Grapevine Fanleaf Virus Coat Protein Research Articles

From a Movement-Deficient Grapevine Fanleaf Virus to the Identification of a New Viral Determinant of Nematode Transmission.

Grapevine fanleaf virus (GFLV) and arabis mosaic virus (ArMV) are nepoviruses responsible for grapevine degeneration. They are specifically transmitted from grapevine to grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two viruses, were previously defined and exchanged to test their involvement in virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in virus transmission, thus representing a new viral determinant of nematode transmission.

Production and Partial Purification of the Grapevine Fanleaf Virus Coat Protein 42 Polyclonal Antibody Against Inclusion Body Expressed in Escherichia coli.

BackgroundExpression of virus coat protein (CP) in Escherichia coli often leads to production of partially folded aggregated proteins which are called inclusion bodies. Grapevine fanleaf virus (GFLV) is one of the most serious and widespread grapevine virus diseases around the world and in Iran.ObjectiveThe main objective of this study was to find a simple and brief method for producing polyclonal antibodies (PAbs) to be used for immunodiagnosis of GFLV.Material and MethodsAn antigenic determinant in GFLV CP gene was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain BL21 (DE3).The recombinant coat protein of GFLV (CP42) was expressed and characterized by SDS-PAGE and western blot analysis using commercial anti-GFLV antibody. Expression of the CP was detected in the form of inclusion bodies in insoluble cytoplasmic fraction. Then, the inclusion bodies were isolated from the bacterial cells and injected into rabbits for PAbs production. The reaction of the antiserum was checked by ELISA assay. In order to analyze efficiency of the produced PAbs, first the infected and uninfected grapevine samples were confirmed based on morphological symptoms then the indirect plate- trapped antigen Enzyme-linked Immunosorbent Assay (IPTA-ELISA) was applied using the commercial anti GFLV antibody. In the next ELISA assay, efficiency of the raised polyclonal antibody was compared with commercial one.ResultsThe expression of recombinant CP42 induced by IPTG was confirmed by the band of 42 kDa in SDS-PAGE and western blot. The antiserum of purified inclusion body immunized rabbit was reacted with CP42 and GFLV infected Grapevine samples. The results revealed an acceptable efficacy for prepared antibodies compared to that of commercial antibody.ConclusionsIt was evident that the recombinant coat protein in the form of inclusion bodies can be prepared and used as the antigen for immunizing animals in order to produce PAbs.

Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

Detection of Grapevine fanleaf virus by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) with recombinant antibody

Grapevine fanleaf virus (GFLV), a member of the genus Nepovirus in the family Secoviridae, is the most widely distributed grapevine virus around the world. In the present research, recombinant antibody to the GFLV coat protein (CP) was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for the detection of GFLV by the use of a pair of primers corresponding to the full length CP gene. In addition, double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was done by the use of recombinant antibody to detect the GFLV isolates. Eventually, an expected band with around 1512 base pair (bp) in length was revealed for three isolates by IC-RT-PCR. Also, DAS-ELISA revealed that the recombinant antibody was able to detect the virus in leaf sample. These results showed that application of recombinant antibody in these detection methods can be useful for rapid detection of viruses in plant tissue because by applying IC-RT-PCR along with recombinant antibody, it is possible to overcome the limitations happened by presence of inhibitors in grapevine trees and non-specific reaction resulting from presence of native proteins in plant sap against conventionally made antibody.

