Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.ES and WAM were financed through a Gutenberg Chair (Région Alsace) grant awarded to WAM. Work in the AEF lab was funded by grants from the Wellcome Trust (088789) and the UK Biotechnology and Biological Research Council (BBSRC) (BB/J007072/1 and BB/J015652/1). WAM was also funded by a Fulbright Foundation Research Scholarship and grant number 5R01GM067104-09 from the NIH Institute of General Medical Sciences.This is the final version. It was first published by PLOS at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004868#ack

The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD

A fluorescent infectious clone of Turnip yellows virus to study the effect of aphid infestation on virus distribution in planta

BGPI : équipe 2Turnip yellows virus (TuYV) is a polerovirus (family Luteoviridae) restricted to phloem tissue and obligatorily transmitted by aphids in a circulative and non-propagative mode. This virus infects several plant species of economic importance. The icosahedral viral particles contain a single positive strand RNA genome. In order to track virus movement in plants and to measure the impact of aphids presence on virus distribution in plants, a GFP-tagged virus was engineered (TuYV-GFP). In this construct, the GFP sequence was inserted in a non-structural protein sequence involved, to some extent, in virus movement. The fluorescent signal is expected to label virus replicating cells. The TuYV-GFP sequence was introduced into a binary vector to inoculate several plant species by agro infiltration. TuYV-GFP was able to replicate and to move in A. thaliana, N.benthamiana and M. perfoliata. Accumulation of TuYV-GFP was however reduced when compared to the wild type virus. The impaired ability of the recombinant virus to reach efficiently non inoculated leaves is likely due to the insertion of the GFP sequence in the viral genome. The TuYVGFP genome was stable in N. benthamiana while it was subjected to partial deletions in A. thalianaand in M. perfoliata. Nevertheless, we observed fluorescent phloem cells in non-inoculated leaves of the three plant species. Importantly, fluorescent phloem cells were also observed after virus acquisition and inoculation by aphids, both in inoculated and non-inoculated leaves. This major breakthrough in the study of poleroviruses persistently transmitted by aphids will now be used to address whether the virus localisation in planta is affected following aphid infestation

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

BGPI : équipe 2International audienceA 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 transmissio

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.This article is from PLoS Pathogens 11 (2015): e1004868, doi: 10.1371/journal.ppat.1004868. Posted with permission.</p

Is glycosylation of viral structural proteins involved in CABYV aphid transmission?

National audienceCucurbit aphid borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) is a plant virus, localized in phloem cells and obligatory transmitted by aphids. Previous studies have shown that post-translational modifications such as N-linked glycosylation can affect polerovirus transmission by aphids. We analyzed the glycosylation status of the 2 structural proteins of CABYV (Coat Protein-CP: major capsid protein, ReadThrough-RT: minor capsid protein) by using different approaches to determine more precisely how this modification may affect aphid transmission. We first constructed four CABYV mutants modified in potential N-linked glycosylation sites (named Ngly-1, -2, -3, -4). Two of them (Ngly-1 and -2) contained a mutation in the major CP, whereas the two others were affected in the RT. When electroporated to plant protoplasts, the four mutants replicated as efficiently as the wild-type virus suggesting that the mutations introduced did not affect virus replication. When introduced in plants, the mutant Ngly-2 was almost totally impeded in long-distance movement whereas Ngly-1, -3, -4 showed a viral accumulation similar to the wild-type virus in systemic leaves. Analysis of the viral progeny in infected plants showed that mutations are maintained and no reverse nor compensatory mutation appeared. Aphid transmission of Ngly-1, -3, -4 mutants was assessed using infected plants as virus source. We observed a reduction in aphid transmission of the 3 mutants using either Aphis gossypii or Myzus persicae, two species known to be efficient vectors of CABYV. In order to conclude if the reduction in transmission efficiency of these viruses was due to a reduction in viral accumulation in plants or to a direct effect in vector interactions, aphid transmission experiments are being performed using purified virus particles as virus source. However, at this stage, we cannot correlate the effects on systemic movement or aphid transmission to modifications of the glycosylation status of the virion. We then analyzed by Mass-Spectrometry if post-translational modifications are present on the two structural proteins of CABYV. So far, our results showed that no glycosylated peptide is present on the viral structural proteins. Glycosylation of virions was also assessed by immunodetection with antibodies specific to complex plants glycanes, and by using the lectin Concanavalin A. No N-glycane could be identified on the viral structural proteins. However, a 90kDa protein (p90), an α-glucosidase, that co-purify with polerovirus was identified and shown to be modified by oligomannosidic and complex N-glycanes. The role in aphid transmission of this plant protein is currently being investigated as well as the potential interaction between p90 and viral particles. We also analysed the behaviour of the virus in A. thaliana mutant, whose homologous p90 protein is not expressed

Is glycosylation of viral structural proteins involved in CABYV aphid transmission?

