in a noninfected plant at both time points analyzed (6 and 72 h) with a more pronounced effect at the later time point of infestation. The metabolic composition of the infected and infested plants was altered in a way that could be beneficial for the vector and the virus transmission. Importantly, these substantial modifications observed in infected and infested plants correlated with a higher TuYV transmission efficiency. This study revealed the capacity of TuYV to alter the plant nutritive content and the defense reaction against the aphid vector to promote the viral transmission.

Bioluminescence Production by Turnip Yellows Virus Infectious Clones: A New Way to Monitor Plant Virus Infection.

We used the NanoLuc luciferase bioluminescent reporter system to detect turnip yellows virus (TuYV) in infected plants. For this, TuYV was genetically tagged by replacing the C-terminal part of the RT protein with full-length NanoLuc (TuYV-NL) or with the N-terminal domain of split NanoLuc (TuYV-N65-NL). Wild-type and recombinant viruses were agro-infiltrated in Nicotiana benthamiana, Montia perfoliata, and Arabidopsis thaliana. ELISA confirmed systemic infection and similar accumulation of the recombinant viruses in N. benthamiana and M. perfoliata but reduced systemic infection and lower accumulation in A. thaliana. RT-PCR analysis indicated that the recombinant sequences were stable in N. benthamiana and M. perfoliata but not in A. thaliana. Bioluminescence imaging detected TuYV-NL in inoculated and systemically infected leaves. For the detection of split NanoLuc, we constructed transgenic N. benthamiana plants expressing the C-terminal domain of split NanoLuc. Bioluminescence imaging of these plants after agro-infiltration with TuYV-N65-NL allowed the detection of the virus in systemically infected leaves. Taken together, our results show that NanoLuc luciferase can be used to monitor infection with TuYV.

[Transmission of plant and vertebrate viruses by arthropods]. / La transmission des virus de plantes et de vertébrés par arthropodes.

Many plant and vertebrate viruses use mobile vectors to be transmitted between hosts. These vectors, mainly arthropods, acquire or inoculate the virus by feeding on plant extract or vertebrate blood. Several virus transmission modes have been characterized based on the tight interactions between the virus and the vector. Some viruses are internalized into cells and migrate through different tissues and organs before being released. In the vector, the virus can replicate in some cases. Other viruses are retained, specifically or non-specifically, on the vector mouthparts. Acquiring knowledge on the molecular mechanisms of virus transmission by arthropods consists in studying (i) virus receptors in the vectors, (ii) the mode of virus uptake into vector cells, (iii) virus localization and transport in the vector, and (iv) viral determinants required for transmission. This review, although non exhaustive, presents a state-of-the-art of plant and vertebrate virus transmission by arthropods, notably by pointing to their similarities and differences.

Impact of Mutations in Arabidopsis thaliana Metabolic Pathways on Polerovirus Accumulation, Aphid Performance, and Feeding Behavior.

During the process of virus acquisition by aphids, plants respond to both the virus and the aphids by mobilizing different metabolic pathways. It is conceivable that the plant metabolic responses to both aggressors may be conducive to virus acquisition. To address this question, we analyze the accumulation of the phloem-limited polerovirus Turnip yellows virus (TuYV), which is strictly transmitted by aphids, and aphid's life traits in six Arabidopsis thaliana mutants (xth33, ss3-2, nata1, myc234, quad, atr1D, and pad4-1). We observed that mutations affecting the carbohydrate metabolism, the synthesis of a non-protein amino acid and the glucosinolate pathway had an effect on TuYV accumulation. However, the virus titer did not correlate with the virus transmission efficiency. Some mutations in A.thaliana affect the aphid feeding behavior but often only in infected plants. The duration of the phloem sap ingestion phase, together with the time preceding the first sap ingestion, affect the virus transmission rate more than the virus titer did. Our results also show that the aphids reared on infected mutant plants had a reduced biomass regardless of the mutation and the duration of the sap ingestion phase.

In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index.

Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family.

The Aphid-Transmitted Turnip yellows virus Differentially Affects Volatiles Emission and Subsequent Vector Behavior in Two Brassicaceae Plants.

Aphids are important pests which cause direct damage by feeding or indirect prejudice by transmitting plant viruses. Viruses are known to induce modifications of plant cues in ways that can alter vector behavior and virus transmission. In this work, we addressed whether the modifications induced by the aphid-transmitted Turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana also apply to the cultivated plant Camelina sativa, both belonging to the Brassicaceae family. In most experiments, we observed a significant increase in the relative emission of volatiles from TuYV-infected plants. Moreover, due to plant size, the global amounts of volatiles emitted by C. sativa were higher than those released by A. thaliana. In addition, the volatiles released by TuYV-infected C. sativa attracted the TuYV vector Myzus persicae more efficiently than those emitted by non-infected plants. In contrast, no such preference was observed for A. thaliana. We propose that high amounts of volatiles rather than specific metabolites are responsible for aphid attraction to infected C. sativa. This study points out that the data obtained from the model pathosystem A. thaliana/TuYV cannot be straightforwardly extrapolated to a related plant species infected with the same virus.

Transmission of Turnip yellows virus by Myzus persicae Is Reduced by Feeding Aphids on Double-Stranded RNA Targeting the Ephrin Receptor Protein.

Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce polerovirus damages.

Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids.

A fluorescent viral clone of the polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmission.

Comparative Analysis of RNAi-Based Methods to Down-Regulate Expression of Two Genes Expressed at Different Levels in Myzus persicae.

With the increasing availability of aphid genomic data, it is necessary to develop robust functional validation methods to evaluate the role of specific aphid genes. This work represents the first study in which five different techniques, all based on RNA interference and on oral acquisition of double-stranded RNA (dsRNA), were developed to silence two genes, ALY and Eph, potentially involved in polerovirus transmission by aphids. Efficient silencing of only Eph transcripts, which are less abundant than those of ALY, could be achieved by feeding aphids on transgenic Arabidopsis thaliana expressing an RNA hairpin targeting Eph, on Nicotiana benthamiana infected with a Tobacco rattle virus (TRV)-Eph recombinant virus, or on in vitro-synthesized Eph-targeting dsRNA. These experiments showed that the silencing efficiency may differ greatly between genes and that aphid gut cells seem to be preferentially affected by the silencing mechanism after oral acquisition of dsRNA. In addition, the use of plants infected with recombinant TRV proved to be a promising technique to silence aphid genes as it does not require plant transformation. This work highlights the need to pursue development of innovative strategies to reproducibly achieve reduction of expression of aphid genes.

A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation.

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RTCter) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RTCter. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells.

Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

Cucurbit aphid-borne yellows virus (CABYV) is a polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

Rack-1, GAPDH3, and actin: proteins of Myzus persicae potentially involved in the transcytosis of beet western yellows virus particles in the aphid.

Beet western yellows virus (BWYV) is a Polerovirus that relies on the aphid Myzus persicae for its transmission, in a persistent-circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary glands (ASG) epithelial barriers in a transcytosis mechanism where vector receptors interact with virions. In this paper, we report in vitro interaction experiments between BWYV and aphid components. Using the M. persicae clone from Colmar, we showed that a set of aphid polypeptides, separated by SDS-PAGE or 2D electrophoresis (2DE), can bind in vitro to purified wild type or mutant particles. Using subcellular fractionation, we showed that the 65-kDa polypeptide identified as symbionin is a soluble protein whereas the other polypeptides seem to be associated more or less strongly to the membrane. We hypothesize that three polypeptides, identified by mass spectrometry as Rack-1, GAPDH3, and actin, may be involved in the epithelial transcytosis of virus particles in the aphid vector.