Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro

Detalhes bibliográficos
Autor(a) principal: Maia, Thiago Andrade
Data de Publicação: 2013
Tipo de documento: Tese
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/1069
Resumo: Coffee leaf rust, caused by the biotrophic fungus Hemileia vastatrix, is the main phytosanitary problem of the coffee plant. The disease may cause yield losses ranging from 35 to 50% if control measures are not implemented. During the interaction with the host plant, rust fungi secrete a variety of effector proteins that modify the structure and function of the host cell allowing the biotrophic interaction. Some of these effector proteins, known as avirulence proteins (Avr), are recognized by plant proteins encoded by resistance genes, which triggers a defense response against pathogen infection. At least nine dominant genes (SH1 to SH9) that confer resistance to coffee leaf rust have been genetically identified, but despite many years of research on this pathosystem, the complementary Avr genes of H. vastatrix have not yet been identified or characterized. One approach that can be used for this purpose is to conduct an transcriptome and bioinformatics analyses in order to identify candidate genes of H. vastatrix that fulfill a set of specific criteria common to effector proteins, and their validation by conducting functional assays for effector activities. Therefore, the objectives of this study were: (i) to identify genes of H. vastatrix that encode proteins secreted during the germination of urediniospores as well as during the biotrophic interaction with the coffee plant, (ii) to express the effector gene candidates from H. vastatrix in the yeast secretion trap system (YST) in order to obtain biological confirmation of the secretion prediction made by bioinformatics tools, (iii) to analyze the genomic structure of a select group of effector gene candidates, (iv) to perform expression analysis of the identified candidates during different stages of infection; and (v) to establish a protocol for transient expression of H. vastatrix secreted proteins in coffee leaves based on the Type III Secretion System of Pseudomonas syringae pv. garcae. Initially, the secretome of germinated urediniospores of H. vastatrix was characterized by constructing a cDNA library from mRNA isolated from urediniospores germinated in vitro during 16 hours. After analysis of the ESTs using bioinformatics tools, 146 ORFs that encode secreted proteins were selected. Annotation of these proteins showed that 20% of the secretome from germinated urediniospores consists of hydrolytic enzymes and that most (67%) of the identified secreted proteins have unknown function. From the latter group of sequences, 35 complete ORFs encoding proteins sharing features with effectors from filamentous fungi and named H. vastatrix effector candidates (HvECs) were selected for further analysed. The genomic sequences of 22 of these HvECs were characterized, confirming the ORFs predicted by bioinformatics. The secretion of eight selected HvECs was confirmed in yeast and their expression analysis revealed a differential expression, which possibly occurs in coordination with the morphological changes of the pathogen during the early stages of infection. The secretome of H. vastatrix during a compatible plant-rust interaction was also characterized by a combination of Sanger sequencing and 454 pyrosequencing. This integrated approach allowed the massive sequencing of a substantial number of sequence tags (ESTs) of genes expressed at four different times during the biotrophic interaction: 48 hours, 72 hours, 9 days and 12 days after inoculation of coffee leaves with H. vastatrix. After bioinformatics analyses of these ESTs, 75 HvECs were selected and an expression analyses based on RT- PCR confirmed the fungal origin for 62 of them. 22 of these HvECs are preferentially expressed during the interaction of the fungus with the coffee plant and show distinct patterns of expression along the progress of infection. Using the pEDV system and the Type III Secretion System of P. syringae pv. garcae it was established a protocol to express transiently secreted proteins from H. vastatrix in coffee leaves established. Transient expression of HvEC-016 inside the cytoplasm of coffee plants carrying the rust resistant gene SH1, triggered a plant defense response indicating that there may have occurred a recognition of the HvEC-016 effector protein mediated by the R protein encoded by SH1. The catalog of effector gene candidates for H. vastatrix expressed during both urediniospore germination and the interaction with the coffee plant and the transient assay established in this study is an important plataform to identify additional avirulence genes from Hemileia vastatrix.