National audienceCucurbit aphid borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) is a plant virus, localized in phloem cells and obligatory transmitted by aphids. Previous studies have shown that post-translational modifications such as N-linked glycosylation can affect polerovirus transmission by aphids. We analyzed the glycosylation status of the 2 structural proteins of CABYV (Coat Protein-CP: major capsid protein, ReadThrough-RT: minor capsid protein) by using different approaches to determine more precisely how this modification may affect aphid transmission. We first constructed four CABYV mutants modified in potential N-linked glycosylation sites (named Ngly-1, -2, -3, -4). Two of them (Ngly-1 and -2) contained a mutation in the major CP, whereas the two others were affected in the RT. When electroporated to plant protoplasts, the four mutants replicated as efficiently as the wild-type virus suggesting that the mutations introduced did not affect virus replication. When introduced in plants, the mutant Ngly-2 was almost totally impeded in long-distance movement whereas Ngly-1, -3, -4 showed a viral accumulation similar to the wild-type virus in systemic leaves. Analysis of the viral progeny in infected plants showed that mutations are maintained and no reverse nor compensatory mutation appeared. Aphid transmission of Ngly-1, -3, -4 mutants was assessed using infected plants as virus source. We observed a reduction in aphid transmission of the 3 mutants using either Aphis gossypii or Myzus persicae, two species known to be efficient vectors of CABYV. In order to conclude if the reduction in transmission efficiency of these viruses was due to a reduction in viral accumulation in plants or to a direct effect in vector interactions, aphid transmission experiments are being performed using purified virus particles as virus source. However, at this stage, we cannot correlate the effects on systemic movement or aphid transmission to modifications of the glycosylation status of the virion. We then analyzed by Mass-Spectrometry if post-translational modifications are present on the two structural proteins of CABYV. So far, our results showed that no glycosylated peptide is present on the viral structural proteins. Glycosylation of virions was also assessed by immunodetection with antibodies specific to complex plants glycanes, and by using the lectin Concanavalin A. No N-glycane could be identified on the viral structural proteins. However, a 90kDa protein (p90), an α-glucosidase, that co-purify with polerovirus was identified and shown to be modified by oligomannosidic and complex N-glycanes. The role in aphid transmission of this plant protein is currently being investigated as well as the potential interaction between p90 and viral particles. We also analysed the behaviour of the virus in A. thaliana mutant, whose homologous p90 protein is not expressed

Phloem-Triggered Virus-Induced Gene Silencing Using a Recombinant Polerovirus

The phloem-limited poleroviruses infect Arabidopsis thaliana without causing noticeable disease symptoms. In order to facilitate visual infection identification, we developed virus-induced gene silencing (VIGS) vectors derived from Turnip yellows virus (TuYV). Short sequences from the host gene AtCHLI1 required for chlorophyll biosynthesis [42 nucleotides in sense or antisense orientation or as an inverted-repeat (IR), or an 81 nucleotide sense fragment] were inserted into the 3′ non-coding region of the TuYV genome to screen for the most efficient and robust silencing vector. All recombinant viruses produced a clear vein chlorosis phenotype on infected Arabidopsis plants due to the expression inhibition of the AtCHLI1 gene. The introduction of a sense-oriented sequence into TuYV genome resulted in a virus exhibiting a more sustainable chlorosis than the virus containing an IR of the same length. This observation was correlated with a higher stability of the sense sequence insertion in the viral genome. In order to evaluate the impact of the TuYV silencing suppressor P0 in the VIGS mechanism a P0 knock-out mutation was introduced into the recombinant TuYV viruses. They induced a similar but milder vein clearing phenotype due to lower viral accumulation. This indicates that P0 does not hinder the performances of the TuYV silencing effect and confirms that in the viral infection context, P0 has no major impact on the production, propagation and action of the short distance silencing signal in phloem cells. Finally, we showed that TuYV can be used to strongly silence the phloem specific AtRTM1 gene. The TuYV-derived VIGS vectors therefore represent powerful tools to easily detect and monitor TuYV in infected plants and conduct functional analysis of phloem-restricted genes. Moreover this example indicates the potential of poleroviruses for use in functional genomic studies of agronomic plants.Fréquence et processus de mise en place des avalanches de débris tsunamigènes de l'arc des Petites Antilles : apport des forages de l'Expédition IODP 340 et impact en terme de risque.Identification de facteurs de l'hôte impliqués dans le mouvement à longue distance des virus de plant