Seeking the Roles for Fungal Small-Secreted Proteins in Affecting Saprophytic Lifestyles.

Small secreted proteins (SSPs) comprise 40-60% of the total fungal secretome and are present in fungi of all phylogenetic groups, representing the entire spectrum of lifestyles. They are characteristically shorter than 300 amino acids in length and have a signal peptide. The majority of SSPs are coded by orphan genes, which lack known domains or similarities to known protein sequences. Effectors are a group of SSPs that have been investigated extensively in fungi that interact with living hosts, either pathogens or mutualistic systems. They are involved in suppressing the host defense response and altering its physiology. Here, we aim to delineate some of the potential roles of SSPs in saprotrophic fungi, that have been bioinformatically predicted as effectors, and termed in this mini-review as "effector-like" proteins. The effector-like Ssp1 from the white-rot fungus Pleurotus ostreatus is presented as a case study, and its potential role in regulating the ligninolytic system, secondary metabolism, development, and fruiting body initiation are discussed. We propose that deciphering the nature of effector-like SSPs will contribute to our understanding of development and communication in saprophytic fungi, as well as help, to elucidate the origin, regulation, and mechanisms of fungal-host, fungal-fungal, and fungal-bacterial interactions.

Identification and manipulation of Neurospora crassa genes involved in sensitivity to furfural.

BACKGROUND: Biofuels derived from lignocellulosic biomass are a viable alternative to fossil fuels required for transportation. Following plant biomass pretreatment, the furan derivative furfural is present at concentrations which are inhibitory to yeasts. Detoxification of furfural is thus important for efficient fermentation. Here, we searched for new genetic attributes in the fungus Neurospora crassa that may be linked to furfural tolerance. The fact that furfural is involved in the natural process of sexual spore germination of N. crassa and that this fungus is highly amenable to genetic manipulations makes it a rational candidate for this study. RESULTS: Both hypothesis-based and unbiased (random promotor mutagenesis) approaches were performed to identify N. crassa genes associated with the response to furfural. Changes in the transcriptional profile following exposure to furfural revealed that the affected processes were, overall, similar to those observed in Saccharomyces cerevisiae. N. crassa was more tolerant (by ~ 30%) to furfural when carboxymethyl cellulose was the main carbon source as opposed to sucrose, indicative of a link between carbohydrate metabolism and furfural tolerance. We also observed increased tolerance in a Δcre-1 mutant (CRE-1 is a key transcription factor that regulates the ability of fungi to utilize non-preferred carbon sources). In addition, analysis of aldehyde dehydrogenase mutants showed that ahd-2 (NCU00378) was involved in tolerance to furfural as well as the predicted membrane transporter NCU05580 (flr-1), a homolog of FLR1 in S. cerevisiae. Further to the rational screening, an unbiased approach revealed additional genes whose inactivation conferred increased tolerance to furfural: (i) NCU02488, which affected the abundance of the non-anchored cell wall protein NCW-1 (NCU05137), and (ii) the zinc finger protein NCU01407. CONCLUSIONS: We identified attributes in N. crassa associated with tolerance or degradation of furfural, using complementary research approaches. The manipulation of the genes involved in furan sensitivity can provide a means for improving the production of biofuel producing strains. Similar research approaches can be utilized in N. crassa and other filamentous fungi to identify additional attributes relevant to other furans or toxic chemicals.

Manipulating the Expression of Small Secreted Protein 1 (Ssp1) Alters Patterns of Development and Metabolism in the White-Rot Fungus Pleurotus ostreatus.

