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Estudo dos mecanismos de resposta à infecção por fungos patogénicos em Pyrus communis
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Orientador(es)
Resumo(s)
As interacções planta-patogéneo compreendem uma série de mecanismos de resposta
de defesa cuja interpretação tem sido alvo de pesquisa com vista ao controlo de muitas
doenças de culturas com interesse comercial. A pereira Rocha é uma cultivar portuguesa de
Pyrus communis L. com elevada importância económica devido à qualidade do seu fruto,
atingindo produções nacionais que ascendem as 180 mil toneladas/ano. No entanto, esta
cultivar é sensível à estenfiliose, uma doença causada pelo fungo patogénico Stemphylium
vesicarium (Wallr.) E. Simmons que provoca danos consideráveis no fruto com consequentes
elevadas perdas económicas visto que pêra Rocha infectada perde o seu valor comercial.
Grande parte do controlo desta doença é efectuado pela aplicação massiva e sistemática de
produtos químicos, o que aumenta os custos de produção e tem um impacto negativo no
ambiente e na saúde pública. Várias patologias afectam plantas economicamente importantes
causando perdas anuais de biliões de euros, com severas consequências para os produtores e
para a economia da região/país. O uso de pesticidas limita a proliferação do patogéneo e reduz
as perdas nas produções, mas a preocupação com o ambiente e saúde pública aliados à
necessidade de alimentos de boa qualidade e o crescente aparecimento de estirpes de
patogéneos resistentes torna cada vez mais comprometedor o uso deste método de combate
a agentes patogénicos. No sentido de diminuir o uso de produtos químicos, a maioria da
investigação tem-se focado no uso plantas resistentes às doenças ou na indução da resistência
em plantas sensíveis.
Torna-se assim urgente a procura de estratégias alternativas envolvendo a
compreensão e manipulação dos mecanismos de defesa do hospedeiro.
O estudo dos mecanismos de infecção e a activação de respostas de defesa de
cultivares resistentes ao fungo S. vesicarium envolveu o recurso à tecnologia de microrrays de
cDNA para identificar genes diferencialmente expressos em pereiras resistentes/tolerantes à
estenfiliose (cultivar Ercolini) comparativamente com pereiras Rocha (sensível à doença),
durante as primeiras 24 horas após contacto com o fungo. Para a construção dos microarrays
de cDNA, foram usados 3257 transcritos, provenientes de duas bibliotecas de cDNA,
elaboradas a partir de folhas infectadas e não infectadas de ambas as cultivares de pereira,
Rocha e Ercolini. Os ensaios de infecção foram efectuados in vivo.
A análise do transcritoma durante a interacção entre o fungo patogénico Stemphylium
vesicarium e Pyrus communis resultaram na obtenção de 137 transcritos diferencialmente
vi
expressos na cultivar Ercolini e 109 transcritos diferencialmente expressos na cultivar Rocha,
com razões de expressão (fold change) superiores as 1,5. Quase todos os transcritos são
homólogos de genes com função conhecida confirmando um total de 88 transcritos não
redundantes com funções relacionadas com stress, patogenicidade e defesa, metabolismo
primário e secundário, transdução de sinal, fotossíntese e fotorespiração, transcrição e
tradução, modulação e degradação proteica.
A análise de cada um dos transcritos com expressão diferencial revelou que os
putativos unigenes codificam para proteínas e metabolitos envolvidos numa sequência de
mecanismos concertados para a activação de respostas à invasão pelo fungo S. vesicarium.
Resumidamente, estes mecanismos envolvem a percepção, por receptores celulares da planta,
de sinais externos que resultam no influxo de cálcio (actua como mensageiro secundário),
activação de cinases/fosfatases, troca de iões K+/H+, conduzindo à acidificação da célula,
abertura de canais aniónicos, activação do complexo NADPH-oxidase, conduzindo à produção
de ROS, peroxidação de lípidos e rearranjo citoplasmático. As ROS induzem o espessamento da
parede celular através da acção de peroxidases. Presentes em níveis moderados, as ROS
podem funcionar como sinais indutores de respostas intracelulares, tais como activação da
transcrição de genes de defesa, biossíntese de ácido salicílico (SA), proteínas relacionadas com
a patogénese (PR-proteins), resposta hipersensível (HR), e sinalização sistémica.
