Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus

Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable pr...

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Autores: Shi, Xiaoqing, Cordero, Teresa, Garrigues, Sandra, Marcos López, José Francisco, Daròs Arnau, José Antonio, Coca, María
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2019
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/184034
Acesso em linha:http://hdl.handle.net/10261/184034
Access Level:acceso abierto
Palavra-chave:Antifungal proteins
Gibson assembl
Nicotiana benthamiana
Plant biofactory
Tobacco mosaic virus
Viral vector
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spelling Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virusShi, XiaoqingCordero, TeresaGarrigues, SandraMarcos López, José FranciscoDaròs Arnau, José AntonioCoca, MaríaAntifungal proteinsGibson assemblNicotiana benthamianaPlant biofactoryTobacco mosaic virusViral vectorFungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.This work was supported by grants BIO2017‐83184‐R, BIO2015‐68790‐C2‐1‐R and BIO2015‐68790‐C2‐2‐R and through the ‘Severo Ochoa Programme for Centres of Excellence in R&D’ (SEV‐2015‐0533) from Spanish Ministerio de Ciencia, Innovación y Universidades (co‐financed FEDER funds) and by the CERCA Programme/Generalitat de Catalunya.Peer reviewedWiley-BlackwellAgencia Estatal de Investigación (España)Ministerio de Ciencia, Innovación y Universidades (España)Ministerio de Economía y Competitividad (España)Generalitat de CatalunyaEuropean CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201920192019info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/184034reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/BIO2017-83184-RBIO2017-83184-R/AEI/10.13039/501100011033info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2015-68790-C2-1-Rinfo:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2015-68790-C2-2-Rinfo:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SEV-2015-0533https://doi.org/10.1111/pbi.13038Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1840342026-05-22T06:33:51Z
dc.title.none.fl_str_mv Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
title Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
spellingShingle Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
Shi, Xiaoqing
Antifungal proteins
Gibson assembl
Nicotiana benthamiana
Plant biofactory
Tobacco mosaic virus
Viral vector
title_short Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
title_full Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
title_fullStr Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
title_full_unstemmed Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
title_sort Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus
dc.creator.none.fl_str_mv Shi, Xiaoqing
Cordero, Teresa
Garrigues, Sandra
Marcos López, José Francisco
Daròs Arnau, José Antonio
Coca, María
author Shi, Xiaoqing
author_facet Shi, Xiaoqing
Cordero, Teresa
Garrigues, Sandra
Marcos López, José Francisco
Daròs Arnau, José Antonio
Coca, María
author_role author
author2 Cordero, Teresa
Garrigues, Sandra
Marcos López, José Francisco
Daròs Arnau, José Antonio
Coca, María
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Agencia Estatal de Investigación (España)
Ministerio de Ciencia, Innovación y Universidades (España)
Ministerio de Economía y Competitividad (España)
Generalitat de Catalunya
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Antifungal proteins
Gibson assembl
Nicotiana benthamiana
Plant biofactory
Tobacco mosaic virus
Viral vector
topic Antifungal proteins
Gibson assembl
Nicotiana benthamiana
Plant biofactory
Tobacco mosaic virus
Viral vector
description Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.
publishDate 2019
dc.date.none.fl_str_mv 2019
2019
2019
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/184034
url http://hdl.handle.net/10261/184034
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/BIO2017-83184-R
BIO2017-83184-R/AEI/10.13039/501100011033
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2015-68790-C2-1-R
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2015-68790-C2-2-R
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SEV-2015-0533
https://doi.org/10.1111/pbi.13038
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Wiley-Blackwell
publisher.none.fl_str_mv Wiley-Blackwell
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,812429

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