The function of small secreted proteins (SSPs) in saprotrophic fungi is, for the most part, unknown. The white-rot mushroom Pleurotus ostreatus produces considerable amounts of SSPs at the onset of secondary metabolism, during colony development, and in response to chemical compounds such as 5-hydroxymethylfurfural and aryl alcohols. Genetic manipulation of Ssp1, by knockdown (KDssp1) or overexpression (OEssp1), indicated that they are, in fact, involved in the regulation of the ligninolytic system. To elucidate their potential involvement in fungal development, quantitative secretome analysis was performed during the trophophase and the idiophase and at a transition point between the two growth phases. The mutations conferred a time shift in the secretion and expression patterns: OEssp1 preceded the entrance to idiophase and secondary metabolism, while KDssp1 was delayed. This was also correlated with expression patterns of selected genes. The KDssp1 colony aged at a slower pace, accompanied by a slower decline in biomass over time. In contrast, the OEssp1 strain exhibited severe lysis and aging of the colony at the same time point. These phenomena were accompanied by variations in yellow pigment production, characteristic of entrance of the wild type into idiophase. The pigment was produced earlier and in a larger amount in the OEssp1 strain and was absent from the KDssp1 strain. Furthermore, the dikaryon harboring OEssp1 exhibited a delay in the initiation of fruiting body formation as well as earlier aging. We propose that Ssp1 might function as a part of the fungal communication network and regulate the pattern of fungal development and metabolism in P. ostreatusIMPORTANCE Small secreted proteins (SSPs) are common in fungal saprotrophs, but their roles remain elusive. As such, they comprise part of a gene pool which may be involved in governing fungal lifestyles not limited to symbiosis and pathogenicity, in which they are commonly referred to as "effectors." We propose that Ssp1 in the white-rot fungus Pleurotus ostreatus regulates the transition from primary to secondary metabolism, development, aging, and fruiting body initiation. Our observations uncover a novel regulatory role of effector-like SSPs in a saprotroph, suggesting that they may act in fungal communication as well as in response to environmental cues. The presence of Ssp1 homologues in other fungal species supports a common potential role in environmental sensing and fungal development.

Effects of cre1 modification in the white-rot fungus Pleurotus ostreatus PC9: altering substrate preference during biological pretreatment.

BACKGROUND: During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation. RESULTS: Pleurotus ostreatus PC9 was genetically modified to either overexpress or eliminate (by gene replacement) the transcriptional regulator CRE1, known to act as a repressor in the process of carbon catabolite repression. The cre1-overexpressing transformant demonstrated lower secreted cellulolytic activity and slightly increased selectivity (based on the chemical composition of pretreated wheat straw), whereas the knockout transformant demonstrated increased cellulolytic activity and significantly reduced residual cellulose, thereby displaying lower selectivity. Pretreatment of wheat straw using the wild-type PC9 resulted in 2.8-fold higher yields of soluble sugar compared to untreated wheat straw. The overexpression transformant showed similar yields (2.6-fold), but the knockout transformant exhibited lower yields (1.2-fold) of soluble sugar. Based on proteomic secretome analysis, production of numerous CAZymes was affected by modification of the expression level of cre1. CONCLUSIONS: The gene cre1 functions as a regulator for expression of fungal CAZymes active against plant cell wall lignocelluloses, hence altering the substrate preference of the fungi tested. While the cre1 knockout resulted in a less efficient biological pretreatment, i.e., less saccharification of the treated biomass, the converse manipulation of cre1 (overexpression) failed to improve efficiency. Despite the inverse nature of the two genetic alterations, the expected "mirror image" (i.e., opposite regulatory response) was not observed, indicating that the secretion level of CAZymes, was not exclusively dependent on CRE1 activity.

Author Correction: A role for small secreted proteins (SSPs) in a saprophytic fungal lifestyle: Ligninolytic enzyme regulation in Pleurotus ostreatus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

A role for small secreted proteins (SSPs) in a saprophytic fungal lifestyle: Ligninolytic enzyme regulation in Pleurotus ostreatus.

Small secreted proteins (SSPs), along with lignocellulose degrading enzymes, are integral components of the secretome of Pleurotus ostreatus, a white rot fungus. In this study, we identified 3 genes (ssp1, 2 and 3) encoding proteins that are annotated as SSPs and that exhibited of ~4,500- fold expression, 24 hr following exposure to the toxic compound 5-hydroxymethylfurfural (HMF). Homologues to genes encoding these SSPs are present in the genomes of other basidiomycete fungi, however the role of SSPs is not yet understood. SSPs, aryl-alcohol oxidases (AAO) and the intracellular aryl-alcohol dehydrogenases (AAD) were also produced after exposure to other aryl-alcohols, known substrates and inducers of AAOs, and during idiophase (after the onset of secondary metabolism). A knockdown strain of ssp1 exhibited reduced production of AAO-and AAD-encoding genes after HMF exposure. Conversely, a strain overexpressing ssp1 exhibited elevated expression of genes encoding AAOs and ADD, resulting in a 3-fold increase in enzymatic activity of AAOs, as well as increased expression and protein abundance of versatile peroxidase 1, which directly degrades lignin. We propose that in addition to symbionts and pathogens, SSPs also have roles in saprophytes and function in P. ostreatus as components of the ligninolytic system.

Detoxification of 5-hydroxymethylfurfural by the Pleurotus ostreatus lignolytic enzymes aryl alcohol oxidase and dehydrogenase.

