The essential role of aggregation for the emulsifying ability of a fungal CYS-rich protein.

Biosurfactants are in demand by the global market as natural commodities suitable for incorporation into commercial products or utilization in environmental applications. Fungi are promising producers of these molecules and have garnered interest also for their metabolic capabilities in efficiently utilizing recalcitrant and complex substrates, like hydrocarbons, plastic, etc. Within this framework, biosurfactants produced by two Fusarium solani fungal strains, isolated from plastic waste-contaminated landfill soils, were analyzed. Mycelia of these fungi were grown in the presence of 5% olive oil to drive biosurfactant production. The characterization of the emulsifying and surfactant capacity of these extracts highlighted that two different components are involved. A protein was purified and identified as a CFEM (common in fungal extracellular membrane) containing domain, revealing a good propensity to stabilize emulsions only in its aggregate form. On the other hand, an unidentified cationic smaller molecule exhibits the ability to reduce surface tension. Based on the 3D structural model of the protein, a plausible mechanism for the formation of very stable aggregates, endowed with the emulsifying ability, is proposed. KEY POINTS: • Two Fusarium solani strains are analyzed for their surfactant production. • A cationic surfactant is produced, exhibiting the ability to remarkably reduce surface tension. • An identified protein reveals a good propensity to stabilize emulsions only in its aggregate form.

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence.

BACKGROUND: Verticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown. RESULTS: Nine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type. CONCLUSIONS: In the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

Structure-function analyses of the Pth11 receptor reveal an important role for CFEM motif and redox regulation in rice blast.

The interaction of Magnaporthe oryzae, the rice blast fungus, and rice begins when M. oryzae establishes contact with the host plant surface. On perception of appropriate surface signals, M. oryzae forms appressoria and initiates host invasion. Pth11, an important G-protein-coupled receptor necessary for appressorium formation in M. oryzae, contains seven transmembrane regions and a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues. We focused on gaining further insight into the role of the CFEM domain in the putative surface sensing/response function of Pth11. Increased/constitutive expression of CFEM resulted in precocious, albeit defective, appressoria formation in wild-type M. oryzae. The Pth11C63A/C65A mutant, probably with disrupted disulfide bonds in the CFEM, showed delayed appressorium formation and reduced virulence. Furthermore, the accumulation of reactive oxygen species (ROS) was found to be altered in the pth11Δ strain. Strikingly, antioxidant treatment induced appressorium formation in pth11Δ. The Gα subunit MagB and the mitogen-activated protein (MAP) kinase Pmk1 were required for the formation of antioxidant-induced appressoria. We conclude that the CFEM domain of Pth11 is required for proper development of the appressoria, appressoria-like structures and pathogenicity. Highly regulated ROS homeostasis is important for Pth11-mediated appressorium formation in M. oryzae.

The three Aspergillus fumigatus CFEM-domain GPI-anchored proteins (CfmA-C) affect cell-wall stability but do not play a role in fungal virulence.

Fungal cell-wall proteins containing the conserved fungal CFEM domain have been implicated in host-pathogen interactions and virulence. To determine the role of these proteins in the mold pathogen Aspergillus fumigatus, we deleted the entire family of three CFEM-containing genes (CfmA-C), singly and in all combinations. We found an additive increase in the susceptibility of the single, double and triple ΔCfm mutants towards the chitin/ß-glucan-microfibril destabilizing compounds Congo Red (CR) and Calcofluor White (CFW), indicating that the A. fumigatus CFEM proteins are involved in stabilizing the cell wall. No defects in growth or germination were observed, indicating that CFEM proteins do not have an essential role in the morphogenesis of A. fumigatus. Unlike in Candida albicans, the A. fumigatus CFEM proteins were not implicated in heme uptake or biofilm formation. The ΔTriple-Cfm deletion strain did not exhibit altered virulence in either insect or murine models of infection, suggesting that cell-wall proteins containing the conserved fungal CFEM domain are not a significant virulence factor in A. fumigatus.

Systematic identification and functional characterization of the CFEM proteins in fishscale bamboo rhombic-spot pathogen Neostagonosporella sichuanensis.

