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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

Schematic representation of the infectious GALV-Nf cDNA clones. GALV-Nf sequences under the control of T7 promoter (T7-GALV-Nf and T7-MCS.GALV-Nf) were linearized with SrfI and used for the in vitro production of infectious transcripts (details of T7 transcription site and SrfI cleavage site are shown in panel A). A polylinker was inserted by BstBI cleavage in the T7-GALV-Nf construct obtaining the T7-MCS.GALV-Nf vector (the BglII and XhoI sites of the polylinker are shown in bold and underlined in the sequence reported in panel B). The viral sequence, placed under the control of the CaMV 35S promoter (35S) and the nos terminator (NOS), was introduced (using SacI/AscI and AsiSI/XbaI sites) between the pK7WG2 left and right borders (RB and LB) producing the pK7WG2-MCS.HRz.GALV-Nf binary vector for A. tumefaciens-mediated infection. To design a functional 5′ viral end, the junction between the 35S and the viral sequence was obtained by ligating blunt-end fragments produced by digesting the 35S sequence with StuI and the viral sequence with DraI (details shown in panel C). The sequence of the Hepatitis delta virus antigenomic ribozyme (HRz) was introduced to allow the production of a correct 3′ viral end following ribozyme autocleavage (panel C). T7: T7 promoter; p33 and p92: RNA-dependent RNA polymerases; p40: coat protein (CP); p24: movement protein (MP); p19: silencing suppressor.
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Fig3: Schematic representation of the infectious GALV-Nf cDNA clones. GALV-Nf sequences under the control of T7 promoter (T7-GALV-Nf and T7-MCS.GALV-Nf) were linearized with SrfI and used for the in vitro production of infectious transcripts (details of T7 transcription site and SrfI cleavage site are shown in panel A). A polylinker was inserted by BstBI cleavage in the T7-GALV-Nf construct obtaining the T7-MCS.GALV-Nf vector (the BglII and XhoI sites of the polylinker are shown in bold and underlined in the sequence reported in panel B). The viral sequence, placed under the control of the CaMV 35S promoter (35S) and the nos terminator (NOS), was introduced (using SacI/AscI and AsiSI/XbaI sites) between the pK7WG2 left and right borders (RB and LB) producing the pK7WG2-MCS.HRz.GALV-Nf binary vector for A. tumefaciens-mediated infection. To design a functional 5′ viral end, the junction between the 35S and the viral sequence was obtained by ligating blunt-end fragments produced by digesting the 35S sequence with StuI and the viral sequence with DraI (details shown in panel C). The sequence of the Hepatitis delta virus antigenomic ribozyme (HRz) was introduced to allow the production of a correct 3′ viral end following ribozyme autocleavage (panel C). T7: T7 promoter; p33 and p92: RNA-dependent RNA polymerases; p40: coat protein (CP); p24: movement protein (MP); p19: silencing suppressor.

Mentions: We developed and tested three different infectious GALV clones (Figure 3A, B, C). First, two GALV-Nf partial sequences were synthesized and manipulated to produce the final T7-GALV-Nf vector (Figure 3A). This clone contains the full-length GALV-Nf sequence under the transcriptional control of the T7 promoter plus a SrfI site for vector linearization, which is required to produce the correct 3′ viral end by in vitro transcription [16, 17]. We added a multiple cloning site (MCS) downstream of the p24 coding region at the unique BstBI restriction site to create vector T7-MCS-GALV-Nf, which contains two new unique restriction sites (BglII and XhoI) thus allowing the further functionalization of this vector in future studies. Three different stop codons, one for each reading frame, were designed upstream the MCS to prevent the formation of aberrant viral proteins (Figure 3B). Finally, the pK7WG2-MCS.HRz.GALV-Nf binary vector was produced (using the SacI/AscI and AsiSI/XbaI restriction sites) placing the GALV-Nf sequence under the control of the CaMV 35S promoter and inserting the HRz ribozyme and nos terminator sequences immediately downstream of the viral 3′ UTR (Figure 3C). This vector can be delivered to plants by agroinfiltration and allows the production of GALV-Nf RNAs almost equivalent to the viral genome. In particular, by using the StuI-DraI ligation strategy described under Materials and Methods, a functional viral 5′ UTR sequence corresponding to the +1 transcriptional start site can be produced, lacking only two nucleotides from the original GALV-Nf genome sequence. The HRz ribozyme can produce the precise viral 3′ terminus by self-cleavage [11].Figure 3


