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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 phenotype obtained with pWSRiA:ChlI show a mosaic of leaf bleaching. Tomato plants were agroinfiltrated with pWSRiA:ChlI to silence the sulfur allele of magnesium chelatase, or with pWSRiA:CAT (negative control). Photographs were taken approximately 4 weeks post-infiltration. (a) Plant treated with pWSRiA:ChlI. (b) Leaves of plant treated with pWSRiA:CAT. (c) Leaves of plant treated with pWSRiA:ChlI showing progressive bleaching beginning from the midrib.
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Figure 6: Silencing phenotype obtained with pWSRiA:ChlI show a mosaic of leaf bleaching. Tomato plants were agroinfiltrated with pWSRiA:ChlI to silence the sulfur allele of magnesium chelatase, or with pWSRiA:CAT (negative control). Photographs were taken approximately 4 weeks post-infiltration. (a) Plant treated with pWSRiA:ChlI. (b) Leaves of plant treated with pWSRiA:CAT. (c) Leaves of plant treated with pWSRiA:ChlI showing progressive bleaching beginning from the midrib.

Mentions: In four independent experiments with a total of 15 inoculated plants, 12 of 15 plants agroinfiltrated at the two to four leaf stage with pWSRiA:ChlI exhibited bleaching indicative of silencing (Table 4). All 16 plants treated with pWSRiA:CAT were identical to untreated control plants. Bleaching was first observed in new leaves of pWSRiA:ChlI treated plants three to four weeks post-infiltration in the petioles and midribs, and spread distally through affected leaves. Bleaching was not uniform across the leaf surface, however, most leaves above the infiltration site were typically affected (Figure 6).


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 phenotype obtained with pWSRiA:ChlI show a mosaic of leaf bleaching. Tomato plants were agroinfiltrated with pWSRiA:ChlI to silence the sulfur allele of magnesium chelatase, or with pWSRiA:CAT (negative control). Photographs were taken approximately 4 weeks post-infiltration. (a) Plant treated with pWSRiA:ChlI. (b) Leaves of plant treated with pWSRiA:CAT. (c) Leaves of plant treated with pWSRiA:ChlI showing progressive bleaching beginning from the midrib.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713212&req=5

Figure 6: Silencing phenotype obtained with pWSRiA:ChlI show a mosaic of leaf bleaching. Tomato plants were agroinfiltrated with pWSRiA:ChlI to silence the sulfur allele of magnesium chelatase, or with pWSRiA:CAT (negative control). Photographs were taken approximately 4 weeks post-infiltration. (a) Plant treated with pWSRiA:ChlI. (b) Leaves of plant treated with pWSRiA:CAT. (c) Leaves of plant treated with pWSRiA:ChlI showing progressive bleaching beginning from the midrib.
Mentions: In four independent experiments with a total of 15 inoculated plants, 12 of 15 plants agroinfiltrated at the two to four leaf stage with pWSRiA:ChlI exhibited bleaching indicative of silencing (Table 4). All 16 plants treated with pWSRiA:CAT were identical to untreated control plants. Bleaching was first observed in new leaves of pWSRiA:ChlI treated plants three to four weeks post-infiltration in the petioles and midribs, and spread distally through affected leaves. Bleaching was not uniform across the leaf surface, however, most leaves above the infiltration site were typically affected (Figure 6).

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