Cerato-platanin Family Protein Research Articles

Partitioning the structural features that underlie expansin-like and elicitor activities of cerato-platanin

Cerato-platanin family (CPF) proteins are produced by fungi and elicit defences when applied to plants, behaving as PAMPs/MAMPs. CPF proteins share structural similarity to plant and bacterial expansins, and have been demonstrated, in some cases, to possess expansin-like loosening activity on cellulose. This is the case of cerato-platanin (CP), the founder of the CPF, which shows both eliciting and cellulose-loosening activities, raising the question as to whether the expansin-like activity may be responsible for defence activation. To pinpoint structural and thermodynamic features underlying eliciting and expansin-like activity of CP, we carried out site-directed mutagenesis targeting separately net charge (N84D mutation), conformational stability (V63A mutation), or conserved position previously shown to affect expansin-like activity in CP (D77A mutation), and characterized wild-type protein and its variants. Removing or adding negative charges on the protein surface led to reducing or increasing, respectively, the expansin-like activity. The activity was instead not affected by mutations affecting protein fold and stability. In contrast, all the mutants showed reduced capacity to elicit defences in plants. We conclude that the expansin-like activity of CP depends on net charge and ability to (weakly) bind cellulose, whereas the eliciting activity on plants does not depend on the cellulose-loosening activity.

Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans.

Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 μg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complemented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.

Secretome of Trichoderma virens induced by banana roots - identification of novel fungal proteins for enhancing plant defence

Trichoderma virens is a plant beneficial fungus that interacts intimately with roots and boosts plant immunity against invading pathogens. Some Trichoderma species have been used to control vascular wilt pathogen Fusarium oxysporum f. sp. cubense by enhancing host plant defence. In order to understand the molecular mechanisms of such interactions of Trichoderma with banana roots, this study analyses the secretome of T. virens induced by tissue culture raised banana cv. Rasthali roots, using a gel-free proteome approach. Tissue cultured banana roots were co-incubated with germinated conidia of T. virens for four days. The filtrate was harvested and proteins were identified using mass spectrometry (trypsin digestion and LC-MS/MS analysis). A total of 77 proteins were identified to be upregulated in T. virens in contact with banana roots, compared with T. virens incubated alone. Among these, 11 and 6 belonged to the glycoside hydrolases and small cysteine-rich secreted proteins (SSCPs), respectively. These two groups of proteins are known to be involved in plant-Trichoderma interactions, facilitating penetration, colonization, and inducing defense responses. Contrary to this, 27 proteins were downregulated in contact with banana roots and 32 did not show any significant changes. This is the first study on proteome analysis of Trichoderma in contact with banana roots leading to the identification of potential elicitor proteins that could be relevant to disease management in banana. A novel cerato-platanin family protein and a Nep-1 like protein were found to be upregulated in contact with banana roots. Since such proteins are known to be involved in plant-microbe interactions, these are being studied for their role in Trichoderma-banana plant interactions especially in induced defence.

A Cerato-Platanin Family Protein FocCP1 Is Essential for the Penetration and Virulence of Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is well-known as the causal agent of Fusarium wilt of banana and is one of the most destructive phytopathogens for banana plants. The molecular mechanisms underlying Foc TR4 virulence remain elusive. Here, we demonstrate that a cerato-platanin (CP) protein, FocCP1, functions as a virulence factor that is required by Foc TR4 for penetration and full virulence. The FocCP1 gene was expressed in every condition studied, showing a high transcript level in planta at the early stage of infection. Infiltration of the recombinant FocCP1 protein induced significant cell death and upregulated defence-related gene expression. FocCP1 knock-out strains showed a significant decrease in aerial growth rather than aqueous growth, which is reminiscent of hydrophobins. Furthermore, deletion of FocCP1 significantly reduced virulence and dramatically reduced infective growth in banana roots, likely resulting from a defective penetration ability. Taken together, the results of this study provide novel insight into the function of the recently identified FocCP1 as a virulence factor in Foc TR4.

SsSm1, a Cerato-platanin family protein, is involved in the hyphal development and pathogenic process of Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum is an important plant pathogen with a worldwide distribution. It can infect a wide variety of plants, causing serious disease in many types of crops, such as rapeseed, sunflower and soybean. Sclerotinia stem rot caused by this fungus affects main crops and has led to great economic loss. Elicitors are a group of compounds that inspire the host plant to produce an immune response against invading pathogens. This study describes a protein that has high homology with the Trichoderma elicitor Sm1 and was found in the genome of S. sclerotiorum. We named this protein SsSm1. To determine whether this protein has an elicitor function like its homology protein, we constructed a heterologous expression vector for SsSm1 and expressed it in Escherichia coli. The protein of heterologous expression led to the formation of lesions in tobacco that closely resemble hypersensitive response lesions. Transient expression of the encoding gene of SsSm1 in tobacco leaves also caused hypersensitive response. Then, RNA silencing was used to identify the function of SsSm1. The hyphal growth and pathogenicity of silenced transformants were shown to be obviously lagging and branched abnormally. Transformants produced less infection cushions and deformed sclerotiorum. In addition, SsSm1 silencing caused weak tolerance to NaCl, sorbitol and SDS, and the sensitivity of mutants to carbendazim was also significantly decreased. Based on the above results, we speculate that this protein may be related to the development of hyphae, infection cushions and sclerotiorum, but the specific molecular mechanism needs to be studied further.