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)

Schematic representation of the infectious GALV-Nf cDNA clones. GALV-Nf sequences under the control of T7 promoter (T7-GALV-Nf and T7-MCS.GALV-Nf) were linearized with SrfI and used for the in vitro production of infectious transcripts (details of T7 transcription site and SrfI cleavage site are shown in panel A). A polylinker was inserted by BstBI cleavage in the T7-GALV-Nf construct obtaining the T7-MCS.GALV-Nf vector (the BglII and XhoI sites of the polylinker are shown in bold and underlined in the sequence reported in panel B). The viral sequence, placed under the control of the CaMV 35S promoter (35S) and the nos terminator (NOS), was introduced (using SacI/AscI and AsiSI/XbaI sites) between the pK7WG2 left and right borders (RB and LB) producing the pK7WG2-MCS.HRz.GALV-Nf binary vector for A. tumefaciens-mediated infection. To design a functional 5′ viral end, the junction between the 35S and the viral sequence was obtained by ligating blunt-end fragments produced by digesting the 35S sequence with StuI and the viral sequence with DraI (details shown in panel C). The sequence of the Hepatitis delta virus antigenomic ribozyme (HRz) was introduced to allow the production of a correct 3′ viral end following ribozyme autocleavage (panel C). T7: T7 promoter; p33 and p92: RNA-dependent RNA polymerases; p40: coat protein (CP); p24: movement protein (MP); p19: silencing suppressor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Schematic representation of the infectious GALV-Nf cDNA clones. GALV-Nf sequences under the control of T7 promoter (T7-GALV-Nf and T7-MCS.GALV-Nf) were linearized with SrfI and used for the in vitro production of infectious transcripts (details of T7 transcription site and SrfI cleavage site are shown in panel A). A polylinker was inserted by BstBI cleavage in the T7-GALV-Nf construct obtaining the T7-MCS.GALV-Nf vector (the BglII and XhoI sites of the polylinker are shown in bold and underlined in the sequence reported in panel B). The viral sequence, placed under the control of the CaMV 35S promoter (35S) and the nos terminator (NOS), was introduced (using SacI/AscI and AsiSI/XbaI sites) between the pK7WG2 left and right borders (RB and LB) producing the pK7WG2-MCS.HRz.GALV-Nf binary vector for A. tumefaciens-mediated infection. To design a functional 5′ viral end, the junction between the 35S and the viral sequence was obtained by ligating blunt-end fragments produced by digesting the 35S sequence with StuI and the viral sequence with DraI (details shown in panel C). The sequence of the Hepatitis delta virus antigenomic ribozyme (HRz) was introduced to allow the production of a correct 3′ viral end following ribozyme autocleavage (panel C). T7: T7 promoter; p33 and p92: RNA-dependent RNA polymerases; p40: coat protein (CP); p24: movement protein (MP); p19: silencing suppressor.
Mentions: We developed and tested three different infectious GALV clones (Figure 3A, B, C). First, two GALV-Nf partial sequences were synthesized and manipulated to produce the final T7-GALV-Nf vector (Figure 3A). This clone contains the full-length GALV-Nf sequence under the transcriptional control of the T7 promoter plus a SrfI site for vector linearization, which is required to produce the correct 3′ viral end by in vitro transcription [16, 17]. We added a multiple cloning site (MCS) downstream of the p24 coding region at the unique BstBI restriction site to create vector T7-MCS-GALV-Nf, which contains two new unique restriction sites (BglII and XhoI) thus allowing the further functionalization of this vector in future studies. Three different stop codons, one for each reading frame, were designed upstream the MCS to prevent the formation of aberrant viral proteins (Figure 3B). Finally, the pK7WG2-MCS.HRz.GALV-Nf binary vector was produced (using the SacI/AscI and AsiSI/XbaI restriction sites) placing the GALV-Nf sequence under the control of the CaMV 35S promoter and inserting the HRz ribozyme and nos terminator sequences immediately downstream of the viral 3′ UTR (Figure 3C). This vector can be delivered to plants by agroinfiltration and allows the production of GALV-Nf RNAs almost equivalent to the viral genome. In particular, by using the StuI-DraI ligation strategy described under Materials and Methods, a functional viral 5′ UTR sequence corresponding to the +1 transcriptional start site can be produced, lacking only two nucleotides from the original GALV-Nf genome sequence. The HRz ribozyme can produce the precise viral 3′ terminus by self-cleavage [11].Figure 3

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