Prevalence and diversity of Grapevine fanleaf virus in southern Spain

The occurrence of Grapevine fanleaf virus (GFLV) in 74 vineyards in grapevine‐growing areas of Andalusia, southern Spain, was investigated, as well as the genetic variability of the coat protein (CP) gene on RNA2. Overall, the prevalence of GFLV (the percentage of vineyards with GFLV) was 24·3%, and it was most common in the Jerez‐Xérès‐Sherry and Manzanilla‐Sanlúcar de Barrameda denomination of origin (DO) (29·4%) and Condado de Huelva DO (24·0%), followed by the Montilla‐Moriles DO (13·3%). GFLV‐infected plants showed fanleaf and yellow mosaic symptoms together with general vine decline. In almost all samples, except one from Jerez‐Xérès‐Sherry and Manzanilla‐Sanlúcar de Barrameda DO and one from Condado de Huelva DO, GFLV‐infected plants showed soil infestation by the virus vector nematode species Xiphinema index and/or the potential vector X. italiae. Reverse transcription‐polymerase chain reaction (RT‐PCR) successfully amplified the partial CP gene product of the expected size (555 bp) in leaves and nematode vectors. A total of 135 clones of the partial GFLV CP gene were analysed for sequence variation using single‐strand conformation polymorphism, yielding 12 different haplotype patterns. Haplotypes were separated among the three different DO. The haplotype patterns MOB, MOC, MMA, MMC and HB were detected in leaf samples and nematode vectors for each locality, except for Jerez de la Frontera‐429. Sequence analysis of the GFLV haplotypes revealed sequence variability within the haplotypes and greater abundance of some variants than others in the same sequenced haplotype pattern. Consequently, GFLV in southern Spain exists as a ‘quasispecies’, as it has been reported in other grapevine‐growing areas of the world. This paper is the first report on the molecular variability of GFLV in Spain.

Safety assessment of transgenic plums and grapevines expressing viral coat protein genes: new insights into real environmental impact of perennial plants engineered for virus resistance.

The potential impact of transgenic plums and grapevines expressing viral coat protein (CP) gene constructs on the diversity and dynamics of virus populations was assessed under open and confined conditions in the frame of a research program sponsored by the European Commission. Across all field trials conducted in different locations (France, Romania, and Spain) and environments (continental and Mediterranean), transgenic plums expressing the CP gene of Plum pox virus (PPV) and transgenic grapevines expressing the CP gene of Grapevine fanleaf virus (GFLV) had no detectable effect on the emergence of recombinant PPV and GFLV species over eight-ten and three years, respectively. Also, no statistically significant difference was found in the number and type of aphids, including viruliferous individuals, and other arthropods that visited transgenic and nontransgenic plum trees. In addition, Apple chlorotic leaf spot virus, Prune dwarf virus, and Prunus necrotic ringspot virus did not influence the stability of the engineered resistance to PPV in co-infected transgenic plums over three dormancy periods. Further, under confined conditions, no recombinant virus was found to detectable level in transgenic grapevines expressing the CP gene of Grapevine virus A (GVA) or Grapevine virus B (GVB) that were challenged with the homologous or heterologous virus, despite high accumulation of transgene transcripts. Also, translocation of transgene-derived products, i.e. protein, mRNAs and siRNAs, did not occur to detectable level from transgenic grapevine rootstocks expressing the GFLV CP gene to nontransgenic scions. Altogether, our transgenic plums and grapevines expressing viral genes had a neutral impact on virus populations and non-target organisms over extended time. These findings provide new insights into the environmental impact of transgenic perennial crops engineered for virus resistance. It is expected that they will assist national and international regulatory authorities in making scientifically based decisions for the release of virus-resistant transgenic crops.

Survey forGrapevine fanleaf virusin vineyards of north-west Iran and genetic diversity of isolates

Grapevine fanleaf virus (genus Nepovirus) was detected by double antibody sandwich enzyme-linked immunosorbant assay (DAS-ELISA) in 31 of 134 grapevine samples from the north-west of Iran. Reverse transcriptase polymerase chain reaction with two primer sets (S2515/A3300 and CP433V/912C) targeting the Grapevine fanleaf virus coat protein gene resulted in amplification of the expected DNA fragments from some but not all DAS-ELISA positive samples. At the nucleotide level, the Iranian isolates were 84–95% identical to each other and 84–91% identical to other isolates from around the world. This is the first comprehensive analysis of the distribution and genetic diversity of Grapevine fanleaf virus in Iran.

