Lara Carresi

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

The ascomcete 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 simlar to proteins of the hydrophobin family.

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 α/β protein with a different percentage of the α-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.

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–β 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.

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 Ophiostomas (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.