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spelling Maia, Thiago Andradehttp://lattes.cnpq.br/6798704880640812Mizubuti, Eduardo Seiti Gomidehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785633J8Queiroz, Marisa Vieira dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785812Z5Brommonschenkel, Sérgio Hermíniohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780948Y4Resende, Mário Lúcio Vilela dehttp://lattes.cnpq.br/8921922898103482Barreto, Robert Weingarthttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783300H6Cunha, Luis Claudio Vieira dahttp://lattes.cnpq.br/9367947173322278Pacheco, Jorge Luis Badelhttp://lattes.cnpq.br/49219325502442952015-03-26T12:42:06Z2015-01-202015-03-26T12:42:06Z2013-02-27MAIA, Thiago Andrade. Indentification and funtional analysis of effectors from Hemileia vastatrix, the causal agent of the coffee rust. 2013. 137 f. Tese (Doutorado em Etiologia; Epidemiologia; Controle) - Universidade Federal de Viçosa, Viçosa, 2013.http://locus.ufv.br/handle/123456789/1069Coffee leaf rust, caused by the biotrophic fungus Hemileia vastatrix, is the main phytosanitary problem of the coffee plant. The disease may cause yield losses ranging from 35 to 50% if control measures are not implemented. During the interaction with the host plant, rust fungi secrete a variety of effector proteins that modify the structure and function of the host cell allowing the biotrophic interaction. Some of these effector proteins, known as avirulence proteins (Avr), are recognized by plant proteins encoded by resistance genes, which triggers a defense response against pathogen infection. At least nine dominant genes (SH1 to SH9) that confer resistance to coffee leaf rust have been genetically identified, but despite many years of research on this pathosystem, the complementary Avr genes of H. vastatrix have not yet been identified or characterized. One approach that can be used for this purpose is to conduct an transcriptome and bioinformatics analyses in order to identify candidate genes of H. vastatrix that fulfill a set of specific criteria common to effector proteins, and their validation by conducting functional assays for effector activities. Therefore, the objectives of this study were: (i) to identify genes of H. vastatrix that encode proteins secreted during the germination of urediniospores as well as during the biotrophic interaction with the coffee plant, (ii) to express the effector gene candidates from H. vastatrix in the yeast secretion trap system (YST) in order to obtain biological confirmation of the secretion prediction made by bioinformatics tools, (iii) to analyze the genomic structure of a select group of effector gene candidates, (iv) to perform expression analysis of the identified candidates during different stages of infection; and (v) to establish a protocol for transient expression of H. vastatrix secreted proteins in coffee leaves based on the Type III Secretion System of Pseudomonas syringae pv. garcae. Initially, the secretome of germinated urediniospores of H. vastatrix was characterized by constructing a cDNA library from mRNA isolated from urediniospores germinated in vitro during 16 hours. After analysis of the ESTs using bioinformatics tools, 146 ORFs that encode secreted proteins were selected. Annotation of these proteins showed that 20% of the secretome from germinated urediniospores consists of hydrolytic enzymes and that most (67%) of the identified secreted proteins have unknown function. From the latter group of sequences, 35 complete ORFs encoding proteins sharing features with effectors from filamentous fungi and named H. vastatrix effector candidates (HvECs) were selected for further analysed. The genomic sequences of 22 of these HvECs were characterized, confirming the ORFs predicted by bioinformatics. The secretion of eight selected HvECs was confirmed in yeast and their expression analysis revealed a differential expression, which possibly occurs in coordination with the morphological changes of the pathogen during the early stages of infection. The secretome of H. vastatrix during a compatible plant-rust interaction was also characterized by a combination of Sanger sequencing and 454 pyrosequencing. This integrated approach allowed the massive sequencing of a substantial number of sequence tags (ESTs) of genes expressed at four different times during the biotrophic interaction: 48 hours, 72 hours, 9 days and 12 days after inoculation of coffee leaves with H. vastatrix. After bioinformatics analyses of these ESTs, 75 HvECs were selected and an expression analyses based on RT- PCR confirmed the fungal origin for 62 of them. 22 of these HvECs are preferentially expressed during the interaction of the fungus with the coffee plant and show distinct patterns of expression along the progress of infection. Using the pEDV system and the Type III Secretion System of P. syringae pv. garcae it was established a protocol to express transiently secreted proteins from H. vastatrix in coffee leaves established. Transient expression of HvEC-016 