Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance

Autores
Debat, Humberto Julio; Grabiele, Mauro; Ducasse, Daniel Adrián; López Lambertini, Paola María
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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.
Fil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina
Fil: Grabiele, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina
Fil: Ducasse, Daniel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina
Fil: López Lambertini, Paola María. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina
Materia
Transient Assay
Gene Silencing
Broad-Spectrum Resistance
Transgene Evaluation
Tobacco
Nicotiana Benthamiana
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/45371
Acceder
id CONICETDig_3555c5498e196c67cbda19dcf24c80cf
oai_identifier_str oai:ri.conicet.gov.ar:11336/45371
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistanceDebat, Humberto JulioGrabiele, MauroDucasse, Daniel AdriánLópez Lambertini, Paola MaríaTransient AssayGene SilencingBroad-Spectrum ResistanceTransgene EvaluationTobaccoNicotiana Benthamianahttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Tospoviruses 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.Fil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Grabiele, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; ArgentinaFil: Ducasse, Daniel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: López Lambertini, Paola María. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaSpringer2015-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/45371Debat, Humberto Julio; Grabiele, Mauro; Ducasse, Daniel Adrián; López Lambertini, Paola María; Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance; Springer; Biologia Plantarum; 59; 4; 12-2015; 715-7250006-3134CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://link.springer.com/article/10.1007/s10535-015-0530-1info:eu-repo/semantics/altIdentifier/doi/10.1007/s10535-015-0530-1info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:03:11Zoai:ri.conicet.gov.ar:11336/45371instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:03:11.914CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv 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
spellingShingle Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance
Debat, Humberto Julio
Transient Assay
Gene Silencing
Broad-Spectrum Resistance
Transgene Evaluation
Tobacco
Nicotiana Benthamiana
title_short 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_sort Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance
dc.creator.none.fl_str_mv Debat, Humberto Julio
Grabiele, Mauro
Ducasse, Daniel Adrián
López Lambertini, Paola María
author Debat, Humberto Julio
author_facet Debat, Humberto Julio
Grabiele, Mauro
Ducasse, Daniel Adrián
López Lambertini, Paola María
author_role author
author2 Grabiele, Mauro
Ducasse, Daniel Adrián
López Lambertini, Paola María
author2_role author
author
author
dc.subject.none.fl_str_mv Transient Assay
Gene Silencing
Broad-Spectrum Resistance
Transgene Evaluation
Tobacco
Nicotiana Benthamiana
topic Transient Assay
Gene Silencing
Broad-Spectrum Resistance
Transgene Evaluation
Tobacco
Nicotiana Benthamiana
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv 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.
Fil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina
Fil: Grabiele, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina
Fil: Ducasse, Daniel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina
Fil: López Lambertini, Paola María. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina
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.
publishDate 2015
dc.date.none.fl_str_mv 2015-12
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/45371
Debat, Humberto Julio; Grabiele, Mauro; Ducasse, Daniel Adrián; López Lambertini, Paola María; Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance; Springer; Biologia Plantarum; 59; 4; 12-2015; 715-725
0006-3134
CONICET Digital
CONICET
url http://hdl.handle.net/11336/45371
identifier_str_mv Debat, Humberto Julio; Grabiele, Mauro; Ducasse, Daniel Adrián; López Lambertini, Paola María; Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance; Springer; Biologia Plantarum; 59; 4; 12-2015; 715-725
0006-3134
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://link.springer.com/article/10.1007/s10535-015-0530-1
info:eu-repo/semantics/altIdentifier/doi/10.1007/s10535-015-0530-1
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Springer
publisher.none.fl_str_mv Springer
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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