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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

Systemic GALV-Nf symptoms in grapevine plants. GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector in different cultivars regenerated from somatic embryos: V. vinifera(A) Brachetto, (B) Syrah, (C) Nebbiolo compared to corresponding healthy plants (D, E, F). Severe stunting and leaf alterations observed on a Nebbiolo plant (G) 5 weeks after infiltration with the GALV-Nf-based binary vector compared to a corresponding healthy plant (H). GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector on different grapevine genotypes: (I)V. riparia cv. Gloire de Montpellier; V. vinifera(J) Sultana, (K) Corvina, compared to corresponding GALV-free plants (L, M, N).
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Fig7: Systemic GALV-Nf symptoms in grapevine plants. GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector in different cultivars regenerated from somatic embryos: V. vinifera(A) Brachetto, (B) Syrah, (C) Nebbiolo compared to corresponding healthy plants (D, E, F). Severe stunting and leaf alterations observed on a Nebbiolo plant (G) 5 weeks after infiltration with the GALV-Nf-based binary vector compared to a corresponding healthy plant (H). GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector on different grapevine genotypes: (I)V. riparia cv. Gloire de Montpellier; V. vinifera(J) Sultana, (K) Corvina, compared to corresponding GALV-free plants (L, M, N).

Mentions: Plantlets representing different grapevine genotypes were inoculated with the binary vector by agroinfiltration. It is important to assess symptom development in completely virus-free plants, so we investigated the outcomes of infection in virus-free plants regenerated from somatic embryos representing the cultivars Brachetto, Syrah and Nebbiolo, to provide solid evidence that GALV-Nf can produce symptoms following artificial infection. Different symptoms were observed in emerging leaves 5 weeks after infiltration (Figure 7A, B, C) and these symptoms were absent in the corresponding healthy plants (Figure 7D, E, F). Regenerated Brachetto plants occasionally developed small chlorotic or necrotic spots along the veins (Figure 7A) whereas Syrah plants showed mild vein clearing or mottling (Figure 7B). No other developmental abnormalities were observed in either cultivar. In contrast, Nebbiolo plants showed malformations of the whole leaf lamina, chlorotic patches and dark green blistering on the leaves (Figure 7C) together with weak and stunted shoot growth (Figure 7G). These symptoms were not observed in healthy controls (Figure 7H).Figure 7


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)

Systemic GALV-Nf symptoms in grapevine plants. GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector in different cultivars regenerated from somatic embryos: V. vinifera(A) Brachetto, (B) Syrah, (C) Nebbiolo compared to corresponding healthy plants (D, E, F). Severe stunting and leaf alterations observed on a Nebbiolo plant (G) 5 weeks after infiltration with the GALV-Nf-based binary vector compared to a corresponding healthy plant (H). GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector on different grapevine genotypes: (I)V. riparia cv. Gloire de Montpellier; V. vinifera(J) Sultana, (K) Corvina, compared to corresponding GALV-free plants (L, M, N).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Systemic GALV-Nf symptoms in grapevine plants. GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector in different cultivars regenerated from somatic embryos: V. vinifera(A) Brachetto, (B) Syrah, (C) Nebbiolo compared to corresponding healthy plants (D, E, F). Severe stunting and leaf alterations observed on a Nebbiolo plant (G) 5 weeks after infiltration with the GALV-Nf-based binary vector compared to a corresponding healthy plant (H). GALV-Nf symptoms induced 5 weeks after infiltration with the GALV-Nf-based binary vector on different grapevine genotypes: (I)V. riparia cv. Gloire de Montpellier; V. vinifera(J) Sultana, (K) Corvina, compared to corresponding GALV-free plants (L, M, N).
Mentions: Plantlets representing different grapevine genotypes were inoculated with the binary vector by agroinfiltration. It is important to assess symptom development in completely virus-free plants, so we investigated the outcomes of infection in virus-free plants regenerated from somatic embryos representing the cultivars Brachetto, Syrah and Nebbiolo, to provide solid evidence that GALV-Nf can produce symptoms following artificial infection. Different symptoms were observed in emerging leaves 5 weeks after infiltration (Figure 7A, B, C) and these symptoms were absent in the corresponding healthy plants (Figure 7D, E, F). Regenerated Brachetto plants occasionally developed small chlorotic or necrotic spots along the veins (Figure 7A) whereas Syrah plants showed mild vein clearing or mottling (Figure 7B). No other developmental abnormalities were observed in either cultivar. In contrast, Nebbiolo plants showed malformations of the whole leaf lamina, chlorotic patches and dark green blistering on the leaves (Figure 7C) together with weak and stunted shoot growth (Figure 7G). These symptoms were not observed in healthy controls (Figure 7H).Figure 7

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