Fungal effectors play a crucial role in the interaction between pathogenic fungi and their hosts. These interactions directly influence the invasion and spread of pathogens, and the development of diseases. Common in fungal extracellular membrane (CFEM) effectors are closely associated with the pathogenicity, cell wall stability, and pathogenic processes of pathogenic fungi. The aim of this study was to investigate the role of CFEM proteins in Neostagonosporella sichuanensis in pathogen-host interactions. We retrieved 19 proteins containing CFEM structural domains from the genome of N. sichuanensis. By systematic analysis, five NsCFEM proteins had signal peptides but lacked transmembrane structural domains, and thus were considered as potential effectors. Among them, NsCFEM1 and NsCFEM2 were successfully cloned and their functions were further investigated. The validation results show that NsCFEM1 was localized in the cell membrane and nucleus, whereas NsCFEM2 was exclusively observed in the cell membrane. Both were identified as secreted proteins. Additionally, NsCFEM1 inhibited Bax-induced programmed cell death in Nicotiana benthamiana, whereas NsCFEM2 did not induce or inhibit this response. NsCFEM1 was implicated as a virulence factor that contributes to fungal growth, development, stress response, and pathogenicity. NsCFEM2 was implicated in maintenance of cell wall stability. This study lays a foundation for elucidating the role of CFEM proteins in the pathogen of fishscale bamboo rhombic-spot caused by N. sichuanensis. In particular, the functional studies of NsCFEM1 and NsCFEM2 revealed their potential roles in the interaction between N. sichuanensis and the host Phyllostachys heteroclada.

Identification of common fungal extracellular membrane (CFEM) proteins in Fusarium sacchari that inhibit plant immunity and contribute to virulence.

IMPORTANCE: Common fungal extracellular membrane (CFEM) domain-containing protein has long been considered an essential effector, playing a crucial role in the interaction of pathogens and plant. Strategies aimed at understanding the pathogenicity mechanism of F. sacchari are eagerly anticipated to ultimately end the spread of pokkah boeng disease. Twenty FsCFEM proteins in the genome of F. sacchari have been identified, and four FsCFEM effector proteins have been found to suppress BCL2-associated X protein-triggered programmed cell death in N. benthamiana. These four effector proteins have the ability to enter plant cells and inhibit plant immunity. Furthermore, the expression of these four FsCFEM effector proteins significantly increases during the infection stage, with the three of them playing an essential role in achieving full virulence. These study findings provide a direction toward further exploration of the immune response in sugarcane. By applying these discoveries, we can potentially control the spread of disease through techniques such as host-induced gene silencing.

A candidate effector protein PstCFEM1 contributes to virulence of stripe rust fungus and impairs wheat immunity.

Common in Fungal Extracellular Membrane (CFEM) domain proteins are considered to be unique to fungi and closely related to pathogenicity. However, the Puccinia striiformis f. sp. tritici (Pst) effector containing the CFEM domain has not been reported. Here, we obtained an effector, PstCFEM1, containing a functional N-terminal signal peptide sequence and the CFEM domain from Pst race CYR31. qRT-PCR assay indicated that the transcript levels of PstCFEM1 were highly induced during the early stages of infection. Overexpression of PstCFEM1 suppressed Pst322 (an elicitor-like protein of Pst)-trigged cell death, reactive oxygen species (ROS) accumulation and callose deposition. Host-induced gene silencing (HIGS) experiments showed that knockdown of PstCFEM1 decreased the virulence of Pst, while ROS accumulation in silenced plants increased near the infection site. In addition, wheat containing the PstCFEM1-silenced construct increased resistance to multiple races of Pst. Our data suggest that PstCFEM1 suppresses wheat defense by inhibiting ROS accumulation and contributes to increased virulence of Pst.

Systematic identification and functional characterization of the CFEM proteins in poplar fungus Marssonina brunnea.

Proteins containing Common in Fungal Extracellular Membrane (CFEM) domains uniquely exist in fungi and play significant roles in their whole life history. In this study, a total of 11 MbCFEM proteins were identified from Marssonina brunnea f. sp. multigermtubi (MULT), a hemibiotrophic pathogenic fungus on poplars that causes severe leaf diseases. Phylogenic analysis showed that the 11 proteins (MbCFEM1-11) were divided into three clades based on the trans-membrane domain and the CFEM domain. Sequence alignment and WebLogo analysis of CFEM domains verified the amino acids conservatism therein. All of them possess eight cysteines except MbCFEM4 and MbCFEM11, which lack two cysteines each. Six MbCFEM proteins with a signal peptide and without trans-membrane domain were considered as candidate effectors for further functional analysis. Three-dimensional (3D) models of their CFEM domains presented a helical-basket structure homologous to the crucial virulence factor Csa2 of Candida albicans. Afterward, four (MbCFEM1, 6, 8, and 9) out of six candidate effectors were successfully cloned and a yeast signal sequence trap (YSST) assay confirmed their secretion activity. Pathogen challenge assays demonstrated that the transient expression of four candidate MbCFEM effectors in Nicotiana benthamiana promoted Fusarium proliferatum infection, respectively. In an N. benthamiana heterogeneous expression system, MbCFEM1, MbCFEM6, and MbCFEM9 appeared to suppress both BAX/INF1-triggered PCD, whereas MbCFEM8 could only defeat BAX-triggered PCD. Additionally, subcellular localization analysis indicated that the four candidate MbCFEM effectors accumulate in the cell membrane, nucleus, chloroplast, and cytosolic bodies. These results demonstrate that MbCFEM1, MbCFEM6, MbCFEM8, and MbCFEM9 are effectors of M. brunnea and provide valuable targets for further dissection of the molecular mechanisms underlying the poplar-M. brunnea interaction.