Molecular characterization of grapevine plants transformed with GFLV resistance genes: II

A collection of 127 putatively transgenic individuals of Vitis vinifera cv. Russalka was characterized by PCR and Southern hybridization. Six different constructs containing the neomycin phosphotransferase (nptII) marker gene and sequences of the Grapevine Fanleaf Virus Coat Protein (GFLV CP) gene including non-translatable and truncated forms were transferred via Agrobacterium-mediated transformation. Detection of transgenic sequences by PCR was positive in all lines. Southern blot analysis revealed that the number of inserted T-DNA copies ranged from 1 to 6. More than 46% of the tested transgenic lines contain one copy of the inserted T-DNA, qualifying them as interesting candidates for further breeding programs. Southern data of one line indicate the presence of an incomplete copy of the T-DNA, thus confirming previous PCR results. Since many putative transgenic lines shared identical hybridization patterns, they were clustered into 39 lines and considered as having originated from independent transformation events. The detection of the tetracycline (TET) resistance genes in 15% of the lines shows that an integration of plasmid backbone sequences beyond the T-DNA borders occurred. Enzyme-linked immunosorbent assay (ELISA) performed on leaf tissue did not show any accumulation of the GFLV CP in the 39 transgenic lines analyzed. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot were carried out; RT-PCR analyses showed that the GFLV CP mRNA was expressed at variable levels.

Transgenic grapevine rootstock clones expressing the coat protein or movement protein genes of Grapevine fanleaf virus: Characterization and reaction to virus infection upon protoplast electroporation

The reaction to Grapevine fanleaf virus (GFLV) infection in 42 independent transgenic grapevine rootstock 41B clones expressing the coat protein (CP) or movement protein (MP) gene of GFLV was assayed by protoplast electroporation. Two of the 26 transgenic clones expressing the CP gene did not support the accumulation of GFLV MP to detectable levels, 12 accumulated substantially lower levels of MP, and 12 accumulated equivalent levels of MP relative to protoplasts of nontransformed controls at 72 h post-electroporation, as shown by Western blots with anti-MP γ-globulins. Interestingly, inhibition of MP accumulation was achieved against virions but not viral RNAs, and was dependent on the inoculum dose. No interference was observed with the multiplication of Arabis mosaic virus, which is closely related to GFLV, likely due to low nucleotide identity between the CP genes. Also, one of the 16 transgenic clones expressing the MP gene significantly reduced the accumulation level of GFLV CP at 72 h post-electroporation, as shown by DAS-ELISA with anti-GFLV γ-globulins. The potential of protoplast electroporation as rapid identification of GFLV-resistant grapevine clones at the cell level will be discussed relative to field screening for resistance at the plant level by nematode-mediated GFLV transmission.

Molecular characterization of grapevine plants transformed with GFLV resistance genes: I

The Grapevine FanLeaf Virus-Coat Protein (GFLV CP) gene was inserted through Agrobacterium-mediated transformation in Vitis vinifera "Nebbiolo", "Lumassina" and "Blaufränkisch". Two plasmids were used: pGA-CP+ (full-length GFLV CP gene with an introduced start codon) and pGA-AS (same gene in antisense orientation). Forty-three transgenic lines were regenerated. As several lines in Southern blots share same hybridization patterns, eight independent line groups resulted for "Nebbiolo", one for "Lumassina", and two for "Blaufränkisch". Inserted T-DNA copies ranged from one to three; one line probably contains an incomplete copy of T-DNA. Except for one "Nebbiolo" line, no evidence for methylation of the transgene at cytosine residues was found by Southern analyses. Specific mRNA was present at variable expression levels; some lines accumulated the coat protein while in others the protein was not detectable by ELISA.

Field safety assessment of recombination in transgenic grapevines expressing the coat protein gene of Grapevine fanleaf virus.