BACKGROUND: Current large-scale pretreatment processes for lignocellulosic biomass are generally accompanied by the formation of toxic degradation products, such as 5-hydroxymethylfurfural (HMF), which inhibit cellulolytic enzymes and fermentation by ethanol-producing yeast. Overcoming these toxic effects is a key technical barrier in the biochemical conversion of plant biomass to biofuels. Pleurotus ostreatus, a white-rot fungus, can efficiently degrade lignocellulose. In this study, we analyzed the ability of P. ostreatus to tolerate and metabolize HMF and investigated relevant molecular pathways associated with these processes. RESULTS: P. ostreatus was capable to metabolize and detoxify HMF 30 mM within 48 h, converting it into 2,5-bis-hydroxymethylfuran (HMF alcohol) and 2,5-furandicarboxylic acid (FDCA), which subsequently allowed the normal yeast growth in amended media. We show that two enzymes groups, which belong to the ligninolytic system, aryl-alcohol oxidases and a dehydrogenase, are involved in this process. HMF induced the transcription and production of these enzymes and was accompanied by an increase in activity levels. We also demonstrate that following the induction of these enzymes, HMF could be metabolized in vitro. CONCLUSIONS: Aryl-alcohol oxidase and dehydrogenase gene family members are part of the transcriptional and subsequent translational response to HMF exposure in P. ostreatus and are involved in HMF transformation. Based on our data, we propose that these enzymatic capacities of P. ostreatus either be integrated in biomass pretreatment or the genes encoding these enzymes may function to detoxify HMF via heterologous expression in fermentation organisms, such as Saccharomyces cerevisiae.

Neurospora crassa protein arginine methyl transferases are involved in growth and development and interact with the NDR kinase COT1.

The protein arginine methyltransferaseas (PRMTs) family is conserved from yeast to human, and regulates stability, localization and activity of proteins. We have characterized deletion strains corresponding to genes encoding for PRMT1/3/5 (designated amt-1, amt-3 and skb-1, respectively) in Neurospora crassa. Deletion of PRMT-encoding genes conferred altered Arg-methylated protein profiles, as determined immunologically. Δamt-1 exhibited reduced hyphal elongation rates (70% of wild type) and increased susceptibility to the ergosterol biosynthesis inhibitor voriconazole. In ▵amt-3, distances between branches were significantly longer than the wild type, suggesting this gene is required for proper regulation of hyphal branching. Deletion of skb-1 resulted in hyper conidiation (2-fold of the wild type) and increased tolerance to the chitin synthase inhibitor polyoxin D. Inactivation of two Type I PRMTs (amt-1 and amt-3) conferred changes in both asymmetric as well as symmetric protein methylation profiles, suggesting either common substrates and/or cross-regulation of different PRMTs. The PRMTs in N. crassa apparently share cellular pathways which were previously reported to be regulated by the NDR (Nuclear DBF2-related) kinase COT1. Using co-immunprecipitation experiments (with MYC-tagged proteins), we have shown that SKB1 and COT1 physically interacted and the abundance of the 75 kDa MYC::COT1 isoform was increased in a Δskb-1 background. On the basis of immunological detection, we propose the possible involvement of PRMTs in Arg-methylation of COT1.

The N-terminal region of the Neurospora†NDR kinase COT1 regulates morphology via its interactions with MOB2A/B.

Nuclear Dbf2p-related (NDR) protein kinases are important for cell differentiation and polar morphogenesis in various organisms, yet some of their functions are still elusive. Dysfunction of the Neurospora crassa NDR kinase COT1 leads to cessation of tip extension and hyperbranching. NDR kinases require the physical interaction between the kinase's N-terminal region (NTR) and the MPS1-binding (MOB) proteins for their activity and functions. To study the interactions between COT1 and MOB2 proteins, we mutated several conserved residues and a novel phosphorylation site within the COT1 NTR. The phenotypes of these mutants suggest that the NTR is required for COT1 functions in regulating hyphal elongation and branching, asexual conidiation and germination. Interestingly, while both MOB2A and MOB2B promote proper hyphal growth, they have distinct COT1-dependent roles in regulation of macroconidiation. Immunoprecipitation experiments indicate physical association of COT1 with both MOB2A and MOB2B, simultaneously. Furthermore, the binding of the two MOB2 proteins to COT1 is mediated by different residues at the COT1 NTR, suggesting a hetero-trimer is formed. Thus, although MOB2A/B may have some overlapping functions in regulating hyphal tip extension, their function is not redundant and they are both required for proper fungal development.