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Development of a gene silencing DNA vector derived from a broad host range geminivirus.

Golenberg EM, Sather DN, Hancock LC, Buckley KJ, Villafranco NM, Bisaro DM - Plant Methods (2009)

Bottom Line: Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application.The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation.However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA. egolenb@biology.biosci.wayne.edu.

ABSTRACT

Background: Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.

Results: The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant.

Conclusion: The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

No MeSH data available.


Related in: MedlinePlus

Silencing phenotypes obtained with pWSRi:rbcS and pWSRi:trnktls display bleached leaves. (a) Leaves from spinach plants treated with pWSRi vector (left) or pWSRi:rbcS (right). (b) Individual leaves from plants treated with pWSRi vector (left) or pWSRi:trnktls (right). The pWSRi:rbcS-treated leaf is yellow-white, whereas pWSRi:trnktls-treated leaf is yellow with residual green color often evident in the veins. (c) Photograph of pWSRi:rbcS treated-plant showing progressive bleaching of the leaves from the petiole, spreading into the blade and distally along the midrib.
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Figure 2: Silencing phenotypes obtained with pWSRi:rbcS and pWSRi:trnktls display bleached leaves. (a) Leaves from spinach plants treated with pWSRi vector (left) or pWSRi:rbcS (right). (b) Individual leaves from plants treated with pWSRi vector (left) or pWSRi:trnktls (right). The pWSRi:rbcS-treated leaf is yellow-white, whereas pWSRi:trnktls-treated leaf is yellow with residual green color often evident in the veins. (c) Photograph of pWSRi:rbcS treated-plant showing progressive bleaching of the leaves from the petiole, spreading into the blade and distally along the midrib.

Mentions: We first chose to test the BCTV vector in cultivated spinach (Spinacea oleracea), a species for which reliable transgenic technology is not yet available. A 407 bp fragment of the spinach rbcS gene and a 245 bp fragment of the spinach transketolase (trnktls) gene were cloned into the NotI-XhoI cloning site within the truncated R1 gene in pWSRi. The two constructs were designated pWSRi:rbcS and pWSRi:trnktls. Both rbcS and transketolase genes have been used as silencing reporter genes in Arabidopsis and Nicotiana systems [7,37,38]. As both genes encode proteins involved in the Calvin cycle, interference with their products results in bleaching of the leaf. In initial trials, six plants were biolistically bombarded at the two leaf-two cotyledon stage with tungsten particles for each treatment listed in Table 2a. There were no observable differences among plants inoculated with empty vector (pWSRi), plants treated with tungsten particles that were not precipitated with DNA, and wild-type untreated plants. However, approximately six to eight weeks after treatment, plants receiving treatments 5, 6, 7, or 8 (pWSRi vectors with target gene inserts with and without the pBCTV.R1 helper plasmid) began to show evidence of bleaching. All plants treated with the rbcS containing vector developed white leaves and there was no readily apparent difference between plants treated with or without pBCTV.R1 in addition to pWSRi:rbcS (Figure 2a). The leaves on all plants receiving treatments with transketolase-containing vectors developed a light yellow color with leaf veins remaining green for another several days (Figure 2b). As with the rbcS treated plants, there was no apparent difference between treatments with or without pBCTV.R1. Thus the pWSRi:trnktls and pWSRi:rbcS constructs successfully generated similar but distinct phenotypes that mimic a photosynthetic mutant. We further concluded that our helper plasmid encoding the viral capsid protein was not necessary and did not obviously enhance vector efficacy. It should be noted that we also did not observe any obvious signs of active viral symptoms, which would be expected if an active BCTV virus were mobile, nor did we assay for capsid protein. Therefore, we cannot conclusively rule out that the pBCTV.R1 was ineffective in expressing the capsid protein.


Development of a gene silencing DNA vector derived from a broad host range geminivirus.

Golenberg EM, Sather DN, Hancock LC, Buckley KJ, Villafranco NM, Bisaro DM - Plant Methods (2009)

Silencing phenotypes obtained with pWSRi:rbcS and pWSRi:trnktls display bleached leaves. (a) Leaves from spinach plants treated with pWSRi vector (left) or pWSRi:rbcS (right). (b) Individual leaves from plants treated with pWSRi vector (left) or pWSRi:trnktls (right). The pWSRi:rbcS-treated leaf is yellow-white, whereas pWSRi:trnktls-treated leaf is yellow with residual green color often evident in the veins. (c) Photograph of pWSRi:rbcS treated-plant showing progressive bleaching of the leaves from the petiole, spreading into the blade and distally along the midrib.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Silencing phenotypes obtained with pWSRi:rbcS and pWSRi:trnktls display bleached leaves. (a) Leaves from spinach plants treated with pWSRi vector (left) or pWSRi:rbcS (right). (b) Individual leaves from plants treated with pWSRi vector (left) or pWSRi:trnktls (right). The pWSRi:rbcS-treated leaf is yellow-white, whereas pWSRi:trnktls-treated leaf is yellow with residual green color often evident in the veins. (c) Photograph of pWSRi:rbcS treated-plant showing progressive bleaching of the leaves from the petiole, spreading into the blade and distally along the midrib.
Mentions: We first chose to test the BCTV vector in cultivated spinach (Spinacea oleracea), a species for which reliable transgenic technology is not yet available. A 407 bp fragment of the spinach rbcS gene and a 245 bp fragment of the spinach transketolase (trnktls) gene were cloned into the NotI-XhoI cloning site within the truncated R1 gene in pWSRi. The two constructs were designated pWSRi:rbcS and pWSRi:trnktls. Both rbcS and transketolase genes have been used as silencing reporter genes in Arabidopsis and Nicotiana systems [7,37,38]. As both genes encode proteins involved in the Calvin cycle, interference with their products results in bleaching of the leaf. In initial trials, six plants were biolistically bombarded at the two leaf-two cotyledon stage with tungsten particles for each treatment listed in Table 2a. There were no observable differences among plants inoculated with empty vector (pWSRi), plants treated with tungsten particles that were not precipitated with DNA, and wild-type untreated plants. However, approximately six to eight weeks after treatment, plants receiving treatments 5, 6, 7, or 8 (pWSRi vectors with target gene inserts with and without the pBCTV.R1 helper plasmid) began to show evidence of bleaching. All plants treated with the rbcS containing vector developed white leaves and there was no readily apparent difference between plants treated with or without pBCTV.R1 in addition to pWSRi:rbcS (Figure 2a). The leaves on all plants receiving treatments with transketolase-containing vectors developed a light yellow color with leaf veins remaining green for another several days (Figure 2b). As with the rbcS treated plants, there was no apparent difference between treatments with or without pBCTV.R1. Thus the pWSRi:trnktls and pWSRi:rbcS constructs successfully generated similar but distinct phenotypes that mimic a photosynthetic mutant. We further concluded that our helper plasmid encoding the viral capsid protein was not necessary and did not obviously enhance vector efficacy. It should be noted that we also did not observe any obvious signs of active viral symptoms, which would be expected if an active BCTV virus were mobile, nor did we assay for capsid protein. Therefore, we cannot conclusively rule out that the pBCTV.R1 was ineffective in expressing the capsid protein.

Bottom Line: Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application.The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation.However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA. egolenb@biology.biosci.wayne.edu.

ABSTRACT

Background: Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.

Results: The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant.

Conclusion: The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

No MeSH data available.


Related in: MedlinePlus