Design of a real-time PCR tool to study cell wall stress in fungi

Autores
Da Cruz Cabral, Lucía Mariana; Delgado, J.; Andrade, M.J.; Patriarca, Andrea Rosana; Asensio, M.A.; Rodrigueiro, Marcela
Año de publicación
2017
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
The cell wall integrity (CWI) pathway is responsible forthe reparation and/or biosynthesis of the cell wall and is activated whenchanges on the cell surface occurred mainly under imposed stress. A new toolable to detect changes in stress-related genes would be useful to understandthe action mechanism of some filamentous fungi against antifungal compounds. Theobjective was to develop a real-time PCR (qPCR) using SYBR Green to monitor changesin the Rho1 gene expression levels inmoulds. Optimization of reaction conditions includedevaluation of different primer pairs, primer concentrations and annealing temperatures/times.The final reaction mixture contained 200nM of each primer and an annealingtemperature of 55ºC. The specificity of primers was demonstrated when amplifieda unique qPCR product with a Tm value of 86ºC. The qPCR showed a R2 value =0.9994 and amplificationefficiency of 97.5%. The method was validated by treating Aspergillus flavus and Penicilliumpolonicum with the antifungal protein PgAFP. The PgAFP-resistant P. polonicum showed an overexpression ofRho1, while the opposite trend wasdetected in A. flavus with theantifungal treatment. This qPCR assay is a valuable tool to analyseintracellular responses linked to CWI pathway activation. This provides data toin-depth understand the ability of fungi to colonise different environments andto develop new antifungals.
Fil: Da Cruz Cabral, Lucía Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Delgado, J.. Universidad de Extremadura; España
Fil: Andrade, M.J.. Universidad de Extremadura; España
Fil: Patriarca, Andrea Rosana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina
Fil: Asensio, M.A.. Universidad de Extremadura; España
Fil: Rodrigueiro, Marcela. Universidad de Extremadura; España
FEMS2017: 7th Congress of European Microbiologists
Valencia
España
Federation of European Microbiological Societies
Materia
FUNGI
WALL
STRESS
QPCR
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:11336/275715
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/275715
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Design of a real-time PCR tool to study cell wall stress in fungiDa Cruz Cabral, Lucía MarianaDelgado, J.Andrade, M.J.Patriarca, Andrea RosanaAsensio, M.A.Rodrigueiro, MarcelaFUNGIWALLSTRESSQPCRhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The cell wall integrity (CWI) pathway is responsible forthe reparation and/or biosynthesis of the cell wall and is activated whenchanges on the cell surface occurred mainly under imposed stress. A new toolable to detect changes in stress-related genes would be useful to understandthe action mechanism of some filamentous fungi against antifungal compounds. Theobjective was to develop a real-time PCR (qPCR) using SYBR Green to monitor changesin the Rho1 gene expression levels inmoulds. Optimization of reaction conditions includedevaluation of different primer pairs, primer concentrations and annealing temperatures/times.The final reaction mixture contained 200nM of each primer and an annealingtemperature of 55ºC. The specificity of primers was demonstrated when amplifieda unique qPCR product with a Tm value of 86ºC. The qPCR showed a R2 value =0.9994 and amplificationefficiency of 97.5%. The method was validated by treating Aspergillus flavus and Penicilliumpolonicum with the antifungal protein PgAFP. The PgAFP-resistant P. polonicum showed an overexpression ofRho1, while the opposite trend wasdetected in A. flavus with theantifungal treatment. This qPCR assay is a valuable tool to analyseintracellular responses linked to CWI pathway activation. This provides data toin-depth understand the ability of fungi to colonise different environments andto develop new antifungals.Fil: Da Cruz Cabral, Lucía Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Delgado, J.. Universidad de Extremadura; EspañaFil: Andrade, M.J.. Universidad de Extremadura; EspañaFil: Patriarca, Andrea Rosana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; ArgentinaFil: Asensio, M.A.. Universidad de Extremadura; EspañaFil: Rodrigueiro, Marcela. Universidad de Extremadura; EspañaFEMS2017: 7th Congress of European MicrobiologistsValenciaEspañaFederation of European Microbiological SocietiesFederation of European Microbiological Societies2017info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectCongresoBookhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/275715Design of a real-time PCR tool to study cell wall stress in fungi; FEMS2017: 7th Congress of European Microbiologists; Valencia; España; 2017; 1-1CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://fems-microbiology.org/wp-content/uploads/2020/05/FEMS2017_abstracts-book.pdfinfo:eu-repo/semantics/altIdentifier/url/https://fems-microbiology.org/about_fems/network-and-activities/congress/past-congresses/Internacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-12-23T14:39:41Zoai:ri.conicet.gov.ar:11336/275715instacron: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:34982025-12-23 14:39:41.562CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Design of a real-time PCR tool to study cell wall stress in fungi
title Design of a real-time PCR tool to study cell wall stress in fungi
spellingShingle Design of a real-time PCR tool to study cell wall stress in fungi
Da Cruz Cabral, Lucía Mariana
FUNGI
WALL
STRESS
QPCR
title_short Design of a real-time PCR tool to study cell wall stress in fungi
title_full Design of a real-time PCR tool to study cell wall stress in fungi
title_fullStr Design of a real-time PCR tool to study cell wall stress in fungi
title_full_unstemmed Design of a real-time PCR tool to study cell wall stress in fungi
title_sort Design of a real-time PCR tool to study cell wall stress in fungi
dc.creator.none.fl_str_mv Da Cruz Cabral, Lucía Mariana
Delgado, J.
Andrade, M.J.
Patriarca, Andrea Rosana
Asensio, M.A.
Rodrigueiro, Marcela
author Da Cruz Cabral, Lucía Mariana
author_facet Da Cruz Cabral, Lucía Mariana
Delgado, J.
Andrade, M.J.
Patriarca, Andrea Rosana
Asensio, M.A.
Rodrigueiro, Marcela
author_role author
author2 Delgado, J.
Andrade, M.J.
Patriarca, Andrea Rosana
Asensio, M.A.
Rodrigueiro, Marcela
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv FUNGI
WALL
STRESS
QPCR
topic FUNGI
WALL
STRESS
QPCR
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The cell wall integrity (CWI) pathway is responsible forthe reparation and/or biosynthesis of the cell wall and is activated whenchanges on the cell surface occurred mainly under imposed stress. A new toolable to detect changes in stress-related genes would be useful to understandthe action mechanism of some filamentous fungi against antifungal compounds. Theobjective was to develop a real-time PCR (qPCR) using SYBR Green to monitor changesin the Rho1 gene expression levels inmoulds. Optimization of reaction conditions includedevaluation of different primer pairs, primer concentrations and annealing temperatures/times.The final reaction mixture contained 200nM of each primer and an annealingtemperature of 55ºC. The specificity of primers was demonstrated when amplifieda unique qPCR product with a Tm value of 86ºC. The qPCR showed a R2 value =0.9994 and amplificationefficiency of 97.5%. The method was validated by treating Aspergillus flavus and Penicilliumpolonicum with the antifungal protein PgAFP. The PgAFP-resistant P. polonicum showed an overexpression ofRho1, while the opposite trend wasdetected in A. flavus with theantifungal treatment. This qPCR assay is a valuable tool to analyseintracellular responses linked to CWI pathway activation. This provides data toin-depth understand the ability of fungi to colonise different environments andto develop new antifungals.
Fil: Da Cruz Cabral, Lucía Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Delgado, J.. Universidad de Extremadura; España
Fil: Andrade, M.J.. Universidad de Extremadura; España
Fil: Patriarca, Andrea Rosana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina
Fil: Asensio, M.A.. Universidad de Extremadura; España
Fil: Rodrigueiro, Marcela. Universidad de Extremadura; España
FEMS2017: 7th Congress of European Microbiologists
Valencia
España
Federation of European Microbiological Societies
description The cell wall integrity (CWI) pathway is responsible forthe reparation and/or biosynthesis of the cell wall and is activated whenchanges on the cell surface occurred mainly under imposed stress. A new toolable to detect changes in stress-related genes would be useful to understandthe action mechanism of some filamentous fungi against antifungal compounds. Theobjective was to develop a real-time PCR (qPCR) using SYBR Green to monitor changesin the Rho1 gene expression levels inmoulds. Optimization of reaction conditions includedevaluation of different primer pairs, primer concentrations and annealing temperatures/times.The final reaction mixture contained 200nM of each primer and an annealingtemperature of 55ºC. The specificity of primers was demonstrated when amplifieda unique qPCR product with a Tm value of 86ºC. The qPCR showed a R2 value =0.9994 and amplificationefficiency of 97.5%. The method was validated by treating Aspergillus flavus and Penicilliumpolonicum with the antifungal protein PgAFP. The PgAFP-resistant P. polonicum showed an overexpression ofRho1, while the opposite trend wasdetected in A. flavus with theantifungal treatment. This qPCR assay is a valuable tool to analyseintracellular responses linked to CWI pathway activation. This provides data toin-depth understand the ability of fungi to colonise different environments andto develop new antifungals.
publishDate 2017
dc.date.none.fl_str_mv 2017
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Congreso
Book
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/275715
Design of a real-time PCR tool to study cell wall stress in fungi; FEMS2017: 7th Congress of European Microbiologists; Valencia; España; 2017; 1-1
CONICET Digital
CONICET
url http://hdl.handle.net/11336/275715
identifier_str_mv Design of a real-time PCR tool to study cell wall stress in fungi; FEMS2017: 7th Congress of European Microbiologists; Valencia; España; 2017; 1-1
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://fems-microbiology.org/wp-content/uploads/2020/05/FEMS2017_abstracts-book.pdf
info:eu-repo/semantics/altIdentifier/url/https://fems-microbiology.org/about_fems/network-and-activities/congress/past-congresses/
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.coverage.none.fl_str_mv Internacional
dc.publisher.none.fl_str_mv Federation of European Microbiological Societies
publisher.none.fl_str_mv Federation of European Microbiological Societies
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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