Bioinformatics Analysis and Functional Characterization of the CFEM Proteins of Metarhizium anisopliae.

The entomopathogen Metarhizium anisopliae is a facultative rhizosphere or endophytic fungus available for managing pests and improving plant growth. The CFEM (common in fungal extracellular membrane) proteins form a unique group in fungi but are rarely reported in entomopathogens. In this study, we cloned and identified 13 CFEM genes from M. anisopliae (MaCFEMs). Sequence alignment and WebLogo analysis showed that eight cysteines were the most conserved amino acids in their CFEM domain. Phylogenic analysis suggested that these 13 proteins could be divided into 4 clades based on the presence of the transmembrane region and the position of CFEM domain in the whole sequence. Six MaCFEM proteins with a signal peptide and without a transmembrane domain were considered candidate effector proteins. According to Phyre2 analysis, the MaCFEM88 and MaCFEM85 have the most homologous to Csa2 in Candida albicans. Subcellular localization analysis revealed that five effectors were located in the plasma membrane, while MaCFEM88 may locate in both plasma membrane and nucleus in the treated Nicotiana benthamiana. Expression pattern analysis showed that MaCFEM81, 85, 88, and 89 expression level was significantly higher in the sporulation stage compared to other growth stages. Furthermore, the yeast secretion assay showed that six candidate effectors were able to secrete out of the cell. All of the MaCFEMs couldn't affect INF1-induced programmed cell death (PCD), but MaCFEM85 and 88 could trigger a slight hypersensitive response both when applied separately or in combination with INF1 in N. benthamiana leaves. These findings showed that six MaCFEM potential effectors with various structures and subcellular localizations in host cells might be used to illustrate the roles of MaCFEM proteins during M. anisopliae-plant interactions.

Systemic Identification and Functional Characterization of Common in Fungal Extracellular Membrane Proteins in Lasiodiplodia theobromae.

Plant pathogenic fungi deploy secreted proteins into apoplastic space or intracellular lumen to promote successful infections during plant-pathogen interactions. In the present study, fourteen CFEM domain-containing proteins were systemically identified in Lasiodiplodia theobromae and eight of them were functionally characterized. All eight proteins were confirmed to be secreted into extracellular space by a yeast signal peptide trapping system. The transcriptional levels of most CFEM genes, except for LtCFEM2 and LtCFEM6, were significantly elevated during infection. In addition, almost all LtCFEM genes, apart from LtCFEM2, LtCFEM3, and LtCFEM6, were transcriptionally up-regulated at 35°C in contrast to that at 25°C and 30°C. As two elicitors, LtCFEM1 induced local yellowish phenotype and LtCFEM4 triggered cell death in Nicotiana benthamiana leaves. Furthermore, these proteins displayed distinct subcellular localizations when expressed transiently in N. benthamiana. Moreover, two genes, LtCFEM7 and LtCFEM8, were found to be spliced alternatively by RT-PCR and sequencing. Therefore, our data suggest that LtCFEM proteins play important roles in multiple aspects, including pathogenicity and plant immune response, which will enhance our understanding of the sophisticated pathogenic mechanisms of plant opportunistic pathogen L. theobromae.

Bioinformatics and Transcriptome Analysis of CFEM Proteins in Fusarium graminearum.

