1H, 15N and 13C resonance assignment of cerato-populin,a fungal PAMP from Ceratocystis populicola

Plant pathogenic fungi secrete several non-catalytic proteins involved in various aspects of the pathogenesis process. Amongst these, cerato-populin (Pop1) produced by Ceratocystis populicola; a protein orthologous of cerato-platanin (CP), the core member of the CP family. These two proteins interact with host and non-host plants. In plane leaves they induce synthesis of phytoalexins, disruption of intercellular and intracellular leaf tissue, cell plasmolysis, programmed cell death, over-expression of defence-related genes, H₂O₂ and NO production, activation of MAPK cascade and plant resistance. All these features point to CP and Pop1 as defence inducers, though Pop1 shows a reduced efficiency. Pop1/CP similarity is 73 %. CD spectroscopy highlights some secondary structure differences between Pop1 and CP. Indeed, the region between the first two cysteines (C20–C57), that in CP includes the β₂-strand and it is involved in GlcNAc (N-acetyl-d-glucosamine) interaction, in Pop1 is predicted to be fully disordered.     

Cerato-platanin shows expansin-like activity on cellulosic materials

Cerato-platanin (CP) is a non-catalytic protein with a double ψβ-barrel fold located in the cell wall of the phytopathogenic fungus Ceratocystis platani. CP is released during growth and induces defence-related responses in plants. CP is also the first member of the “cerato-platanin family” (CPF) (Pfam PF07249). In the CPF, the molecular mechanism of action on plants and above all the biological role in fungal life are little-known aspects. However, an expansin-like function has recently been suggested concerning CP. Expansin-like proteins have the ability to act non-hydrolytically on cellulose. In the present work, the expansin-like activity of CP and Pop1, a CP family member, was investigated. Like expansins, CP and Pop1 were able to weaken filter paper in a concentration-dependent manner and without the production of reducing sugars. A metal-dependent polysaccharide monooxygenase-like activity was excluded. The optimum of activity was pH?5.0, 38 °C. CP was also able to cause fragmentation of the crystalline cellulose Avicel and the breakage and defibration of cotton fibres. However, the interaction did not involve a stable bond with the substrates and CP did not significantly enhance the hydrolytic activity of cellulase. On the other hand, CP and Pop1 bound quickly to chitin. We consider CP as a novel one-domain expansin-like protein. We propose a structural role for CP in the fungal cell wall due to the ability to bind chitin, and hypothesize a functional role in the interaction of the fungus with the plant for the weakening activity shown on cellulose.     

Atomic force microscopy images suggest aggregation mechanism in cerato-platanin

Cerato-platanin (CP), the first member of the "cerato-platanin family", is a moderately hydrophobic protein produced by Ceratocystis fimbriata, the causal agent of a severe plant disease called "canker stain". The protein is localized in the cell wall of the fungus and it seems to be involved in the host-plane interaction and induces both cell necrosis and phytoalexin synthesis (one of the first plant defence-related events). Recently, it has been determined that CP, like other fungal surface protein, is able to self assemble in vitro. In this paper we characterize the aggregates of CP by Atomic Force Microscopy (AFM) images. We observe that CP tends to form early annular-shaped oligomers that seem to constitute the fundamental bricks of a hierarchical aggregation process, eventually resulting in large macrofibrillar assemblies. A simple model, based on the hypothesis that the aggregation is energetically favourable when the exposed surface is reduced, is compatible with the measured aggregates' shape and size. The proposed model can help to understand the mechanism by which CP and many other fungal surface proteins exert their effects.     

Cerato-platanin, the first member of a new fungal protein family

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.     

The structure of the elicitor Cerato-platanin (CP), the first member of the CP fungal protein family, reveals a double ψβ-barrel fold and carbohydrate binding

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψβ-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the β-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψβ-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.     

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-β 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 <Emphasis Type="Italic">Ceratocystis</Emphasis> species and the purification and characterization of cerato-populin from <Emphasis Type="Italic">Ceratocystis populicola</Emphasis>

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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Sm2, a paralog of the Trichoderma cerato-platanin elicitor Sm1, is also highly important for plant protection conferred by the fungal-root interaction of Trichoderma with maize

Background

The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated.

Results

In order to analyze the function of the cerato-platanin protein Sm2, sm1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains.

Conclusions

Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0333-0) contains supplementary material, which is available to authorized users.     

PUF proteins bind Pop2p to regulate messenger RNAs

PUF proteins, a family of RNA-binding proteins, interact with the 3' untranslated regions (UTRs) of specific mRNAs to control their translation and stability. PUF protein action is commonly correlated with removal of the poly(A) tail of target mRNAs. Here, we focus on how PUF proteins enhance deadenylation and mRNA decay. We show that a yeast PUF protein physically binds Pop2p, which is a component of the Ccr4p-Pop2p-Not deadenylase complex, and that Pop2p is required for PUF repression activity. By binding Pop2p, the PUF protein simultaneously recruits the Ccr4p deadenylase and two other enzymes involved in mRNA regulation, Dcp1p and Dhh1p. We reconstitute regulated deadenylation in vitro and demonstrate that the PUF-Pop2p interaction is conserved in yeast, worms and humans. We suggest that the PUF-Pop2p interaction underlies regulated deadenylation, mRNA decay and repression by PUF proteins.     

