Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance
Tospoviruses are devastating plant viruses causing severe economic losses in a diverse range of crops worldwide. Here, we describe the development and evaluation of an RNA interference (RNAi) broad-spectrum virus resistance strategy based on a unique and short hairpin-RNA-generating construct (pNhpR...
| Autores principales: | , , , |
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| Formato: | Artículo |
| Lenguaje: | Inglés |
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2018
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| Acceso en línea: | https://link.springer.com/article/10.1007/s10535-015-0530-1 http://hdl.handle.net/20.500.12123/2240 https://doi.org/10.1007/s10535-015-0530-1 |
| _version_ | 1855483019931942912 |
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| author | Debat, Humberto Julio Grabiele, Mauro Ducasse, Daniel Adrian Lopez Lambertini, Paola Maria |
| author_browse | Debat, Humberto Julio Ducasse, Daniel Adrian Grabiele, Mauro Lopez Lambertini, Paola Maria |
| author_facet | Debat, Humberto Julio Grabiele, Mauro Ducasse, Daniel Adrian Lopez Lambertini, Paola Maria |
| author_sort | Debat, Humberto Julio |
| collection | INTA Digital |
| description | Tospoviruses are devastating plant viruses causing severe economic losses in a diverse range of crops worldwide. Here, we describe the development and evaluation of an RNA interference (RNAi) broad-spectrum virus resistance strategy based on a unique and short hairpin-RNA-generating construct (pNhpRNA). This construct was designed from a region of the nucleocapsid gene (N) of Tomato spotted wilt virus (TSWV) that showed a high sequence identity to the corresponding region in the related species Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV). To test the effectiveness of the pNhpRNA construct, we developed a silencing reporter assay based on three fusion proteins in which the complete viral N gene sequence from each of the three tospoviruses was fused in frame to the green fluorescent protein (GFP) sequence. Co-agroinoculation of these constructs with pNhpRNA into leaves of Nicotiana benthamiana resulted in a strong silencing phenotype determined by GFP decay and suppression of the three N genes at the RNA and protein levels. To test the potential of the pNhpRNA construct to generate virus-resistant plants, we infiltrated the whole shoots of N. benthamiana with pNhpRNA. When these infiltrated plants were mechanically inoculated with the mentioned viruses 100, 70, and 60 % resistance phenotypes to TSWV, GRSV, and TCSV, respectively, were observed. The induction of a broad tospovirus resistance with a simple construct and a minimized off-target effect are the main contributions of pNhpRNA. |
| format | Artículo |
| id | INTA2240 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| record_format | dspace |
| spelling | INTA22402018-04-13T12:53:07Z Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance Debat, Humberto Julio Grabiele, Mauro Ducasse, Daniel Adrian Lopez Lambertini, Paola Maria ARN Resistencia a la Enfermedad Virus de las Plantas RNA Disease Resistance Plant Viruses Transgenes Tospovirus Tospoviruses are devastating plant viruses causing severe economic losses in a diverse range of crops worldwide. Here, we describe the development and evaluation of an RNA interference (RNAi) broad-spectrum virus resistance strategy based on a unique and short hairpin-RNA-generating construct (pNhpRNA). This construct was designed from a region of the nucleocapsid gene (N) of Tomato spotted wilt virus (TSWV) that showed a high sequence identity to the corresponding region in the related species Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV). To test the effectiveness of the pNhpRNA construct, we developed a silencing reporter assay based on three fusion proteins in which the complete viral N gene sequence from each of the three tospoviruses was fused in frame to the green fluorescent protein (GFP) sequence. Co-agroinoculation of these constructs with pNhpRNA into leaves of Nicotiana benthamiana resulted in a strong silencing phenotype determined by GFP decay and suppression of the three N genes at the RNA and protein levels. To test the potential of the pNhpRNA construct to generate virus-resistant plants, we infiltrated the whole shoots of N. benthamiana with pNhpRNA. When these infiltrated plants were mechanically inoculated with the mentioned viruses 100, 70, and 60 % resistance phenotypes to TSWV, GRSV, and TCSV, respectively, were observed. The induction of a broad tospovirus resistance with a simple construct and a minimized off-target effect are the main contributions of pNhpRNA. Instituto de Patología Vegetal Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Grabiele, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; Argentina Fil: Ducasse, Daniel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Lopez Lambertini, Paola Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina 2018-04-13T12:26:52Z 2018-04-13T12:26:52Z 2015-12 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion https://link.springer.com/article/10.1007/s10535-015-0530-1 http://hdl.handle.net/20.500.12123/2240 0006-3134 (Print) 1573-8264 (Online) https://doi.org/10.1007/s10535-015-0530-1 eng info:eu-repo/semantics/restrictedAccess application/pdf Biologia Plantarum 59 (4) : 715–725 (December 2015) |
| spellingShingle | ARN Resistencia a la Enfermedad Virus de las Plantas RNA Disease Resistance Plant Viruses Transgenes Tospovirus Debat, Humberto Julio Grabiele, Mauro Ducasse, Daniel Adrian Lopez Lambertini, Paola Maria Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance |
| title | Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance |
| title_full | Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance |
| title_fullStr | Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance |
| title_full_unstemmed | Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance |
| title_short | Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance |
| title_sort | use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hprna construct to induce multiple tospovirus resistance |
| topic | ARN Resistencia a la Enfermedad Virus de las Plantas RNA Disease Resistance Plant Viruses Transgenes Tospovirus |
| url | https://link.springer.com/article/10.1007/s10535-015-0530-1 http://hdl.handle.net/20.500.12123/2240 https://doi.org/10.1007/s10535-015-0530-1 |
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