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Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

Lovato A, Faoro F, Gambino G, Maffi D, Bracale M, Polverari A, Santi L - Virol. J. (2014)

Bottom Line: Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species.The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples.This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy. annalisa.polverari@univr.it.

ABSTRACT

Background: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts.

Methods: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species.

Results: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe.

Conclusions: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

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Related in: MedlinePlus

Phylogenetic tree of predicted coat proteins derived from different GALV isolates. Predicted protein sequences of different isolates were aligned and the phylogenetic three was constructed using the neighbor-joining method (from Algeria: GALV-Vv.1, GALV-Vv.2; from Germany (DE): Water Doss, Schunter River, Gyp 2; from Netherlands (NL): Lim 3, Lim 4 and from Japan: GALV-Nf, Limo-08).
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Fig2: Phylogenetic tree of predicted coat proteins derived from different GALV isolates. Predicted protein sequences of different isolates were aligned and the phylogenetic three was constructed using the neighbor-joining method (from Algeria: GALV-Vv.1, GALV-Vv.2; from Germany (DE): Water Doss, Schunter River, Gyp 2; from Netherlands (NL): Lim 3, Lim 4 and from Japan: GALV-Nf, Limo-08).

Mentions: The nucleotide and deduced amino acid sequences of the CP genes were aligned using ClustalW2 and a Percent Identity Matrix was produced in order to evaluate sequence similarities. The CP sequences from different isolates showed amino acid sequence identities ranging from 91.49 to 99.73% whereas the nucleotide sequence identities ranged from 84.03 to 99.73% (Table 1). The CP sequence lengths were also variable, comprising 376 amino acids in most cases but increasing to 378 and 381 residues for the Shunter River and GALV-Vv.1 isolates, respectively (Figure 1). Based on these available CP amino acid sequences, a phylogenetic tree was constructed, showing the sequences clustered in three distinct groups with high bootstrap values (Figure 2). The Water Doss, Lim 3 and GALV-Vv isolates clustered in group I. The Lim 4, Gyp 2, Limo-08 and GALV-Nf isolates clustered in group II. Finally, the Shunter River isolate was the sole representative of group III.Table 1


Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

Lovato A, Faoro F, Gambino G, Maffi D, Bracale M, Polverari A, Santi L - Virol. J. (2014)

Phylogenetic tree of predicted coat proteins derived from different GALV isolates. Predicted protein sequences of different isolates were aligned and the phylogenetic three was constructed using the neighbor-joining method (from Algeria: GALV-Vv.1, GALV-Vv.2; from Germany (DE): Water Doss, Schunter River, Gyp 2; from Netherlands (NL): Lim 3, Lim 4 and from Japan: GALV-Nf, Limo-08).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289286&req=5

Fig2: Phylogenetic tree of predicted coat proteins derived from different GALV isolates. Predicted protein sequences of different isolates were aligned and the phylogenetic three was constructed using the neighbor-joining method (from Algeria: GALV-Vv.1, GALV-Vv.2; from Germany (DE): Water Doss, Schunter River, Gyp 2; from Netherlands (NL): Lim 3, Lim 4 and from Japan: GALV-Nf, Limo-08).
Mentions: The nucleotide and deduced amino acid sequences of the CP genes were aligned using ClustalW2 and a Percent Identity Matrix was produced in order to evaluate sequence similarities. The CP sequences from different isolates showed amino acid sequence identities ranging from 91.49 to 99.73% whereas the nucleotide sequence identities ranged from 84.03 to 99.73% (Table 1). The CP sequence lengths were also variable, comprising 376 amino acids in most cases but increasing to 378 and 381 residues for the Shunter River and GALV-Vv.1 isolates, respectively (Figure 1). Based on these available CP amino acid sequences, a phylogenetic tree was constructed, showing the sequences clustered in three distinct groups with high bootstrap values (Figure 2). The Water Doss, Lim 3 and GALV-Vv isolates clustered in group I. The Lim 4, Gyp 2, Limo-08 and GALV-Nf isolates clustered in group II. Finally, the Shunter River isolate was the sole representative of group III.Table 1

Bottom Line: Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species.The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples.This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy. annalisa.polverari@univr.it.

ABSTRACT

Background: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts.

Methods: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species.

Results: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe.

Conclusions: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

Show MeSH
Related in: MedlinePlus