Cerato-populin and cerato-platanin, two non-catalytic proteins from phytopathogenic fungi, interact with hydrophobic inanimate surfaces and leaves.

Based on sequence homology, several fungal Cys-rich secreted proteins have been grouped in the cerato-platanin (CP) family, which comprises at least 40 proteins involved mainly in eliciting defense-related responses. The core member of this family is cerato-platanin, a moderately hydrophobic protein with a double ψ-ß barrel fold. CP and the recently identified orthologous cerato-populin (Pop1) are involved in host-fungus interaction, and can be considered non-catalytic fungal PAMPs. CP is more active in inducing defense when in an aggregated conformation than in its native form, but little is known about other CP-orthologous proteins. Here, we cloned, expressed, and purified recombinant Pop1, which was used to characterize the protein aggregates. Our results suggest that the unfolded, self-assembled Pop1 is more active in inducing defense, and that the unfolding process can be induced by interaction with hydrophobic inanimate surfaces such as Teflon, treated mica, and gold sheets. In vivo, we found that both CP and Pop1 interact with the hydrophobic cuticle of leaves. Therefore, we propose that the interaction of these proteins with host cuticle waxes could induce unfolding and consequently trigger their PAMP-like activity.

Identification and characterization of GEO1, a new class II hydrophobin from Geosmithia spp.

In the present paper we describe a new noncatalytic protein belonging to the hydrophobin family, designated GEO1, purified from the culture filtrate of Geosmithia pallida (Ascomycota: Hypocreales), and the corresponding gene sequence. In the fungal genome, GEO1 was encoded by a single-copy gene with a 450 bp open reading frame interrupted by 2 small introns whose primary translation product was 109 amino acids long and included a 23 amino acids signal peptide. The mature protein had a molecular mass of 8111.75 Da and a theoretical pI of 4.33. The deduced amino acid sequence showed similarity to class II hydrophobins and contained 8 conserved cysteine residues, present in all hydrophobins isolated so far. Biochemical properties, such as foam-forming ability and trapezoid-like shape of a GEO1 drop, also resembled the typical features of the class II hydrophobins. Expression of the geo1 gene was assessed after 2, 4, 7, 9, and 11 days of culture and showed that the geo1 transcript appeared after 7 days and increased up to 11 days.

Characterization of ordered aggregates of cerato-platanin and their involvement in fungus-host interactions.

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.

New proteins orthologous to cerato-platanin in various Ceratocystis species and the purification and characterization of cerato-populin from Ceratocystis populicola.

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured alpha/beta protein with a different percentage of the alpha-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth.

Isolation of the orthologue of the cerato-ulmin gene in Ophiostoma quercus and characterization of the purified protein.

Ophiostoma quercus is an ophiostomatoid fungus strictly related to the Ophiostoma's (O. ulmi, O. novo-ulmi, and O. himal-ulmi) that cause Dutch elm disease (DED). O. quercus has a number of morphological characteristics in common with the DED pathogens, and is a well-known and economically important sapstaining fungus occurring worldwide on hardwoods and commercially produced pines, and causes typical cankers on oak stems. In elm trees O. quercus can survive for months without causing any disease symptoms. DED fungi produce cerato-ulmin (CU), a class II hydrophobin, which is generally considered as the main toxin potentially involved in various phases of the DED pathogenesis. In the present work we isolated and sequenced the orthologue of the cu gene in the O. quercus isolates H988, H1042, and H2053. Moreover the CU protein from O. quercus isolate H988 was also purified and characterized. Sequence analysis showed that there is a pronounced difference between the whole cu gene region of O. quercus and the homologous fragments of the DED-causing species O. ulmi, O. novo-ulmi, and O. himal-ulmi. It also appeared that differences in the structural conformation of the promoter were unlikely to play a role in the modulation of the transcript level and that, for O. quercus, differences in CU production did not result from the potential different regulation levels. Clear differences were shown in the transcriptional unit of the cu genes and in the amino acid sequences among all the CUs. The purified O. quercus CU was separated using matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) spectrometry into seven forms of increasing molecular weight from 7190 to 7724Da. The hydrophobicity profiles indicated that two regions of the O. quercus CU protein were more hydrophobic than the corresponding regions of the CUs of the DED fungi. The O. quercus CUs had theoretical isoelectric point values similar to those of the DED fungi. Finally, the contradiction between the consistent differences between these four Ophiostoma species in the cu gene region and in the CU proteins and their strict phylogenetic relationship is discussed.

Cerato-platanin, a phytotoxic protein from Ceratocystis fimbriata: expression in Pichia pastoris, purification and characterization.

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.

Cerato-platanin, the first member of a new fungal protein family: cloning, expression, and characterization.

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.

Characterization of the major allergens purified from the venom of the paper wasp Polistes gallicus.

Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules. In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.