Ivan Baccelli

Cerato-platanin family proteins: one function for multiple biological roles?

Despite the increasing number of sequenced genomes, cerato-platanin family proteins (CPPs) seem to exist only in fungi. Bacteria, oomycota, plants, and animals do not in fact possess CP homologs, whereas genes codifying for CPPs have been found, so far, in more than 50 fungal genomes (Chen et al., 2013). Among these fungi there are plant pathogens, such as Botrytis cinerea and Magnaporthe grisea (Ascomycota), Heterobasidion irregulare and Moniliophthora perniciosa (Basidiomycota), but also biocontrol agents (e.g., Trichoderma spp.), mycorrhizal fungi, saprotrophs, and human pathogens. Experimental data suggest that CPPs play a role during fungus-plant interactions. When T. virens and T. harzianum were respectively co-cultured with cotton or tomato roots, the expression of CPPs increased compared to the culture obtained in the absence of the plant (Djonovic et al., 2006; Samolski et al., 2009); in B. cinerea and M. grisea, knockout mutants for CPP genes showed reduced virulence on their plant hosts (see Table ​Table1)1) (Jeong et al., 2007; Frias et al., 2011). Table 1 Summary table of the mutants obtained up to date for genes encoding cerato-platanin family proteins (CPPs). Plants have developed the ability to recognize CPPs and to activate defense responses when in contact with them. In fact, CPPs have been reported to act as microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) (for reviews, see Gaderer et al., 2014; Pazzagli et al., 2014). The secretion of these small proteins (120–130 a.a.) seems to be universal in fungi. CPPs are often the most present in the culture filtrates, but in some studies they have been found localized in the fungal cell walls (Boddi et al., 2004; Seidl et al., 2006; Shah et al., 2009; Frias et al., 2014). Gene knockout experiments have not yet permitted the identification of a clear biological function, and to date it is not easy to answer the question of why fungi produce CPPs. Gene expression data and studies on their biochemical properties have led to the formulation of various hypotheses that may appear different for each homolog. In summary, it is generally believed that CPPs play at least two roles in fungi: one in growth and development (generally referred to as the primary role), which should justify their presence in the cell wall, and one, more elusive, that should explain their secretion and the interaction with plants (Gaderer et al., 2014; Pazzagli et al., 2014). But what is their function? This opinion article will attempt to answer these questions: can we hypothesize a basal function which characterizes CPPs and allows them to have multiple biological roles depending on the context (fungal cell wall or extracellular environment)? Can this function imply a role in plant colonization?

Cerato-Platanin Induces Resistance in Arabidopsis Leaves through Stomatal Perception, Overexpression of Salicylic Acid- and Ethylene-Signalling Genes and Camalexin Biosynthesis

Microbe-associated molecular patterns (MAMPs) lead to the activation of the first line of plant defence. Few fungal molecules are universally qualified as MAMPs, and proteins belonging to the cerato-platanin protein (CPP) family seem to possess these features. Cerato-platanin (CP) is the name-giving protein of the CPP family and is produced by Ceratocystis platani, the causal agent of the canker stain disease of plane trees (Platanus spp.). On plane tree leaves, the biological activity of CP has been widely studied. Once applied on the leaf surface, CP acts as an elicitor of defence responses. The molecular mechanism by which CP elicits leaves is still unknown, and the protective effect of CP against virulent pathogens has not been clearly demonstrated. In the present study, we tried to address these questions in the model plant Arabidopsis thaliana. Our results suggest that stomata rapidly sense CP since they responded to the treatment with ROS signalling and stomatal closure, and that CP triggers salicylic acid (SA)- and ethylene (ET)-signalling pathways, but not the jasmonic acid (JA)-signalling pathway, as revealed by the expression pattern of 20 marker genes. Among these, EDS1, PAD4, NPR1, GRX480, WRKY70, ACS6, ERF1a/b, COI1, MYC2, PDF1.2a and the pathogenesis-related (PR) genes 1–5. CP rapidly induced MAPK phosphorylation and induced the biosynthesis of camalexin within 12 hours following treatment. The induction of localised resistance was shown by a reduced susceptibility of the leaves to the infection with Botrytis cinerea and Pseudomonas syringae pv. tomato. These results contribute to elucidate the key steps of the signalling process underlying the resistance induction in plants by CP and point out the central role played by the stomata in this process.

