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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 in systemically-infectedN. benthamianaleaves. (A) Epidermal cell filled with virus particles (V) and showing some multivesicular bodies (arrows), one of them enlarged in (B). Virions are distributed throughout the cytoplasm, or aggregated in a large crystal (C), or enclosed in membrane-bound enclaves (D). A plastid, enlarged in (E), shows thylakoid disorganization and vesiculation. Similar chloroplast alterations are visible also in parenchyma mesophyll cells (F) and, in some instances, vesicles similar to those forming the multivesicular bodies are observed (arrows). In cells at a late stage of infection, the chloroplast structure appears completely disrupted (G) and electron-dense tubular structures (T) are visible in the stroma. These structures are sometimes present also in mitochondria (H). N, nucleus; P, peroxisome; L, lipid globule. Black bars = 200 nm; white bars = 400 nm.
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Fig6: GALV-Nf cytopathology in systemically-infectedN. benthamianaleaves. (A) Epidermal cell filled with virus particles (V) and showing some multivesicular bodies (arrows), one of them enlarged in (B). Virions are distributed throughout the cytoplasm, or aggregated in a large crystal (C), or enclosed in membrane-bound enclaves (D). A plastid, enlarged in (E), shows thylakoid disorganization and vesiculation. Similar chloroplast alterations are visible also in parenchyma mesophyll cells (F) and, in some instances, vesicles similar to those forming the multivesicular bodies are observed (arrows). In cells at a late stage of infection, the chloroplast structure appears completely disrupted (G) and electron-dense tubular structures (T) are visible in the stroma. These structures are sometimes present also in mitochondria (H). N, nucleus; P, peroxisome; L, lipid globule. Black bars = 200 nm; white bars = 400 nm.

Mentions: In systemically-infected leaves, icosahedral virus particles were present in almost all tissues, in the cytoplasm and vacuoles but not in other organelles (Figure 6). In some heavily-infected cells, large virus crystals occupied most of the cell lumen (Figure 6A, C) together with aggregates of virions in membrane-bound enclaves (Figure 6D). The other major cytopathic features of infected cells included the presence of multivesicular bodies formed by stacked vesicles containing a fibrillar network (Figure 6B) and altered chloroplasts showing thylakoid disorganization and vesiculation (Figure 6E). Thylakoid disorganization was observed in almost all chloroplasts of the parenchymal mesophyll cells. In some of these chloroplasts, vesicles similar to those forming the multivesicular bodies were also visible, either singly or in small groups, and stacked on the thylakoid membranes (Figure 6F). The analysis of multiple sections through the multivesicular bodies did not reveal the origin of these structures. In cells from leaves at the late stage of infection, the chloroplast structure was completely disrupted (Figure 6G) and electron-dense tubular structures were visible in the stroma. These structures were also present in some mitochondria (Figure 6H), although the morphology of the peroxisomes and mitochondria did not show significant changes in most of the infected cells.Figure 6


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 in systemically-infectedN. benthamianaleaves. (A) Epidermal cell filled with virus particles (V) and showing some multivesicular bodies (arrows), one of them enlarged in (B). Virions are distributed throughout the cytoplasm, or aggregated in a large crystal (C), or enclosed in membrane-bound enclaves (D). A plastid, enlarged in (E), shows thylakoid disorganization and vesiculation. Similar chloroplast alterations are visible also in parenchyma mesophyll cells (F) and, in some instances, vesicles similar to those forming the multivesicular bodies are observed (arrows). In cells at a late stage of infection, the chloroplast structure appears completely disrupted (G) and electron-dense tubular structures (T) are visible in the stroma. These structures are sometimes present also in mitochondria (H). N, nucleus; P, peroxisome; L, lipid globule. Black bars = 200 nm; white bars = 400 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: GALV-Nf cytopathology in systemically-infectedN. benthamianaleaves. (A) Epidermal cell filled with virus particles (V) and showing some multivesicular bodies (arrows), one of them enlarged in (B). Virions are distributed throughout the cytoplasm, or aggregated in a large crystal (C), or enclosed in membrane-bound enclaves (D). A plastid, enlarged in (E), shows thylakoid disorganization and vesiculation. Similar chloroplast alterations are visible also in parenchyma mesophyll cells (F) and, in some instances, vesicles similar to those forming the multivesicular bodies are observed (arrows). In cells at a late stage of infection, the chloroplast structure appears completely disrupted (G) and electron-dense tubular structures (T) are visible in the stroma. These structures are sometimes present also in mitochondria (H). N, nucleus; P, peroxisome; L, lipid globule. Black bars = 200 nm; white bars = 400 nm.
Mentions: In systemically-infected leaves, icosahedral virus particles were present in almost all tissues, in the cytoplasm and vacuoles but not in other organelles (Figure 6). In some heavily-infected cells, large virus crystals occupied most of the cell lumen (Figure 6A, C) together with aggregates of virions in membrane-bound enclaves (Figure 6D). The other major cytopathic features of infected cells included the presence of multivesicular bodies formed by stacked vesicles containing a fibrillar network (Figure 6B) and altered chloroplasts showing thylakoid disorganization and vesiculation (Figure 6E). Thylakoid disorganization was observed in almost all chloroplasts of the parenchymal mesophyll cells. In some of these chloroplasts, vesicles similar to those forming the multivesicular bodies were also visible, either singly or in small groups, and stacked on the thylakoid membranes (Figure 6F). The analysis of multiple sections through the multivesicular bodies did not reveal the origin of these structures. In cells from leaves at the late stage of infection, the chloroplast structure was completely disrupted (Figure 6G) and electron-dense tubular structures were visible in the stroma. These structures were also present in some mitochondria (Figure 6H), although the morphology of the peroxisomes and mitochondria did not show significant changes in most of the infected cells.Figure 6

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