A mechanistic model may explain the dissimilar biological efficiency of the fungal elicitors cerato-platanin and cerato-populin

Among their various functions, the members of the cerato-platanin family can stimulate plants' defense responses and induce resistance against microbial pathogens. Recent results suggest that conserved loops, also involved in chitin binding, might be a structural motif central for their eliciting activity. Here, we focus on cerato-platanin and its orthologous cerato-populin, searching for a rationale of their diverse efficiency to elicit plants' defense and to interact with oligosaccharides. A 3D model of cerato-populin has been generated by homology modeling using the NMR-derived cerato-platanin structure as template, and it has been validated by fitting with residual dipolar couplings. Loops β1-β2 and β2-β3 have been indicated as important for some CPPs members to express their biological function. When compared to cerato-platanin, in cerato-populin they present two mutations and an insertion that significantly modify their electrostatic surface. NMR relaxation experiments point to a reduced conformational plasticity of cerato-populin loops with respect to the ones of cerato-platanin. The different electrostatic surface of the loops combined with a distinct network of intra-molecular interactions are expected to be factors that, by leading to a diverse spatial organization and dissimilar collective motions, can regulate the eliciting efficacy of the two proteins and their affinity for oligosaccharides.     

Purification, characterization, and amino acid sequence of cerato-platanin, a new phytotoxic protein from Ceratocystis fimbriata f. sp. platani.

A new phytotoxic protein (cerato-platanin) of about 12.4 kDa has been identified in culture filtrates of the Ascomycete Ceratocystis fimbriata f. sp. platani, the causal agent of canker stain disease. The toxicity of the pure protein was bioassayed by detecting the inducing necrosis in tobacco leaves. The pure protein also elicited host synthesis of fluorescent substances in tobacco and plane (Platanus acerifolia) leaves. We purified the protein from culture medium to homogeneity. Its complete amino acid sequence was determined; this protein consists of 120 amino acid residues, contains 4 cysteines (S-S-bridged), and has a high percentage of hydrophobic residues. The molecular weight calculated from the amino acid sequence agrees with that determined by mass spectrometry, suggesting that no post-transnational modification occurs. Searches performed by the BLAST program in data banks (Swiss-Prot, EBI, and GenBank(TM)) revealed that this protein is highly homologous with two proteins produced by other Ascomycete fungi. One, produced during infection of wheat leaves, is codified by the snodprot1 gene of Phaeosphaeria nodorum (the causal agent of glume blotch of wheat), whereas the other is the rAsp f13 allergen from Aspergillus fumigatus. Furthermore, the N terminus of cerato-platanin is homologous with that of cerato-ulmin, a phytotoxic protein belonging to the hydrophobin family and produced by Ophiostoma (Ceratocystis) ulmi, a fungus responsible for Dutch elm disease.     

Cloning, in silico characterization and interaction of cysteine protease and cystatin for establishing their role in early blight disease in tomato

Cysteine protease (CP) and Cysteine protease inhibitor (CPI) or cystatin constitute a critical point in programmed cell death (PCD), a basic biological phenomenon which takes place in the plants, when they are exposed to varying biotic and abiotic stresses. In the present study we isolated and cloned cDNAs encoding cysteine protease and cystatin from early blight infected tomato plants. Using computational biology tools the sequence-structure-function relationships for the tomato cystatin and cysteine protease were elucidated. Interaction between the cystatin and cysteine protease of host and pathogen is higher as compared to interaction shown by cystatin and cysteine protease within the host. The interaction energy of (a)tomato cystatin—tomato cysteine protease, (b)tomato cystatin—fungal cysteine protease and (c)tomato cysteine protease—fungal cystatin are ?319.33 Kcal/mol, ?504.71 Kcal/mol and ?373.731 Kcal/mol respectively. Comparative protein sequence analysis with different plant cystatins and cysteine protease were also done with the sequences of cystatin and cysteine protease isolated from tomato. Structures for all the cystatin and cysteine protease were modeled along with their interactions with fungal cystatin and cysteine protease in order to explore the structural variability and its manifestation at the functional level. This helped to relate the already known functions of these proteins with their sequences as well as the predicted structures. This also served to better understand the CP-CPI interaction operational in developing this protein family and its implication in plant defense during fungal pathogenesis in tomato plants.     

Phylogeny and molecular dating of the cerato-platanin-encoding genes

The cerato-platanin family consists of proteins that can induce immune responses, cause necrosis, change chemotaxis and locomotion and may be related to the growth and development of various fungi. In this work, we analyzed the phylogenetic relationships among genes encoding members of the cerato-platanin family and computed the divergence times of the genes and corresponding fungi. The results showed that cerato-platanin-encoding genes could be classified into 10 groups but did not cluster according to fungal classes or their functions. The genes transferred horizontally and showed duplication. Molecular dating and adaptive evolution analyses indicated that the cerato-platanin gene originated with the appearance of saprophytes and that the gene was under positive selection. This finding suggests that cerato-platanin-encoding genes evolved with the development of fungal parasitic characteristics.     

Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica

Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact-dependent killing of host cells via membrane-active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N-terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica , which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three-dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non-pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica .