Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis

Detalhes bibliográficos
Autor(a) principal: Hayashibara, Carolina Alessandra de Almeida
Data de Publicação: 2021
Tipo de documento: Tese
Idioma: eng
Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
Texto Completo: https://www.teses.usp.br/teses/disponiveis/11/11137/tde-09092021-154121/
Resumo: Myrtle rust is a disease caused by the biotrophic fungus Austropuccinia psidii. Biotrophic fungi require living tissues to obtain nutrients and to develop, establishing a close relationship with the host. Since the first contact between pathogen and host, several molecules of both are involved. Information on the interaction between A. psidii and the host is scarce, due to a large number of infected Myrtaceae species, in addition to the biotrophic lifestyle of A. psidii, which limits the in vitro manipulation. To overcome the defense mechanisms of plants, fungi can release effectors during the infection. In general, effectors are molecules secreted by the pathogen that may alter the host\'s physiology for successful infection and colonization. Thus, the characterization of secreted effectors is a crucial strategy to understand the mechanisms involved in the host infection by the pathogen. Chapter 1 shows the first study on effector candidates of A. psidii biotype MF-1. First, 255 effector candidates were identified, and in silico prediction of localization and functions was performed, the most effector candidates were predicted as apoplastic and as hypothetical proteins with unknown functions. By RT-qPCR, we performed in vitro expression validation of seven randomly selected effector candidates, using as stimulus cuticular waxes extracted from leaves of resistant and susceptible Eucalyptus species to the pathogen, E. urophylla and E. grandis, respectively. The seven effector candidate\'s expression was modulated according to cuticular waxes. Two effector candidates, Ap28303 and Ap30385, were cloned into binary vectors containing the G3GFP (Green Fluorescent Protein) protein, and agroinfiltrated in leaves of Nicotiana benthamiana. Agroinfiltration was performed to observe the subcellular localization of the effector candidates, however the effector candidate Ap30385 was not observed in any compartment, and the effector candidate Ap28303 was observed accumulating in the nucleus. For the first time, A. psidii effector candidates were identified, as well as the subcellular localization of one effector candidate. In Chapter 2, the identification and comparison of effectors from three A. psidii biotypes, a pandemic biotype from Australia, a biotype from South Africa, and the MF-1 biotype from Brazil were carried out. It was observed a greater number of effectors from pandemic biotype compared with the others, however, it was not found differences in the predicted localization of effector candidates. By homology analysis, it was observed that the Australian biotype was more distant from the other two, regarding the effector candidates, showing the highest number of exclusive proteins. From PCR validation, the most effector candidates showed conserved among the tested biotypes, and polymorphisms were found. Therefore, deep studies are required for a better understanding of variation importance on host range. Certainly, this pioneering study will enable new studies about effector candidates from A. psidii, improving the comprehension of effector evolution and host relationship, as well offering a profile to further detailed characterization of other effector candidates.
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spelling Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysisEstudo dos efetores candidatos de Austropuccina psidii: identificação, caracterização e análise de genômica comparativaAgroinfiltraçãoAgroinfiltrationBiótiposBiotypesEfetorômicaEffectoromicFerrugem das mirtáceasMyrtle rustMyrtle rust is a disease caused by the biotrophic fungus Austropuccinia psidii. Biotrophic fungi require living tissues to obtain nutrients and to develop, establishing a close relationship with the host. Since the first contact between pathogen and host, several molecules of both are involved. Information on the interaction between A. psidii and the host is scarce, due to a large number of infected Myrtaceae species, in addition to the biotrophic lifestyle of A. psidii, which limits the in vitro manipulation. To overcome the defense mechanisms of plants, fungi can release effectors during the infection. In general, effectors are molecules secreted by the pathogen that may alter the host\'s physiology for successful infection and colonization. Thus, the characterization of secreted effectors is a crucial strategy to understand the mechanisms involved in the host infection by the pathogen. Chapter 1 shows the first study on effector candidates of A. psidii biotype MF-1. First, 255 effector candidates were identified, and in silico prediction of localization and functions was performed, the most effector candidates were predicted as apoplastic and as hypothetical proteins with unknown functions. By RT-qPCR, we performed in vitro expression validation of seven randomly selected effector candidates, using as stimulus cuticular waxes extracted from leaves of resistant and susceptible Eucalyptus species to the pathogen, E. urophylla and E. grandis, respectively. The seven effector candidate\'s expression was modulated according to cuticular waxes. Two effector candidates, Ap28303 and Ap30385, were cloned into binary vectors