Identificación y análisis funcional de genes de etapas tempranas del ciclo biológico de botrytis cinerea
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Fecha
2009
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Profesor/a Guía
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
El hongo Botrytis cinerea es uno de los principales problemas
fitosanitarios de la industria hortofrutícola en Chile, causando importantes
pérdidas económicas antes, durante y después de la cosecha en cultivos como
vides, tomates, frutillas, arándanos, duraznos, etc. La infección comienza
cuando las esporas se adhieren a la superficie de la planta donde germinan y
forman la hifa de infección. Esta estructura es capaz de atravesar la cutícula e
invadir al hospedero por degradación química de la pared celular mediante la
producción de enzimas hidrolíticas que permiten la invasión y posterior
colonización del tejido.
En este trabajo se han caracterizado diferentes asilados de B. cinerea
disponibles en el laboratorio, lo que permitió identificar aislados con diferentes
grados de virulencia. Posteriormente, mediante la generación de una genoteca
de ADNc y el uso de herramientas bioinformáticas, se identificó genes
involucrados en etapas tempranas de la infección e importantes en la
patogenicidad de B. cinerea. Adicionalmente, el diseño y análisis preliminar de
expresión génica, mediante microarreglos, permitió la identificación de 171
genes específicos de la espora, 92 genes específicos de la germinación y 544
relacionados con el crecimiento de la hifa de B. cinerea.
De un total de 180 genes ortólogos identificados, el 40% de ellos están
involucrados en etapas iniciales como adhesión, conidiogénesis, germinación y
crecimiento de la hifa. Algunos de estos genes se aislaron, clonaron y
secuenciaron y se analizó su expresión. Entre estos, el gen hex-1 que codifica
para la proteína principal del cuerpo de Woronin, presentó un patrón de
expresión diferencial al comparar dos aislados con diferente velocidad de
crecimiento. El análisis funcional de hex-1 permitió revelar su importancia como
gen patogénico y sugirió una participación, no sólo en el crecimiento vegetativo
sino también en la germinación y en la capacidad de establecer la infección por
B. cinerea. Otro gen estudiado fue cox-5, el cual codifica para una subunidad
de la citocromo C oxidasa. Análisis funcionales de cox-5 permitió identificar un
fenotipo letal producto de la inactivación del gen. En virtud de lo anterior, la
identificación y caracterización de hex-1 y cox-5 proporcionan dos nuevos
blancos para desarrollar estrategias de defensa en la planta que apunte a
inactivar la capacidad del hongo de crecer sobre superficies vegetales.
En resumen, este trabajo representa un aporte importante al
conocimiento de la maquinaría génica que dispone este patógeno para invadir
su hospedero y define un importante inventario de genes de B. cinerea para
futuros estudios acerca de la interacción planta-patógeno.
Botrytis cinerea, also known as "gray mold fungus," is considered a serious problem far the Chilean agricultura! industry. This pathogen causes pre- and post-harvest diseases in at least two hundred plant species, including agriculturally important soft commodities, such as grapes, tomatoes, strawberries, cucumbers, bulb flowers, cut flowers and ornamental plants. The broad host range of B. cinerea results in great economic losses not only during growth but also during storage and transport. The interaction of this fungus with plants begins with conidia attachment to the host surface and continues with conidial germination, formation of infection structures and finally penetration and colonization into the host. B. cinerea penetrates host tissue via wounds or through natural openings; nevertheless this fungus also can directly penetrates the cuticle of plants through its enzymatic degradation. In this thesis, different native B. cinerea isolates were characterized in terms of fungal virulence parameters. Subsequently, a cDNA library from mycelia was constructed and sequenced. In the ESTs analysis several genes showed expression during vegetative growth including putative virulence factors. Additionally, signal transduction components and secondary metabolism enzymes were also identified. In addition, the design and preliminary gene expression analysis by microarray technology permitted the identification of genes differentially expressed during the infection allowing the identification of 171 conidia-specific genes, 92 genes specifically involved in germination and 544 genes related to hyphal growth of B. cinerea. Notably, 40% of characterized genes participate in early steps of the infection process such as adhesion, conidiogénesis, germination and hyphal growth and represents a substantial increase in information available on B. cinerea genes. In addition, sorne genes involved in hyphal growth and germination were experimentally analyzed. The hex-1 gene which encades the majar protein of the Woronin body was isolated, cloned and sequenced from the cDNA library. The functional studies of hex-1 were facilitated by the fact the gene presents a single copy in the B. cinerea genome. Hex-1 mutants exhibited a low growth and germination rate and were unable to establish infection. Another characterized gen was cox-5 which encades cytochrome C oxidase subunit. Cloning, mRNA expression studies and functional analysis were carried out far this gene. The cox-5 inactivation leads to a lethal phenotype. These results suggest hex-1 and cox-5 genes as candidates far novel antifungicide development. In summary, this work provides an important inventory of B. cinerea genes which might be crucial far future studies on the cellular events related to hostpathogen interaction.
Botrytis cinerea, also known as "gray mold fungus," is considered a serious problem far the Chilean agricultura! industry. This pathogen causes pre- and post-harvest diseases in at least two hundred plant species, including agriculturally important soft commodities, such as grapes, tomatoes, strawberries, cucumbers, bulb flowers, cut flowers and ornamental plants. The broad host range of B. cinerea results in great economic losses not only during growth but also during storage and transport. The interaction of this fungus with plants begins with conidia attachment to the host surface and continues with conidial germination, formation of infection structures and finally penetration and colonization into the host. B. cinerea penetrates host tissue via wounds or through natural openings; nevertheless this fungus also can directly penetrates the cuticle of plants through its enzymatic degradation. In this thesis, different native B. cinerea isolates were characterized in terms of fungal virulence parameters. Subsequently, a cDNA library from mycelia was constructed and sequenced. In the ESTs analysis several genes showed expression during vegetative growth including putative virulence factors. Additionally, signal transduction components and secondary metabolism enzymes were also identified. In addition, the design and preliminary gene expression analysis by microarray technology permitted the identification of genes differentially expressed during the infection allowing the identification of 171 conidia-specific genes, 92 genes specifically involved in germination and 544 genes related to hyphal growth of B. cinerea. Notably, 40% of characterized genes participate in early steps of the infection process such as adhesion, conidiogénesis, germination and hyphal growth and represents a substantial increase in information available on B. cinerea genes. In addition, sorne genes involved in hyphal growth and germination were experimentally analyzed. The hex-1 gene which encades the majar protein of the Woronin body was isolated, cloned and sequenced from the cDNA library. The functional studies of hex-1 were facilitated by the fact the gene presents a single copy in the B. cinerea genome. Hex-1 mutants exhibited a low growth and germination rate and were unable to establish infection. Another characterized gen was cox-5 which encades cytochrome C oxidase subunit. Cloning, mRNA expression studies and functional analysis were carried out far this gene. The cox-5 inactivation leads to a lethal phenotype. These results suggest hex-1 and cox-5 genes as candidates far novel antifungicide development. In summary, this work provides an important inventory of B. cinerea genes which might be crucial far future studies on the cellular events related to hostpathogen interaction.
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
Botrytis Cinerea, Industria hortofrutícola, Hifa de infección, Herramientas bioinformáticas, Conocimiento de la maquinaría génica, Chile