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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

Serological GALV-Nf detection in systemically-infectedN. benthamianaleaves and analysis of viral particles by electron microscopy. Tissue-print immunoassay using a GALV-specific antibody on systemically-infected leaves of (A) agroinfiltrated and (B) healthy plants. Purified viral particles from (C) T7-GALV-Nf infected or (D) agroinfiltrated N. benthamiana plants detected by immunosorbent electron microscopy.
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Fig5: Serological GALV-Nf detection in systemically-infectedN. benthamianaleaves and analysis of viral particles by electron microscopy. Tissue-print immunoassay using a GALV-specific antibody on systemically-infected leaves of (A) agroinfiltrated and (B) healthy plants. Purified viral particles from (C) T7-GALV-Nf infected or (D) agroinfiltrated N. benthamiana plants detected by immunosorbent electron microscopy.

Mentions: We developed the GALV-Nf binary vector described above to improve the efficiency of infection, and delivered the vector to N. benthamiana plants by leaf agroinfiltration. Upper non-infiltrated leaves displayed light mottling with some necrotic spots, and apical leaf necrosis at 12 dpi (Figure 4C). The efficiency of infection in agroinfiltrated plants was ~90% in almost all experiments. Systemic spreading was confirmed in all symptomatic plants by RT-PCR analysis (Figure 4D) and by tissue-print immunoassays in the same plants (Figure 5A, B). GALV-Nf particles were purified from systemically infected leaves of N. benthamiana plants rubbed with the T7-GALV-Nf transcripts (Figure 5C) or following agroinfiltration with the binary vector (Figure 5D). Immunosorbent electron microscopy (ISEM) revealed in both cases the presence of correctly-assembled 32-nm particles with the expected icosahedral morphology.Figure 5


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)

Serological GALV-Nf detection in systemically-infectedN. benthamianaleaves and analysis of viral particles by electron microscopy. Tissue-print immunoassay using a GALV-specific antibody on systemically-infected leaves of (A) agroinfiltrated and (B) healthy plants. Purified viral particles from (C) T7-GALV-Nf infected or (D) agroinfiltrated N. benthamiana plants detected by immunosorbent electron microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Serological GALV-Nf detection in systemically-infectedN. benthamianaleaves and analysis of viral particles by electron microscopy. Tissue-print immunoassay using a GALV-specific antibody on systemically-infected leaves of (A) agroinfiltrated and (B) healthy plants. Purified viral particles from (C) T7-GALV-Nf infected or (D) agroinfiltrated N. benthamiana plants detected by immunosorbent electron microscopy.
Mentions: We developed the GALV-Nf binary vector described above to improve the efficiency of infection, and delivered the vector to N. benthamiana plants by leaf agroinfiltration. Upper non-infiltrated leaves displayed light mottling with some necrotic spots, and apical leaf necrosis at 12 dpi (Figure 4C). The efficiency of infection in agroinfiltrated plants was ~90% in almost all experiments. Systemic spreading was confirmed in all symptomatic plants by RT-PCR analysis (Figure 4D) and by tissue-print immunoassays in the same plants (Figure 5A, B). GALV-Nf particles were purified from systemically infected leaves of N. benthamiana plants rubbed with the T7-GALV-Nf transcripts (Figure 5C) or following agroinfiltration with the binary vector (Figure 5D). Immunosorbent electron microscopy (ISEM) revealed in both cases the presence of correctly-assembled 32-nm particles with the expected icosahedral morphology.Figure 5

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