One of the major environmental safety issues over transgenic crops containing virus-derived genes relates to the outcome of recombination events between viral transgene transcripts and RNAs from indigenous virus populations. We addressed this issue by assessing the emergence of viable Grapevine fanleaf virus (GFLV) recombinants in transgenic grapevines expressing the GFLV coat protein (CP) gene. Test plants consisted of nontransgenic scions grafted onto transgenic and nontransgenic rootstocks that were exposed over 3 years to nematode-mediated GFLV infection in two distinct vineyard sites. The CP gene of challenging GFLV isolates was amplified from scions by IC-RT-PCR, and characterized by RFLP and nucleotide sequencing using strain F13 as reference since it provided the CP transgene. Analysis of EcoRI and StyI RFLP banding patterns from 347 challenging GFLV isolates and sequence data from 85 variants revealed no characteristics similar to strain F13 and no difference in the molecular variability among isolates from 190 transgenic and 157 nontransgenic plants, or from plants within (253 individuals) or outside (94 individuals) of the two sites. Interestingly, five GFLV recombinants were identified in three nontransgenic plants located outside of the two field settings. This survey indicates that transgenic grapevines did not assist the emergence of viable GFLV recombinants to detectable levels nor did they affect the molecular diversity of indigenous GFLV populations during the trial period. This is the first report on safety assessment of recombination with a transgenic crop expressing a CP gene under field conditions of heavy disease pressure but low, if any, selection pressure against recombinant viruses.

Bermuda Grass as a Potential Reservoir Host for Grapevine fanleaf virus.

Grapevine fanleaf virus (GFLV) was detected in samples of Bermuda grass (BG) from Iran by reverse transcription-polymerase chain reaction (RT-PCR) using two different pairs of GFLV-specific primers, and also by enzyme-linked immunosorbent assay (ELISA) using antiserum specific for a North American isolate of the virus. RT-PCR detected GFLV in both fresh and dried BG tissues and in virus preparations purified from these plants. Cloning and sequencing of the RT-PCR products confirmed that the amplified sequences were sections of the GFLV coat protein gene. Similar results were obtained when random and oligo(dT) primers were used on viral RNA templates recovered from BG to synthesize cDNA for cloning and sequencing. The virus induced few or no symptoms in BG, but could nonetheless be transmitted from BG to Chenopodium quinoa by mechanical inoculation. Some isolates induced systemic chlorotic spots and leaf deformation; others remained symptomless in this plant. Both symptomatic and symptomless C. quinoa plants were found to be infected with GFLV, giving positive ELISA and RT-PCR tests. A North American isolate of GFLV was found to be mechanically transmissible to BG as indicated by positive RT-PCR results from root samples of inoculated plants. GFLV-infected BG was widely distributed in the Fars province of Iran.

Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens

To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia ‘Gloire de Montpellier’, Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N6-benzyladenine (BA) per L, and 0.8% Noble agar). The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocultivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of GLRaV-3 (43K), and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3–4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.

High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene

Genetically transformed grapevines were obtained through co-cultivation of embryogenic cell suspensions with an engineered A. tumefaciens strain. Two economically important rootstocks, 41B and SO4, as well as a well-known grapevine vinifera variety, Chardonnay were regenerated. For the first time transformation of a scion variety is reported. A chimeric coat protein gene (CP) was integrated in order to protect grapevine against grapevine fanleaf virus (GFLV) infection. A neomycin phosphotransferase II (NPT II) gene allowed the selection of large number of transformed embryogenic calli and plants for the three varieties. Percentages of transformed material were first estimated with GUS activity. Presence of the CP gene was assessed by PCR and Southern analysis and gene expression by ELISA. Transformed calli have now been subcultured in vitro for 3 years without losing their embryogenic ability. GUS activity assays on leaves and roots of acclimatized plants showed transformation to be stable.

Detection of a Region of the Coat Protein Gene of Grapevine Fanleaf Virus by RT-PCR in the Nematode VectorXiphinema index

A region of the coat protein (CP) gene of grapevine fanleaf virus (GFLV) was detected by RT-PCR in the nematode vector Xiphinema index. The reverse transcription step was performed with total RNA extracted from viruliferous X. index. From the synthesized first cDNA strand, an 810-nucleotide fragment (nt 2515-3324 of GFLV-RNA2) was amplified by two 25-mer primers designed in the CP gene. Specific detection was successfully accomplished with samples of one or 10 viruliferous nematodes. Preliminary analysis and comparison of the 207-nt sequence of a stretch (nt 2549-2755 of GFLV-RNA2) of five different RT-PCR amplified fragments, obtained from 10 nematodes of a same sample in a diseased vineyard, indicated substantial variations in the GFLV CP gene