Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris

Autores
Conforte, Valeria Paola; Otero, Lisandro Horacio; Toum, Laila; Sirigu, Serena; Antelo, Giuliano Tomás; Rinaldi, Jimena Julieta; Klinke, Sebastian; Chavas, Leonardo; Goldbaum, Fernando Alberto; Malamud, Florencia; Bonomi, Hernán Ruy
Año de publicación
2021
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Photoreceptors are able to detect light and transduce that signal generating a cellular response. Among them are the red/far red light sensing bacteriophytochromes (BphP). These bilin-binding proteins have the ability to photoswitch between two states, a red-absorbing (Pr) and a far-red-absorbing (Pfr), by the isomerization of the bilin chromophore and generating structural changes that result in the transduction of the light signal into biochemical signaling. The genome of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, codes for a functional BphP (XccBphP) which was extensively described in previous studies. It has been defined as a negative regulator of several light-mediated mechanisms involved in its virulence. Here, we deepened the analysis of the XccBphP structure and function. In this work, we designed and constructed three different variants with single amino acid changes that affect XccBphP photocycle favoring its Pr state: L193Q, L193N and D199A. We purified the recombinants full-length mutants, crystalized them and solved their crystal structures, showing a Pr conformation almost identical to the wild-type previously obtained. We also examined their UV-Vis absorption spectroscopic properties, proving that Pr is their preferred state. After establishing the effect of each mutation on the structure of XccBphP, we tested the effects of altering the XccBphP photocycle using exopolysaccharide (EPS) production and stomatal aperture assays as indicators of its bacterial signaling pathway. Null mutant complementation assays showed that L193Q or L193N Pr-stabilized versions decreased bacterial EPS production in darkness or under far-red light and increased it under red light in an amplified manner compared to the complementation with the wild-type version. Furthermore, strains expressing Pr-favored XccBphP versions could not promote stomatal reopening at Xcc null mutant levels when tested in Arabidopsis epidermis. Taken together, our results highlight the relevance of the XccBphP Pr-Pfr balance in physiological processes in Xcc.
Fil: Conforte, Valeria Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Sirigu, Serena. Synchrotron Soleil; Francia
Fil: Antelo, Giuliano Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Chavas, Leonardo. Synchrotron Soleil; Francia
Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Malamud, Florencia. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Bonomi, Hernán Ruy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology
Argentina
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Materia
PHYTOCHROME
BPHP
SIGNALING
X-RAY CRYSTALLOGRAPHY
VIRULENCE
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:11336/167534
Acceder
id CONICETDig_525f70dd90859b10921b4628010ac9c6
oai_identifier_str oai:ri.conicet.gov.ar:11336/167534
network_acronym_str CONICETDig
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network_name_str CONICET Digital (CONICET)
spelling Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestrisConforte, Valeria PaolaOtero, Lisandro HoracioToum, LailaSirigu, SerenaAntelo, Giuliano TomásRinaldi, Jimena JulietaKlinke, SebastianChavas, LeonardoGoldbaum, Fernando AlbertoMalamud, FlorenciaBonomi, Hernán RuyPHYTOCHROMEBPHPSIGNALINGX-RAY CRYSTALLOGRAPHYVIRULENCEhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Photoreceptors are able to detect light and transduce that signal generating a cellular response. Among them are the red/far red light sensing bacteriophytochromes (BphP). These bilin-binding proteins have the ability to photoswitch between two states, a red-absorbing (Pr) and a far-red-absorbing (Pfr), by the isomerization of the bilin chromophore and generating structural changes that result in the transduction of the light signal into biochemical signaling. The genome of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, codes for a functional BphP (XccBphP) which was extensively described in previous studies. It has been defined as a negative regulator of several light-mediated mechanisms involved in its virulence. Here, we deepened the analysis of the XccBphP structure and function. In this work, we designed and constructed three different variants with single amino acid changes that affect XccBphP photocycle favoring its Pr state: L193Q, L193N and D199A. We purified the recombinants full-length mutants, crystalized them and solved their crystal structures, showing a Pr conformation almost identical to the wild-type previously obtained. We also examined their UV-Vis absorption spectroscopic properties, proving that Pr is their preferred state. After establishing the effect of each mutation on the structure of XccBphP, we tested the effects of altering the XccBphP photocycle using exopolysaccharide (EPS) production and stomatal aperture assays as indicators of its bacterial signaling pathway. Null mutant complementation assays showed that L193Q or L193N Pr-stabilized versions decreased bacterial EPS production in darkness or under far-red light and increased it under red light in an amplified manner compared to the complementation with the wild-type version. Furthermore, strains expressing Pr-favored XccBphP versions could not promote stomatal reopening at Xcc null mutant levels when tested in Arabidopsis epidermis. Taken together, our results highlight the relevance of the XccBphP Pr-Pfr balance in physiological processes in Xcc.Fil: Conforte, Valeria Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Sirigu, Serena. Synchrotron Soleil; FranciaFil: Antelo, Giuliano Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Chavas, Leonardo. Synchrotron Soleil; FranciaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Malamud, Florencia. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bonomi, Hernán Ruy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaLVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General MicrobiologyArgentinaSociedad Argentina de Investigación en Bioquímica y Biología MolecularTech Science Press2021info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciatext/plainapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/167534Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris; LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology; Argentina; 2020; 104-1040327-95451667-5746CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.saib.org.ar/sites/default/files/BIOCELL-SAIB-2020-version-final.pdfNacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:44:23Zoai:ri.conicet.gov.ar:11336/167534instacron: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:34982025-10-15 15:44:23.512CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
title Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
spellingShingle Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
Conforte, Valeria Paola
PHYTOCHROME
BPHP
SIGNALING
X-RAY CRYSTALLOGRAPHY
VIRULENCE
title_short Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
title_full Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
title_fullStr Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
title_full_unstemmed Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
title_sort Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris
dc.creator.none.fl_str_mv Conforte, Valeria Paola
Otero, Lisandro Horacio
Toum, Laila
Sirigu, Serena
Antelo, Giuliano Tomás
Rinaldi, Jimena Julieta
Klinke, Sebastian
Chavas, Leonardo
Goldbaum, Fernando Alberto
Malamud, Florencia
Bonomi, Hernán Ruy
author Conforte, Valeria Paola
author_facet Conforte, Valeria Paola
Otero, Lisandro Horacio
Toum, Laila
Sirigu, Serena
Antelo, Giuliano Tomás
Rinaldi, Jimena Julieta
Klinke, Sebastian
Chavas, Leonardo
Goldbaum, Fernando Alberto
Malamud, Florencia
Bonomi, Hernán Ruy
author_role author
author2 Otero, Lisandro Horacio
Toum, Laila
Sirigu, Serena
Antelo, Giuliano Tomás
Rinaldi, Jimena Julieta
Klinke, Sebastian
Chavas, Leonardo
Goldbaum, Fernando Alberto
Malamud, Florencia
Bonomi, Hernán Ruy
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv PHYTOCHROME
BPHP
SIGNALING
X-RAY CRYSTALLOGRAPHY
VIRULENCE
topic PHYTOCHROME
BPHP
SIGNALING
X-RAY CRYSTALLOGRAPHY
VIRULENCE
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Photoreceptors are able to detect light and transduce that signal generating a cellular response. Among them are the red/far red light sensing bacteriophytochromes (BphP). These bilin-binding proteins have the ability to photoswitch between two states, a red-absorbing (Pr) and a far-red-absorbing (Pfr), by the isomerization of the bilin chromophore and generating structural changes that result in the transduction of the light signal into biochemical signaling. The genome of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, codes for a functional BphP (XccBphP) which was extensively described in previous studies. It has been defined as a negative regulator of several light-mediated mechanisms involved in its virulence. Here, we deepened the analysis of the XccBphP structure and function. In this work, we designed and constructed three different variants with single amino acid changes that affect XccBphP photocycle favoring its Pr state: L193Q, L193N and D199A. We purified the recombinants full-length mutants, crystalized them and solved their crystal structures, showing a Pr conformation almost identical to the wild-type previously obtained. We also examined their UV-Vis absorption spectroscopic properties, proving that Pr is their preferred state. After establishing the effect of each mutation on the structure of XccBphP, we tested the effects of altering the XccBphP photocycle using exopolysaccharide (EPS) production and stomatal aperture assays as indicators of its bacterial signaling pathway. Null mutant complementation assays showed that L193Q or L193N Pr-stabilized versions decreased bacterial EPS production in darkness or under far-red light and increased it under red light in an amplified manner compared to the complementation with the wild-type version. Furthermore, strains expressing Pr-favored XccBphP versions could not promote stomatal reopening at Xcc null mutant levels when tested in Arabidopsis epidermis. Taken together, our results highlight the relevance of the XccBphP Pr-Pfr balance in physiological processes in Xcc.