containing the G3GFP (Green Fluorescent Protein) protein, and agroinfiltrated in leaves of Nicotiana benthamiana. Agroinfiltration was performed to observe the subcellular localization of the effector candidates, however the effector candidate Ap30385 was not observed in any compartment, and the effector candidate Ap28303 was observed accumulating in the nucleus. For the first time, A. psidii effector candidates were identified, as well as the subcellular localization of one effector candidate. In Chapter 2, the identification and comparison of effectors from three A. psidii biotypes, a pandemic biotype from Australia, a biotype from South Africa, and the MF-1 biotype from Brazil were carried out. It was observed a greater number of effectors from pandemic biotype compared with the others, however, it was not found differences in the predicted localization of effector candidates. By homology analysis, it was observed that the Australian biotype was more distant from the other two, regarding the effector candidates, showing the highest number of exclusive proteins. From PCR validation, the most effector candidates showed conserved among the tested biotypes, and polymorphisms were found. Therefore, deep studies are required for a better understanding of variation importance on host range. Certainly, this pioneering study will enable new studies about effector candidates from A. psidii, improving the comprehension of effector evolution and host relationship, as well offering a profile to further detailed characterization of other effector candidates.A ferrugem das mirtáceas é uma doença causada pelo fungo biotrófico Austropuccinia psidii. Fungos biotróficos necessitam dos tecidos vivos do hospedeiro para obtenção de nutrientes e desenvolvimento, criando uma relação íntima com o hospedeiro. Desde o primeiro contato entre patógeno e hospedeiro diversas moléculas estão envolvidas nesta interação. Informações sobre a interação A. psidii e o hospedeiro são escassas, devido ao grande número de espécies de Mirtáceas infectadas, além do estilo de vida biotrófico de A. psidii, que limita a sua manipulação. Para superar os mecanismos de defesa das plantas, os fungos podem liberar efetores durante a infecção do hospedeiro. Em geral, efetores são moléculas secretadas pelo patógeno que podem alterar a fisiologia do hospedeiro para uma bem-sucedida infecção e colonização. Dessa forma, a caracterização de efetores é uma importante estratégia para compreender os mecanismos envolvidos durante a infecção do hospedeiro pelo patógeno. No Capítulo 1 foi realizado o primeiro estudo sobre candidatos a efetores do biótipo MF-1 de A. psidii. Primeiramente foi realizada a identificação de 255 efetores candidatos, e a predição da localização e funções dos mesmos, sendo a maioria dos candidatos preditos como apoplásticos e com funções hipotéticas. Foi realizada uma validação de expressão in vitro por RT-qPCR de sete efetores candidatos selecionados aleatoriamente, utilizando como estímulo ceras cuticulares extraídas de folhas de espécies de eucalipto resistentes e suscetíveis ao patógeno, Eucalyptus urophylla e E. grandis, respectivamente. Foi observado que a expressão dos sete efetores candidatos foi modulada de acordo com a espécie, as quais as ceras cuticulares eram provenientes. Dois efetores candidatos, Ap28303 e Ap30385, foram clonados em vetores binários contendo a proteína G3GFP (proteína verde fluorescente), e agroinfiltrados em folhas de Nicotiana benthamiana. A agroinfiltração foi realizada para observar a localização subcelular dos efetores candidatos, porém o efetor candidato Ap30385 não foi observado em nenhum compartimento, enquanto foi observado um acúmulo do efetor candidato Ap28303 no núcleo. Pela primeira vez foram identificados efetores candidatos de A. psidii, bem como a localização subcelular de um efetor candidato. No Capítulo 2, foi realizada a identificação e comparação de efetores de três biótipos de A. psidii, um biótipo pandêmico originário da Austrália (Au), um biótipo da África do Sul (SA) e o biótipo MF-1 do Brasil. Foi observado um número maior de efetores do biótipo pandêmico, em comparação aos demais, no entanto, não foram encontradas diferenças nas localizações preditas dos efetores candidatos. Nas análises de homologia, também se observou que o biótipo australiano se mostrou mais distante dos outros dois, em relação aos efetores candidatos, possuindo um maior número de proteínas exclusivas. A partir da validação por PCR, a maioria dos efetores candidatos mostraram-se conservados entre os biótipos testados, mas também foram encontrados polimorfismos. Portanto, estudos mais aprofundados sobre esses polimorfismos encontrados serão necessários para um melhor entendimento da importância dessas variações na amplitude de hospedeiros. Este estudo pioneiro certamente possibilita novos estudos acerca de efetores candidatos de A. psidii, em relação a evolução dos efetores quanto ao hospedeiro, bem como uma detalhada caracterização de outros efetores candidatos.Biblioteca Digitais de Teses e Dissertações da USPVerdi, Maria Carolina QuecineHayashibara, Carolina Alessandra de Almeida2021-06-10info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttps://www.teses.usp.br/teses/disponiveis/11/11137/tde-09092021-154121/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPLiberar o conteúdo para acesso público.info:eu-repo/semantics/openAccesseng2023-09-09T13:00:13Zoai:teses.usp.br:tde-09092021-154121Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212023-09-09T13:00:13Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false