inside the cytoplasm of coffee plants carrying the rust resistant gene SH1, triggered a plant defense response indicating that there may have occurred a recognition of the HvEC-016 effector protein mediated by the R protein encoded by SH1. The catalog of effector gene candidates for H. vastatrix expressed during both urediniospore germination and the interaction with the coffee plant and the transient assay established in this study is an important plataform to identify additional avirulence genes from Hemileia vastatrix.A ferrugem do cafeeiro, causada pelo fungo biotrófico Hemileia vastatrix, é o principal problema fitossanitário da cafeicultura, podendo ocasionar perdas da ordem de 35 a 50% da produção se não forem implantadas medidas de controle. Durante a interação com a planta hospedeira, os fungos causadores de ferrugens secretam um arsenal de proteínas efetoras que modificam a estrutura e a função da célula hospedeira, permitindo o estabelecimento da interação biotrófica. Algumas dessas proteínas efetoras, denominadas proteínas de avirulência (Avr), são reconhecidas por proteínas codificadas por genes de resistência, o que desencadeia uma resposta de defesa da planta contra a infecção pelo patógeno. Já foram identificados geneticamente pelo menos nove genes dominantes (SH1 a SH9) que conferem resistência à ferrugem do cafeeiro, no entanto, apesar de muitos anos de pesquisa neste patossistema, os genes Avr complementares de H. vastatrix ainda não foram identificados ou caracterizados. Uma abordagem que pode ser realizada com esta finalidade é a análise do transcriptôma e de bioinformática para identificar genes candidatos de H. vastatrix que cumpram uma lista de critérios específicos em banco de dados de sequências, seguida da validação por meio de ensaios funcionais para atividades de efetores. Diante do exposto, os objetivos desse estudo foram: (i) identificar genes de H. vastatrix que codificam proteínas secretadas, expressas durante a germinação dos urediniósporos e também durante a fase biotrófica da interação com o cafeeiro; (ii) expressar genes candidatos a efetores de H. vastatrix, utilizando o sistema armadilha de secreção em levedura YST (Yeast Secretion Trap) para confirmação biológica da predição de secreção realizada por ferramentas de bioinformática; (iii) analisar a estrutura genômica dos genes candidatos a efetores selecionados; (iv) efetuar a análise da expressão temporal dos genes identificados durante as diferentes fases da patogênese; (v) estabelecer um protocolo de expressão transiente de proteínas secretadas de H. vastatrix em folhas de cafeeiro, com base no Sistema de Secreção Tipo III de Pseudomonas syringae pv. garcae. Inicialmente, caracterizou-se o secretoma de urediniósporos germinados de H. vastatrix por meio da construção de uma biblioteca de cDNA a partir de mRNA isolado de urediniósporos germinados in vitro por 16 horas. Após análises das sequências por ferramentas de bioinformática, foram selecionadas 146 ORFs que codificam proteínas secretadas. Durante a anotação dessas sequências constatou-se que 20% do secretoma de urediniósporos germinados são constituídos por enzimas hidrolíticas e que a maioria (67%) das proteínas secretadas identificadas possui função desconhecida. A partir dessas sequências foram selecionadas 35 ORFs completas que codificam proteínas com características comuns aos efetores de fungos filamentosos, denominados genes candidatos a efetores de H. vastatrix (HvECs H. vastatrix effector candidates). Desses, 22 HvECs tiveram suas sequências genômicas caracterizadas, confirmando as ORFs preditas por bioinformática. A secreção de oito HvECs selecionados foi comprovada em levedura, e sua expressão diferenciada, que possivelmente ocorre com as mudanças morfológicas do desenvolvimento do patógeno nos estágios inicias de infecção, foi comprovada por meio RT-qPCR. Posteriormente, caracterizou-se o secretoma de H. vastatrix expresso durante uma interação compatível com o cafeeiro, por meio da combinação de sequenciamento Sanger e pirossequenciamento 454. Esta abordagem integrada permitiu o sequenciamento massivo de um substancial número de etiquetas de sequências expressas (ESTs) em folhas de café infectadas com H. vastatrix, amostradas em quatro tempos diferentes durante a fase biotrófica da interação: 48 horas, 72 horas, 9 dias e 12 dias após a inoculação. Após análises de bioinformática foram selecionados 75 genes e as análises de RT-PCR confirmaram a origem fúngica de 62 genes HvECs. Desses, 22 HvECs são preferencialmente expressos durante a interação com o cafeeiro, apresentando padrões distintos de expressão durante a fase biotrófica da interação. Adicionalmente, desenvolveu-se um protocolo de expressão transiente de proteínas secretadas de H. vastatrix em folhas de cafeeiro, por meio do sistema pEDV e do Sistema de Secreção Tipo III de P. syrigae pv. garcae. A expressão transiente do gene HvEC-016 no citoplasma de cafeeiros resistentes à ferrugem, portadores do gene SH1, desencadeou uma resposta de defesa das plantas, indicando que pode ter ocorrido o reconhecimento da proteína efetora HvEC-016 mediada pela proteína R codificada pelo gene SH1. Esse sistema de expressão transiente e o catálogo de genes candidatos a efetores de H. vastatrix expressos tanto durante a germinação dos urediniósporos como na interação com o cafeeiro, disponibilizados por esse estudo, constituem uma importante plataforma para a identificação de outros genes de avirulência de Hemileia vastatrix.