Differential timing of defense-related responses induced by cerato-platanin and cerato-populin, two non-catalytic fungal elicitors.

The cerato-platanin (CP) family consists of fungal-secreted proteins involved in various stages of the host-fungus interaction and acting as phytotoxins and elicitors of defense responses. The founder member of this family is CP, a non-catalytic protein with a six-stranded double-ψβ-barrel fold. Cerato-populin (Pop1) is an ortholog showing low sequence identity with CP. CP is secreted by Ceratocystis platani, the causal agent of the canker stain of plane. Pop1 is secreted by Ceratocystis populicola, a pathogen of poplar. CP and Pop1 have been suggested to act as PAMPs (pathogen-associated molecular patterns) because they induce phytoalexin synthesis, transcription of defense-related genes, restriction of conidia growth and cell death in various plants. Here, we treated plane leaves with CP or Pop1, and monitored defense responses to define the role of these elicitors in the plant interactions. Both CP and Pop1 were able to induce mitogen-activated protein kinases (MAPKs) phosphorylation, production of reactive oxygen species and nitric oxide, and overexpression of defense related genes. The characteristic DNA fragmentation and the cytological features indicate that CP and Pop1 induce cell death by a mechanism of programmed cell death. Therefore, CP and Pop1 can be considered as two novel, non-catalytic fungal PAMPs able to enhance primary defense. Of particular interest is the observation that CP showed faster activity compared to Pop1. The different timing in defense activation could potentially be due to the structural differences between CP and Pop1 (i.e. different hydrophobic index and different helix content) therefore constituting a starting point in unraveling their structure-function relationships.

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.

Early transcription of defence-related genes in Platanus × acerifolia leaves following treatment with cerato-platanin

The protein elicitor cerato-platanin (CP) is known to induce defence-related responses in various plants. Some of these responses occur very quickly. In the present work, transcriptional changes caused by CP in leaves from Platanus × acerifolia (Aiton) Willd. were studied. With a cDNA microarray, 131 differentially regulated transcripts were identified as responsive to CP after 24 h of treatment. Eighty-six of these were cold-or ozone-modulated transcripts, thus revealing a significant overlap between genes responsive to CP and to cold/ozone stress. The transcriptional changes caused by CP were compared with the CP-orthologous protein Pop1 in a time-course analysis performed after 3, 6, 12, and 24 h of treatment by real-time RT-PCR on five defence-related genes. Despite some differences, CP and Pop1 were both able to induce early transcriptional changes (WRKY was overexpressed after only 3 h) confirming that pathogenassociated molecular patterns (PAMPs) act very quickly on gene transcription.

Gene expression analyses reveal a relationship between conidiation and cerato-platanin in homokaryotic and heterokaryotic strains of the fungal plant pathogen Heterobasidion irregulare

The Basidiomycete Heterobasidion irregulare was recently sequenced and three cerato-platanin encoding genes were found in its genome (HiCPs). Cerato-platanin family proteins (CPPs) are produced by both plant pathogenic and non-pathogenic fungi, and can act both as virulence factors and elicitors of defence responses. In fungal life, these proteins seem to play a dual role, in the fungal cell wall and in the fungus–plant interaction, but most data available to date on CPPs derive from studies performed on Ascomycetes. In the present study, we investigated the expression of HiCPs in three homokaryotic isolates and two heterokaryotic isolates of the forest pathogen H. irregulare. Transcription of HiCPs was analysed both at the edge and at the centre of the fungal colony and compared between homokaryon and heterokaryon. The results showed that only HiCP1 and HiCP2 are likely to be translated in H. irregulare and that, under the tested conditions, HiCP1 is the gene with by far the highest transcript abundance among HiCPs. HiCP1 did not show any preferential expression in different sections of the fungal colony, while HiCP2 was significantly more expressed at the colony centre, thus suggesting a link with the production of conidia. The level of expression of HiCPs in heterokaryons was generally comparable to that of one or both the parental homokaryons, irrespective of the colony section, thus demonstrating that HiCPs are not transcriptionally influenced by the heterokaryotic stage.