Fil: Conforte, Valeria Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina
Fil: Sirigu, Serena. Synchrotron Soleil; Francia
Fil: Antelo, Giuliano Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Chavas, Leonardo. Synchrotron Soleil; Francia
Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Malamud, Florencia. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Bonomi, Hernán Ruy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology
Argentina
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
description Photoreceptors are able to detect light and transduce that signal generating a cellular response. Among them are the red/far red light sensing bacteriophytochromes (BphP). These bilin-binding proteins have the ability to photoswitch between two states, a red-absorbing (Pr) and a far-red-absorbing (Pfr), by the isomerization of the bilin chromophore and generating structural changes that result in the transduction of the light signal into biochemical signaling. The genome of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, codes for a functional BphP (XccBphP) which was extensively described in previous studies. It has been defined as a negative regulator of several light-mediated mechanisms involved in its virulence. Here, we deepened the analysis of the XccBphP structure and function. In this work, we designed and constructed three different variants with single amino acid changes that affect XccBphP photocycle favoring its Pr state: L193Q, L193N and D199A. We purified the recombinants full-length mutants, crystalized them and solved their crystal structures, showing a Pr conformation almost identical to the wild-type previously obtained. We also examined their UV-Vis absorption spectroscopic properties, proving that Pr is their preferred state. After establishing the effect of each mutation on the structure of XccBphP, we tested the effects of altering the XccBphP photocycle using exopolysaccharide (EPS) production and stomatal aperture assays as indicators of its bacterial signaling pathway. Null mutant complementation assays showed that L193Q or L193N Pr-stabilized versions decreased bacterial EPS production in darkness or under far-red light and increased it under red light in an amplified manner compared to the complementation with the wild-type version. Furthermore, strains expressing Pr-favored XccBphP versions could not promote stomatal reopening at Xcc null mutant levels when tested in Arabidopsis epidermis. Taken together, our results highlight the relevance of the XccBphP Pr-Pfr balance in physiological processes in Xcc.
publishDate 2021
dc.date.none.fl_str_mv 2021
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
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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/11336/167534
Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris; LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology; Argentina; 2020; 104-104
0327-9545
1667-5746
CONICET Digital
CONICET
url http://hdl.handle.net/11336/167534
identifier_str_mv Structural and functional analyses of Pr-induced variants of XccBphP bacteriophytochrome from Xanthomas campestris; LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology; Argentina; 2020; 104-104
0327-9545
1667-5746
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://www.saib.org.ar/sites/default/files/BIOCELL-SAIB-2020-version-final.pdf
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dc.format.none.fl_str_mv text/plain
application/pdf
application/pdf
dc.coverage.none.fl_str_mv Nacional
dc.publisher.none.fl_str_mv Tech Science Press
publisher.none.fl_str_mv Tech Science Press
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repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
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