Comparison of cerato-platanin family protein BcSpl1 produced in Pichia pastoris and Escherichia coli

The Botrytis cinerea BcSpl1 protein is a member of the cerato-platanin family, and consists of 137 amino acids and two disulfide bridges. This protein induces the onset of necrosis in infiltrated plant hosts. Recombinant BcSpl1 proteins produced in Pichia pastoris (pBcSpl1) and Escherichia coli (eBcSpl1) were initially compared regarding their abilities to induce necrosis and systemic acquired resistance (SAR). The pBcSpl1 and eBcSpl1 treatments led to the development of necrotic lesions on tomato leaves, and provided tomato plants with SAR to B. cinerea. The lesion area of leaves infiltrated with the BcSpl1 proteins decreased by 22.7% (pBcSpl1) and 21.8% (eBcSpl1). Additionally, eBcSpl1 up-regulated the expression levels of some defense-related genes, including PR-1a, prosystemin, PI I, and PI II, as well as SIPK and TPK1b, which encode two protein kinases. Furthermore, eBcSpl1 exhibited chitin-binding properties. Our data revealed that the E. coli expression system produces higher BcSpl1 yields than the P. pastoris system. This high-yield expression of BcSpl1 may be relevant for future large-scale applications of this elicitor to improve crop production.

Magnaporthe oryzae-Secreted Protein MSP1 Induces Cell Death and Elicits Defense Responses in Rice.

The Magnaporthe oryzae snodprot1 homolog (MSP1), secreted by M. oryzae, is a cerato-platanin family protein. msp1-knockout mutants have reduced virulence on barley leaves, indicating that MSP1 is required for the pathogenicity of rice blast fungus. To investigate the functional roles of MSP1 and its downstream signaling in rice, recombinant MSP1 was produced in Escherichia coli and was assayed for its functionality. Application of MSP1 triggered cell death and elicited defense responses in rice. MSP1 also induced H2O2 production and autophagic cell death in both suspension-cultured cells and rice leaves. One or more protein kinases triggered cell death, jasmonic acid and abscisic acid enhanced cell death, while salicylic acid suppressed it. We demonstrated that the secretion of MSP1 into the apoplast is a prerequisite for triggering cell death and activating defense-related gene expression. Furthermore, pretreatment of rice with a sublethal MSP1 concentration potentiated resistance to the pathogen. Taken together, our results showed that MSP1 induces a high degree of cell death in plants, which might be essential for its virulence. Moreover, rice can recognize MSP1, resulting in the induction of pathogen-associated molecular pattern-triggered immunity.

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.

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?

The phytotoxic activity of the cerato‐platanin BcSpl1 resides in a two‐peptide motif on the protein surface

Cerato-platanin family proteins are secreted and have been found in both the fungal cell wall and the extracellular medium. They elicit defence responses in a variety of plants and have been proposed to be perceived as pathogen-associated molecular patterns (PAMPs) by the plant immune system, although, in the case of the necrotroph Botrytis cinerea, the cerato-platanin BcSpl1 contributes to fungal virulence instead of plant resistance. In this study, we report that BcSpl1, which was previously found in the secretome as an abundant protein, is even more abundant in the fungal cell wall. By fusion to green fluorescent protein (GFP), we also show that BcSpl1 associates with the plant plasma membrane causing rapid morphological changes at the cellular level, such as the disorganization of chloroplasts, prior to macroscopic necrosis in the treated tissue. By a combination of serial deletion studies, synthetic peptides and chimeric proteins, we mapped the eliciting activity to a two-peptide motif in the protein surface. The expression of a chimeric protein displaying this motif in B. cinerea mutants lacking BcSpl1 undoubtedly showed that the motif is responsible for the contribution of BcSpl1 to virulence.

The Botrytis cinerea cerato‐platanin BcSpl1 is a potent inducer of systemic acquired resistance (SAR) in tobacco and generates a wave of salicylic acid expanding from the site of application

Systemic acquired resistance (SAR) is a potent plant defence system that, in response to a first contact with a plant pathogen, prepares the whole plant for subsequent attacks, so that it becomes more resistant to the same and to other pathogens. BcSpl1, a cerato-platanin family protein abundantly secreted by Botrytis cinerea, is required for full virulence and elicits the hypersensitive response in the host. Here, we report that BcSpl1 is also able to induce in tobacco systemic resistance to two plant pathogens, Pseudomonas syringae and B. cinerea, which correlates with the induction of two pathogenesis-related genes, PR-1a and PR-5. Levels of salicylic acid were quantified in situ on BcSpl1 infiltration, and a wave of salicylic acid departing from the point of infiltration and running through the leaf was observed, as well as the appearance of this plant hormone in the neighbouring leaves as early as 3 days after infiltration.

BcSpl1, a cerato-platanin family protein, contributes to Botrytis cinerea virulence and elicits the hypersensitive response in the host

Proteins belonging to the cerato-platanin family are small proteins with phytotoxic activity. A member of this family, BcSpl1, is one of the most abundant proteins in the Botrytis cinerea secretome. Expression analysis of the bcspl1 gene revealed that the transcript is present in every condition studied, showing the highest level in planta at the late stages of infection. Expression of a second cerato-platanin gene found in the B. cinerea genome, bcspl2, was not detected in any condition. Two bcspl1 knock-out mutants were generated and both showed reduced virulence in a variety of hosts. • bcspl1 was expressed in Pichia pastoris and the recombinant protein was able to cause a fast and strong necrosis when infiltrated in tomato, tobacco and Arabidopsis leaves, in a dose-dependent manner. The BcSpl1-treated plant tissues showed symptoms of the hypersensitive response such as induction of reactive oxygen species, electrolyte leakage, cytoplasm shrinkage, and cell autofluorescence, as well as the induction of defense genes considered to be markers of the hypersensitive response. The Arabidopsis bak1 mutation partially prevented the induction of necrosis in this plant by BcSpl1. Two different BcSpl1-derived 40-amino acids peptides were also active in inducing necrosis.