RESUMEN
BACKGROUND: The "cerato-platanin family" consists of fungal-secreted proteins that are involved in various stages of the host-fungus interaction and act as phytotoxins, elicitors of defense responses and allergens. Cerato-platanin (CP) is a moderately hydrophobic protein secreted and localized in the cell wall of Ceratocystis platani, the causal agent of a severe disease of Platanus. These properties make CP like the hydrophobins: these are self-assembling proteins that form a surface coating which is involved in the formation of aerial hyphae and in adherence to surfaces. METHODS: CP aggregation was monitored by ThT, circular dichroism, and AFM. The eliciting activity of CP aggregates was assayed on leaves and cells. RESULTS: The CP self-assembles forming amyloid-like aggregates via a nucleated growth mechanism which is joined up with a cleavage of the N-terminus. The ovoidal shape and the lack of a clear transition toward an all-beta structure distinguish these aggregates from typical amyloid fibrils. Moreover, CP aggregates interact with hydrophobic surfaces and enhance the hypersensitive response of Platanus. CONCLUSION AND GENERAL SIGNIFICANCE: CP forms "ordered aggregates" for which the soluble prefibrillar structures are the end point of the aggregation process, and do not evolve to insoluble fibrils. An involvement in host-microbe interaction is also suggested.
Asunto(s)
Ascomicetos/fisiología , Proteínas Fúngicas/química , Enfermedades de las Plantas/microbiología , Plantas/microbiología , Secuencia de Aminoácidos , Ascomicetos/química , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacología , Interacciones Huésped-Patógeno , Cinética , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Células Vegetales , Conformación Proteica , Pliegue de ProteínaRESUMEN
The ascomycete Ceratocystis fimbriata, the causal agent of "canker stain disease," secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP similar to proteins of the hydrophobin family.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Expresión Génica/genética , Secuencia de Aminoácidos , Secuencia de Bases/genética , Mapeo Cromosómico/métodos , Clonación Molecular/métodos , Escherichia coli/genética , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Hifa/metabolismo , Datos de Secuencia Molecular , Extractos Vegetales/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Sesquiterpenos , Esporas Fúngicas/metabolismo , Terpenos , FitoalexinasRESUMEN
Cerato-platanin (CP) is a phytotoxic protein secreted by the Ascomycete Ceratocystis fimbriata f.sp. platani. This Ascomycete causes canker stain which is a severe disease with a high incidence in the European Platanus acerifolia. CP probably plays a role in the disease, eliciting defence-related responses in the host plants. CP is a 120 amino acid protein, containing 40% hydrophobic residues and two S-S bridges. In the EMBL data bank CP is the first member of a new fungal protein family known as the Cerato-Platanin Family. The N-terminal region of CP shows a high similarity with that of cerato-ulmin, a phytotoxic protein produced by the Ophiostoma species and that belongs to the hydrophobin family. Hydrophobins are hydrophobic proteins secreted by many saprophytic or pathogenic fungi and have a remarkable ability to self-assemble into a rodlet structure takes part in physiological and/or pathological processes. The methyltrophic yeast Pichia pastoris was used to obtain a high-level expression of recombinant CP (rCP) and the pPIC9 vector was chosen to bring about extra-cellular secretion of the protein. The preliminary structural and functional characterization presented here reveals no significant differences between the native and the recombinant protein. We also show that CP self-assembles in solution. The availability of rCP will allow its three-dimensional structure to be determined, facilitating an understanding of the role of CP in the pathogenesis of canker stain. It is also an excellent model for investigating the mechanism of action of the other proteins related to CP.