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaDoutorado em FitopatologiaUFVBREtiologia; Epidemiologia; ControleFerrugem-do-cafeeiroHemileia vastatrixInteração planta-patógenoCafé - Resistência a doenças e pragasRust in coffeeHemileia vastatrixPlant-pathogenCoffee - Resistance to diseases and pestsCNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::FITOPATOLOGIAIdentificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiroIndentification and funtional analysis of effectors from Hemileia vastatrix, the causal agent of the coffee rustinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1536759https://locus.ufv.br//bitstream/123456789/1069/1/texto%20completo.pdfedfc663b086d5a927204773326c9803eMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain285741https://locus.ufv.br//bitstream/123456789/1069/2/texto%20completo.pdf.txt4e840c8a825b5fa8b31f95a41e93a463MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3628https://locus.ufv.br//bitstream/123456789/1069/3/texto%20completo.pdf.jpg7eaec012db9dfd328e607c2e0309f5f3MD53123456789/10692016-04-06 23:18:01.951oai:locus.ufv.br:123456789/1069Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-07T02:18:01LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
dc.title.alternative.eng.fl_str_mv Indentification and funtional analysis of effectors from Hemileia vastatrix, the causal agent of the coffee rust
title Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
spellingShingle Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
Maia, Thiago Andrade
Ferrugem-do-cafeeiro
Hemileia vastatrix
Interação planta-patógeno
Café - Resistência a doenças e pragas
Rust in coffee
Hemileia vastatrix
Plant-pathogen
Coffee - Resistance to diseases and pests
CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::FITOPATOLOGIA
title_short Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
title_full Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
title_fullStr Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
title_full_unstemmed Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
title_sort Identificação e análise funcional de efetores de Hemileia vastatrix, agente causal da ferrugem do cafeeiro
author Maia, Thiago Andrade
author_facet Maia, Thiago Andrade
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/6798704880640812
dc.contributor.author.fl_str_mv Maia, Thiago Andrade
dc.contributor.advisor-co1.fl_str_mv Mizubuti, Eduardo Seiti Gomide
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785633J8
dc.contributor.advisor-co2.fl_str_mv Queiroz, Marisa Vieira de
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785812Z5
dc.contributor.advisor1.fl_str_mv Brommonschenkel, Sérgio Hermínio
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780948Y4
dc.contributor.referee1.fl_str_mv Resende, Mário Lúcio Vilela de
dc.contributor.referee1Lattes.fl_str_mv http://lattes.cnpq.br/8921922898103482
dc.contributor.referee2.fl_str_mv Barreto, Robert Weingart
dc.contributor.referee2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783300H6
dc.contributor.referee3.fl_str_mv Cunha, Luis Claudio Vieira da
dc.contributor.referee3Lattes.fl_str_mv http://lattes.cnpq.br/9367947173322278
dc.contributor.referee4.fl_str_mv Pacheco, Jorge Luis Badel
dc.contributor.referee4Lattes.fl_str_mv http://lattes.cnpq.br/4921932550244295
contributor_str_mv Mizubuti, Eduardo Seiti Gomide
Queiroz, Marisa Vieira de
Brommonschenkel, Sérgio Hermínio
Resende, Mário Lúcio Vilela de
Barreto, Robert Weingart
Cunha, Luis Claudio Vieira da
Pacheco, Jorge Luis Badel
dc.subject.por.fl_str_mv Ferrugem-do-cafeeiro
Hemileia vastatrix
Interação planta-patógeno
Café - Resistência a doenças e pragas
topic Ferrugem-do-cafeeiro
Hemileia vastatrix
Interação planta-patógeno
Café - Resistência a doenças e pragas
Rust in coffee
Hemileia vastatrix
Plant-pathogen
Coffee - Resistance to diseases and pests
CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::FITOPATOLOGIA
dc.subject.eng.fl_str_mv Rust in coffee
Hemileia vastatrix
Plant-pathogen
Coffee - Resistance to diseases and pests
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::FITOPATOLOGIA