O presente trabalho contem informação sobre os mecanismos moleculares envolvidos
nas interacções planta-patogéneo em particular na estenfiliose em pereira, permitindo a
definição de vias comuns de resistência/tolerância ao fungo em pomoideas, assim como
noutras espécies de plantas sensíveis, proporcionando conhecimento de produtos de
expressão de genes e metabolitos comuns que estão na base das respostas induzidas pelo
patogéneo. Este conhecimento permitirá o estabelecimento de novas estratégias de combate
a agentes patogénios para a protecção e produção integradas de culturas com interesse
económico.
Plant-pathogen interactions include a number of defense response mechanisms whose interpretation has been subject of research in order to control diseases of many crops with commercial interest. Rocha pear is a Pyrus communis L. portuguese cultivar with great economic importance due to its fruit quality with national productions amounting to 180 thousand tons / year. However, this cultivar is sensitive to the pathogenic fungus Stemphylium vesicarium (Wallr.) E. Simmons, the brown spot agent, causing considerable damage on fruit and yield production losses since infected Rocha pear loses its commercial value. Brown spot disease field control is carried out by massive and systematic application of antifungal chemicals which increases production costs and has a negative impact on the environment and public health. Several diseases affect economically important crops, causing annual losses of billions of euros, with severe consequences for producers and the economy of the region / country. Pesticide application limits the proliferation of the pathogen and reduces crops losses, but environment and public health concern along with the need for good quality food and the increasing emergence of pathogen resistant strains is increasingly compromising the use of this method to combat pathogens. In order to reduce the use of chemicals, most research has focused on using plants resistant to diseases or to induce resistance in sensitive plants. Thus becomes urgent to look for alternative strategies involving the understanding and manipulation of host defense mechanisms. The study of the infection mechanisms and activation of defense responses of cultivars resistant to S. vesicarium fungus involved the use of cDNA microrrays technology to identify differentially expressed genes in pear trees resistant / tolerant to brown spot disease (Ercolini cultivar) compared to Rocha pear tree (sensitive to the disease) during the first 24 hours after contact with the fungus. For the construction of cDNA microarrays 3257 transcripts from two cDNA libraries, prepared from infected and uninfected leaves of both pear trees cultivars, Rocha and Ercolini were used. The infection assays of pear trees were carried out in vivo. The analysis of the transcriptome during the interaction between Stemphylium vesicarium pathogenic fungus and Pyrus communis resulted on 137 differentially expressed transcripts in Ercolini cultivar and 109 differentially expressed transcripts in Rocha cultivar, with expression ratios (fold change) higher than the 1.5. Almost all transcripts are homologous to genes with known function confirming a total of 88 non-redundant transcripts with functions related to stress, pathogenicity and defense, primary and secondary metabolism, signal transduction, photosynthesis and photorespiration, transcription and translation, modulation and protein degradation. The analysis of each of the differentially expressed transcripts revealed that the putative unigenes encode proteins and metabolites involved in a series of concerted mechanisms for the activation of responses to S. vesicarium fungus invasion. Briefly, these mechanisms involve the perception by plant cell receptors of external signals that result in calcium influx (acts as a second messenger), activation of kinases / phosphatases, ion exchange K+ / H+, leading to acidification of the cell, anionic channels opening, activation of the NADPH oxidase complex, leading to ROS production, lipid peroxidation and cytoplasmic rearrangement. ROS induce cell wall thickening by the action of peroxidases. Present at moderate levels, ROS may function as signals that induce intracellular responses such as activation of transcription of defense genes, biosynthesis of salicylic acid (SA), pathogenesis related proteins (PR-proteins), hypersensitive response (HR) and systemic signaling. This work contains information on the molecular mechanisms involved in plantpathogen interactions namely in pear brown spot disease, allowing the definition of common pathways of resistance / tolerance to fungi in Pomoideae fruit trees, as well as other sensitive plant species, providing knowledge on the expression of genes and metabolites that are commonly in the basis of the responses induced by pathogens. This knowledge will allow the establishment of new strategies to combat the pathogen agents for the protection and integrated production of crops with economic interest.