Fusarium blight of wheat is usually caused by Fusarium graminearum, and the pathogenic fungi will secrete effectors into the host plant tissue to affect its normal physiological process, so as to make it pathogenic. The CFEM (Common in Fungal Extracellular Membrane) protein domain is unique to fungi, but it is not found in all fungi. The CFEM protein contained in F. graminearum may be closely related to pathogenicity. In this study, 23 FgCFEM proteins were identified from the F. graminearum genome. Then, features of these proteins, such as signal peptide, subcellular localization, and transmembrane domains, etc., were analyzed and candidate effectors were screened out. Sequence alignment results revealed that each FgCFEM protein contains one CFEM domain. The amino acids of the CFEM domain are highly conserved and contain eight spaced cysteines, with the exception that FgCFEM8, 9, and 15 lack two cysteines and three cysteines were missed in FgCFEM18 and FgCFEM22. A recently identified CFEM_DR motif was detected in 11 FgCFEMs, and importantly we identified two new conserved motifs containing about 29 and 18 amino acids (CFEM_WR and CFEM_KF), respectively, in some of FgCFEM proteins. Transcriptome analysis of the genes encoding CFEM proteins indicated that all the CFEM-containing genes were expressed during wheat infection, with seven and six genes significantly up- and down-regulated, respectively, compared with in planta and in vitro. Based on the above analysis, FgCFEM11 and FgCFEM23 were predicted to be F. graminearum effectors. This study provides the basis for future functional analyses of CFEM proteins in F. graminearum.

Insights of the Neofusicoccum parvum-Liquidambar styraciflua Interaction and Identification of New Cysteine-Rich Proteins in Both Species.

Neofusicoccum parvum belongs to the Botryosphaeriaceae family, which contains endophytes and pathogens of woody plants. In this study, we isolated 11 strains from diseased tissue of Liquidambar styraciflua. Testing with Koch's postulates-followed by a molecular approach-revealed that N. parvum was the most pathogenic strain. We established an in vitro pathosystem (L. styraciflua foliar tissue-N. parvum) in order to characterize the infection process during the first 16 days. New CysRPs were identified for both organisms using public transcriptomic and genomic databases, while mRNA expression of CysRPs was analyzed by RT-qPCR. The results showed that N. parvum caused disease symptoms after 24 h that intensified over time. Through in silico analysis, 5 CysRPs were identified for each organism, revealing that all of the proteins are potentially secreted and novel, including two of N. parvum proteins containing the CFEM domain. Interestingly, the levels of the CysRPs mRNAs change during the interaction. This study reports N. parvum as a pathogen of L. styraciflua for the first time and highlights the potential involvement of CysRPs in both organisms during this interaction.

Comparative genomics of Sporothrix species and identification of putative pathogenic-gene determinants.

Aim: To understand the phylogenomics, pathogenic/virulence-associated genes and genomic evolution of pathogenic Sporothrix species. Materials & methods: We performed in silico comparative genome analysis of Sporothrix species using ab initio tools and in-house scripts. We predicted genes and repeats, compared genomes based on synteny, identified orthologous clusters, assessed genes family expansion/contraction, predicted secretory proteins and finally searched for similar sequences from various databases. Results: The phylogenomics revealed that Sporothrix species are closely related to Ophiostoma species. The gene family evolutionary analysis revealed the expansion of genes related to virulence (CFEM domain, iron acquisition genes, lysin motif domain), stress response (Su[var]3-9, Enhancer-of-zeste and Trithorax domain and Domain of unknown function 1996), proteases (aspartic protease, x-pro dipeptidyl-peptidase), cell wall composition associated genes (chitin deacetylase, chitinase) and transporters (major facilitator superfamily transporter, oligo-peptide transporter family) in Sporothrix species. Conclusion: The present study documents the putative pathogenic/virulence-associated genes in the Sporothrix species.

BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea.

We experimentally isolated and characterized a CFEM protein with putative GPI-anchored site BcCFEM1 in Botrytis cinerea. BcCFEM1 contains a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues at N terminus, and a predicted GPI modification site at C terminus. BcCFEM1 was significantly up-regulated during early stage of infection on bean leaves and induced chlorosis in Nicotiana benthamiana leaves using Agrobacterium infiltration method. Targeted deletion of BcCFEM1 in B. cinerea affected virulence, conidial production and stress tolerance, but not growth rate, conidial germination, colony morphology, and sclerotial formation. However, over expression of BcCFEM1 did not make any observable phenotype change. Therefore, our data suggested that BcCFEM1 contributes to virulence, conidial production, and stress tolerance. These findings further enhance our understanding on the sophisticated pathogenicity of B. cinerea beyond necrotrophic stage, highlighting the importance of CFEM protein to B. cinerea and other broad-host-range necrotrophic pathogens.