description Coffee leaf rust, caused by the biotrophic fungus Hemileia vastatrix, is the main phytosanitary problem of the coffee plant. The disease may cause yield losses ranging from 35 to 50% if control measures are not implemented. During the interaction with the host plant, rust fungi secrete a variety of effector proteins that modify the structure and function of the host cell allowing the biotrophic interaction. Some of these effector proteins, known as avirulence proteins (Avr), are recognized by plant proteins encoded by resistance genes, which triggers a defense response against pathogen infection. At least nine dominant genes (SH1 to SH9) that confer resistance to coffee leaf rust have been genetically identified, but despite many years of research on this pathosystem, the complementary Avr genes of H. vastatrix have not yet been identified or characterized. One approach that can be used for this purpose is to conduct an transcriptome and bioinformatics analyses in order to identify candidate genes of H. vastatrix that fulfill a set of specific criteria common to effector proteins, and their validation by conducting functional assays for effector activities. Therefore, the objectives of this study were: (i) to identify genes of H. vastatrix that encode proteins secreted during the germination of urediniospores as well as during the biotrophic interaction with the coffee plant, (ii) to express the effector gene candidates from H. vastatrix in the yeast secretion trap system (YST) in order to obtain biological confirmation of the secretion prediction made by bioinformatics tools, (iii) to analyze the genomic structure of a select group of effector gene candidates, (iv) to perform expression analysis of the identified candidates during different stages of infection; and (v) to establish a protocol for transient expression of H. vastatrix secreted proteins in coffee leaves based on the Type III Secretion System of Pseudomonas syringae pv. garcae. Initially, the secretome of germinated urediniospores of H. vastatrix was characterized by constructing a cDNA library from mRNA isolated from urediniospores germinated in vitro during 16 hours. After analysis of the ESTs using bioinformatics tools, 146 ORFs that encode secreted proteins were selected. Annotation of these proteins showed that 20% of the secretome from germinated urediniospores consists of hydrolytic enzymes and that most (67%) of the identified secreted proteins have unknown function. From the latter group of sequences, 35 complete ORFs encoding proteins sharing features with effectors from filamentous fungi and named H. vastatrix effector candidates (HvECs) were selected for further analysed. The genomic sequences of 22 of these HvECs were characterized, confirming the ORFs predicted by bioinformatics. The secretion of eight selected HvECs was confirmed in yeast and their expression analysis revealed a differential expression, which possibly occurs in coordination with the morphological changes of the pathogen during the early stages of infection. The secretome of H. vastatrix during a compatible plant-rust interaction was also characterized by a combination of Sanger sequencing and 454 pyrosequencing. This integrated approach allowed the massive sequencing of a substantial number of sequence tags (ESTs) of genes expressed at four different times during the biotrophic interaction: 48 hours, 72 hours, 9 days and 12 days after inoculation of coffee leaves with H. vastatrix. After bioinformatics analyses of these ESTs, 75 HvECs were selected and an expression analyses based on RT- PCR confirmed the fungal origin for 62 of them. 22 of these HvECs are preferentially expressed during the interaction of the fungus with the coffee plant and show distinct patterns of expression along the progress of infection. Using the pEDV system and the Type III Secretion System of P. syringae pv. garcae it was established a protocol to express transiently secreted proteins from H. vastatrix in coffee leaves established. Transient expression of HvEC-016 inside the cytoplasm of coffee plants carrying the rust resistant gene SH1, triggered a plant defense response indicating that there may have occurred a recognition of the HvEC-016 effector protein mediated by the R protein encoded by SH1. The catalog of effector gene candidates for H. vastatrix expressed during both urediniospore germination and the interaction with the coffee plant and the transient assay established in this study is an important plataform to identify additional avirulence genes from Hemileia vastatrix.
publishDate 2013
dc.date.issued.fl_str_mv 2013-02-27
dc.date.accessioned.fl_str_mv 2015-03-26T12:42:06Z
dc.date.available.fl_str_mv 2015-01-20
2015-03-26T12:42:06Z
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dc.identifier.citation.fl_str_mv MAIA, Thiago Andrade. Indentification and funtional analysis of effectors from Hemileia vastatrix, the causal agent of the coffee rust. 2013. 137 f. Tese (Doutorado em Etiologia; Epidemiologia; Controle) - Universidade Federal de Viçosa, Viçosa, 2013.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/1069
identifier_str_mv MAIA, Thiago Andrade. Indentification and funtional analysis of effectors from Hemileia vastatrix, the causal agent of the coffee rust. 2013. 137 f. Tese (Doutorado em Etiologia; Epidemiologia; Controle) - Universidade Federal de Viçosa, Viçosa, 2013.
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dc.publisher.department.fl_str_mv Etiologia; Epidemiologia; Controle
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