dc.title.none.fl_str_mv Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
Estudo dos efetores candidatos de Austropuccina psidii: identificação, caracterização e análise de genômica comparativa
title Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
spellingShingle Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
Hayashibara, Carolina Alessandra de Almeida
Agroinfiltração
Agroinfiltration
Biótipos
Biotypes
Efetorômica
Effectoromic
Ferrugem das mirtáceas
Myrtle rust
title_short Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
title_full Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
title_fullStr Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
title_full_unstemmed Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
title_sort Insights on effector candidates of Austropuccinia psidii: identification, characterization and comparative genomic analysis
author Hayashibara, Carolina Alessandra de Almeida
author_facet Hayashibara, Carolina Alessandra de Almeida
author_role author
dc.contributor.none.fl_str_mv Verdi, Maria Carolina Quecine
dc.contributor.author.fl_str_mv Hayashibara, Carolina Alessandra de Almeida
dc.subject.por.fl_str_mv Agroinfiltração
Agroinfiltration
Biótipos
Biotypes
Efetorômica
Effectoromic
Ferrugem das mirtáceas
Myrtle rust
topic Agroinfiltração
Agroinfiltration
Biótipos
Biotypes
Efetorômica
Effectoromic
Ferrugem das mirtáceas
Myrtle rust
description Myrtle rust is a disease caused by the biotrophic fungus Austropuccinia psidii. Biotrophic fungi require living tissues to obtain nutrients and to develop, establishing a close relationship with the host. Since the first contact between pathogen and host, several molecules of both are involved. Information on the interaction between A. psidii and the host is scarce, due to a large number of infected Myrtaceae species, in addition to the biotrophic lifestyle of A. psidii, which limits the in vitro manipulation. To overcome the defense mechanisms of plants, fungi can release effectors during the infection. In general, effectors are molecules secreted by the pathogen that may alter the host\'s physiology for successful infection and colonization. Thus, the characterization of secreted effectors is a crucial strategy to understand the mechanisms involved in the host infection by the pathogen. Chapter 1 shows the first study on effector candidates of A. psidii biotype MF-1. First, 255 effector candidates were identified, and in silico prediction of localization and functions was performed, the most effector candidates were predicted as apoplastic and as hypothetical proteins with unknown functions. By RT-qPCR, we performed in vitro expression validation of seven randomly selected effector candidates, using as stimulus cuticular waxes extracted from leaves of resistant and susceptible Eucalyptus species to the pathogen, E. urophylla and E. grandis, respectively. The seven effector candidate\'s expression was modulated according to cuticular waxes. Two effector candidates, Ap28303 and Ap30385, were cloned into binary vectors containing the G3GFP (Green Fluorescent Protein) protein, and agroinfiltrated in leaves of Nicotiana benthamiana. Agroinfiltration was performed to observe the subcellular localization of the effector candidates, however the effector candidate Ap30385 was not observed in any compartment, and the effector candidate Ap28303 was observed accumulating in the nucleus. For the first time, A. psidii effector candidates were identified, as well as the subcellular localization of one effector candidate. In Chapter 2, the identification and comparison of effectors from three A. psidii biotypes, a pandemic biotype from Australia, a biotype from South Africa, and the MF-1 biotype from Brazil were carried out. It was observed a greater number of effectors from pandemic biotype compared with the others, however, it was not found differences in the predicted localization of effector candidates. By homology analysis, it was observed that the Australian biotype was more distant from the other two, regarding the effector candidates, showing the highest number of exclusive proteins. From PCR validation, the most effector candidates showed conserved among the tested biotypes, and polymorphisms were found. Therefore, deep studies are required for a better understanding of variation importance on host range. Certainly, this pioneering study will enable new studies about effector candidates from A. psidii, improving the comprehension of effector evolution and host relationship, as well offering a profile to further detailed characterization of other effector candidates.
publishDate 2021
dc.date.none.fl_str_mv 2021-06-10
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
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dc.identifier.uri.fl_str_mv https://www.teses.usp.br/teses/disponiveis/11/11137/tde-09092021-154121/
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dc.rights.driver.fl_str_mv Liberar o conteúdo para acesso público.
info:eu-repo/semantics/openAccess
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eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
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dc.publisher.none.fl_str_mv Biblioteca Digitais de Teses e Dissertações da USP
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reponame:Biblioteca Digital de Teses e Dissertações da USP
instname:Universidade de São Paulo (USP)
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