Plant-pathogen interactions include a number of defense response mechanisms whose interpretation has been subject of research in order to control diseases of many crops with commercial interest. Rocha pear is a Pyrus communis L. portuguese cultivar with great economic importance due to its fruit quality with national productions amounting to 180 thousand tons / year. However, this cultivar is sensitive to the pathogenic fungus Stemphylium vesicarium (Wallr.) E. Simmons, the brown spot agent, causing considerable damage on fruit and yield production losses since infected Rocha pear loses its commercial value. Brown spot disease field control is carried out by massive and systematic application of antifungal chemicals which increases production costs and has a negative impact on the environment and public health. Several diseases affect economically important crops, causing annual losses of billions of euros, with severe consequences for producers and the economy of the region / country. Pesticide application limits the proliferation of the pathogen and reduces crops losses, but environment and public health concern along with the need for good quality food and the increasing emergence of pathogen resistant strains is increasingly compromising the use of this method to combat pathogens. In order to reduce the use of chemicals, most research has focused on using plants resistant to diseases or to induce resistance in sensitive plants. Thus becomes urgent to look for alternative strategies involving the understanding and manipulation of host defense mechanisms. The study of the infection mechanisms and activation of defense responses of cultivars resistant to S. vesicarium fungus involved the use of cDNA microrrays technology to identify differentially expressed genes in pear trees resistant / tolerant to brown spot disease (Ercolini cultivar) compared to Rocha pear tree (sensitive to the disease) during the first 24 hours after contact with the fungus. For the construction of cDNA microarrays 3257 transcripts from two cDNA libraries, prepared from infected and uninfected leaves of both pear trees cultivars, Rocha and Ercolini were used. The infection assays of pear trees were carried out in vivo. The analysis of the transcriptome during the interaction between Stemphylium vesicarium pathogenic fungus and Pyrus communis resulted on 137 differentially expressed transcripts in Ercolini cultivar and 109 differentially expressed transcripts in Rocha cultivar, with expression ratios (fold change) higher than the 1.5. Almost all transcripts are homologous to genes with known function confirming a total of 88 non-redundant transcripts with functions related to stress, pathogenicity and defense, primary and secondary metabolism, signal transduction, photosynthesis and photorespiration, transcription and translation, modulation and protein degradation. The analysis of each of the differentially expressed transcripts revealed that the putative unigenes encode proteins and metabolites involved in a series of concerted mechanisms for the activation of responses to S. vesicarium fungus invasion. Briefly, these mechanisms involve the perception by plant cell receptors of external signals that result in calcium influx (acts as a second messenger), activation of kinases / phosphatases, ion exchange K+ / H+, leading to acidification of the cell, anionic channels opening, activation of the NADPH oxidase complex, leading to ROS production, lipid peroxidation and cytoplasmic rearrangement. ROS induce cell wall thickening by the action of peroxidases. Present at moderate levels, ROS may function as signals that induce intracellular responses such as activation of transcription of defense genes, biosynthesis of salicylic acid (SA), pathogenesis related proteins (PR-proteins), hypersensitive response (HR) and systemic signaling. This work contains information on the molecular mechanisms involved in plantpathogen interactions namely in pear brown spot disease, allowing the definition of common pathways of resistance / tolerance to fungi in Pomoideae fruit trees, as well as other sensitive plant species, providing knowledge on the expression of genes and metabolites that are commonly in the basis of the responses induced by pathogens. This knowledge will allow the establishment of new strategies to combat the pathogen agents for the protection and integrated production of crops with economic interest.
Descrição
Tese de doutoramento, Biologia (Biologia Molecular), Universidade de Lisboa, Faculdade de Ciências, 2011
Palavras-chave
Genómica funcional Microarray Fungos patogénicos Pyrus communis Resistência a antifúngicos Stemphylium vesicarium Teses de doutoramento - 2011