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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

GALV-Nf cytopathology of systemically-infected grapevine leaves. (A) Virus infected cells are easily identified even under a light microscope close to small veins (X) and show different stages of plasmolysis and membrane rupture. Chloroplasts of spongy mesophyll (arrow) appear swollen, in comparison with those visible in an uninfected control leaf (B). At the ultrastructural level (C), swollen chloroplasts (Ch) show thylakoid disorganization, and numerous virus particles (V) are visible in the surrounding cytoplasm (D); mitochondria (M) are also swollen, apparently without stroma and almost devoid of cristae. An uninfected control cell is visible in (E) for comparison. Cells around small veins sometimes show incipient necrosis (F) with dense cytoplasm in which virus-like particles are still distinguishable (G). Infected parenchyma cells of small veins (H) show mitochondria with vacuolization and loss of cristae and small vesicles are sometimes present in apparently dilated ER cisternae (I). N, nucleus; P, peroxisome; Ps, plasmodesmata. Black bars = 400 nm; white bars = 100 nm, if not otherwise stated.
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Fig9: GALV-Nf cytopathology of systemically-infected grapevine leaves. (A) Virus infected cells are easily identified even under a light microscope close to small veins (X) and show different stages of plasmolysis and membrane rupture. Chloroplasts of spongy mesophyll (arrow) appear swollen, in comparison with those visible in an uninfected control leaf (B). At the ultrastructural level (C), swollen chloroplasts (Ch) show thylakoid disorganization, and numerous virus particles (V) are visible in the surrounding cytoplasm (D); mitochondria (M) are also swollen, apparently without stroma and almost devoid of cristae. An uninfected control cell is visible in (E) for comparison. Cells around small veins sometimes show incipient necrosis (F) with dense cytoplasm in which virus-like particles are still distinguishable (G). Infected parenchyma cells of small veins (H) show mitochondria with vacuolization and loss of cristae and small vesicles are sometimes present in apparently dilated ER cisternae (I). N, nucleus; P, peroxisome; Ps, plasmodesmata. Black bars = 400 nm; white bars = 100 nm, if not otherwise stated.

Mentions: Ultrastructural analysis of systemically-infected leaves (mainly in the Syrah plants showing vein clearing and mottling) confirmed that the most severely affected cells were those adjacent to small veins, particularly in the spongy mesophyll (Figure 9A). These cells showed different stages of plasmolysis and membrane rupture, and the chloroplasts appeared swollen compared to matched, uninfected control leaves (Figure 9B, E). At the ultrastructural level, the altered chloroplasts showed evidence of thylakoid disorganization, although without vesiculation (Figure 9C), and numerous virus particles were spread throughout the surrounding cytoplasm (Figure 9D). The mitochondria were occasionally swollen, and almost devoid of cristae and stroma (Figure 9C). Cells around small veins sometimes showed incipient necrosis (Figure 9F), and contained dense cytoplasm in which it was still possible to distinguish virus-like particles (Figure 9G). The mitochondria of infected parenchyma cells in small veins were vacuolated and devoid of cristae (Figure 9H) and small vesicles were sometimes present in dilated cisternae derived from the endoplasmic reticulum (Figure 9I). However, multivesicular bodies like those observed in N. benthamiana were not observed in the infected grapevine cells. All the ultrastructural alterations described above were also present in the symptomatic leaves of Nebbiolo plants. The only ultrastructural feature associated with the peculiar symptoms observed in Nebbiolo plants was the significant reduction in chloroplast number in cells representing the light-green areas of the leaf (data not shown).Figure 9


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)

GALV-Nf cytopathology of systemically-infected grapevine leaves. (A) Virus infected cells are easily identified even under a light microscope close to small veins (X) and show different stages of plasmolysis and membrane rupture. Chloroplasts of spongy mesophyll (arrow) appear swollen, in comparison with those visible in an uninfected control leaf (B). At the ultrastructural level (C), swollen chloroplasts (Ch) show thylakoid disorganization, and numerous virus particles (V) are visible in the surrounding cytoplasm (D); mitochondria (M) are also swollen, apparently without stroma and almost devoid of cristae. An uninfected control cell is visible in (E) for comparison. Cells around small veins sometimes show incipient necrosis (F) with dense cytoplasm in which virus-like particles are still distinguishable (G). Infected parenchyma cells of small veins (H) show mitochondria with vacuolization and loss of cristae and small vesicles are sometimes present in apparently dilated ER cisternae (I). N, nucleus; P, peroxisome; Ps, plasmodesmata. Black bars = 400 nm; white bars = 100 nm, if not otherwise stated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig9: GALV-Nf cytopathology of systemically-infected grapevine leaves. (A) Virus infected cells are easily identified even under a light microscope close to small veins (X) and show different stages of plasmolysis and membrane rupture. Chloroplasts of spongy mesophyll (arrow) appear swollen, in comparison with those visible in an uninfected control leaf (B). At the ultrastructural level (C), swollen chloroplasts (Ch) show thylakoid disorganization, and numerous virus particles (V) are visible in the surrounding cytoplasm (D); mitochondria (M) are also swollen, apparently without stroma and almost devoid of cristae. An uninfected control cell is visible in (E) for comparison. Cells around small veins sometimes show incipient necrosis (F) with dense cytoplasm in which virus-like particles are still distinguishable (G). Infected parenchyma cells of small veins (H) show mitochondria with vacuolization and loss of cristae and small vesicles are sometimes present in apparently dilated ER cisternae (I). N, nucleus; P, peroxisome; Ps, plasmodesmata. Black bars = 400 nm; white bars = 100 nm, if not otherwise stated.
Mentions: Ultrastructural analysis of systemically-infected leaves (mainly in the Syrah plants showing vein clearing and mottling) confirmed that the most severely affected cells were those adjacent to small veins, particularly in the spongy mesophyll (Figure 9A). These cells showed different stages of plasmolysis and membrane rupture, and the chloroplasts appeared swollen compared to matched, uninfected control leaves (Figure 9B, E). At the ultrastructural level, the altered chloroplasts showed evidence of thylakoid disorganization, although without vesiculation (Figure 9C), and numerous virus particles were spread throughout the surrounding cytoplasm (Figure 9D). The mitochondria were occasionally swollen, and almost devoid of cristae and stroma (Figure 9C). Cells around small veins sometimes showed incipient necrosis (Figure 9F), and contained dense cytoplasm in which it was still possible to distinguish virus-like particles (Figure 9G). The mitochondria of infected parenchyma cells in small veins were vacuolated and devoid of cristae (Figure 9H) and small vesicles were sometimes present in dilated cisternae derived from the endoplasmic reticulum (Figure 9I). However, multivesicular bodies like those observed in N. benthamiana were not observed in the infected grapevine cells. All the ultrastructural alterations described above were also present in the symptomatic leaves of Nebbiolo plants. The only ultrastructural feature associated with the peculiar symptoms observed in Nebbiolo plants was the significant reduction in chloroplast number in cells representing the light-green areas of the leaf (data not shown).Figure 9

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