Altered Expression of Two Small Secreted Proteins (ssp4 and ssp6) Affects the Degradation of a Natural Lignocellulosic Substrate by Pleurotus ostreatus.

Pleurotus ostreatus is a white-rot fungus that can degrade lignin in a preferential manner using a variety of extracellular enzymes, including manganese and versatile peroxidases (encoded by the vp1-3 and mnp1-6 genes, respectively). This fungus also secretes a family of structurally related small secreted proteins (SSPs) encoded by the ssp1-6 genes. Using RNA sequencing (RNA-seq), we determined that ssp4 and ssp6 are the predominant members of this gene family that were expressed by P. ostreatus during the first three weeks of growth on wheat straw. Downregulation of ssp4 in a strain harboring an ssp RNAi construct (KDssp1) was then confirmed, which, along with an increase in ssp6 transcript levels, coincided with reduced lignin degradation and the downregulation of vp2 and mnp1. In contrast, we observed an increase in the expression of genes related to pectin and side-chain hemicellulose degradation, which was accompanied by an increase in extracellular pectin-degrading capacity. Genome-wide comparisons between the KDssp1 and the wild-type strains demonstrated that ssp silencing conferred accumulated changes in gene expression at the advanced cultivation stages in an adaptive rather than an inductive mode of transcriptional response. Based on co-expression networking, crucial gene modules were identified and linked to the ssp knockdown genotype at different cultivation times. Based on these data, as well as previous studies, we propose that P. ostreatus SSPs have potential roles in modulating the lignocellulolytic and pectinolytic systems, as well as a variety of fundamental biological processes related to fungal growth and development.

Lineage-specific genes are clustered with HET-domain genes and respond to environmental and genetic manipulations regulating reproduction in Neurospora.

Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. In the genome of Neurospora crassa, most of the 670 Neurospora LSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, some of which regulate self-recognition and define vegetative incompatibility groups. In contrast, the LSG-encoding proteins possess few to no domains that would help to identify potential functional roles. Possible functional roles of LSGs were further assessed by performing transcriptomic profiling in genetic mutants and in response to environmental alterations, as well as examining gene knockouts for phenotypes. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural, a wildfire-produced chemical that is a strong inducer of sexual development, and the structurally-related furan 5-hydroxymethyl furfural (HMF). Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that also regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts of adv-1 and pp-1 that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. These observations encouraged further investigation of the roles of clustered lineage-specific and HET-domain genes in ecology and reproduction regulation in Neurospora, especially the regulation of the switch from the asexual growth to sexual reproduction, in response to dramatic environmental conditions changes.

Origins of lineage-specific elements via gene duplication, relocation, and regional rearrangement in Neurospora crassa.

The origin of new genes has long been a central interest of evolutionary biologists. However, their novelty means that they evade reconstruction by the classical tools of evolutionary modelling. This evasion of deep ancestral investigation necessitates intensive study of model species within well-sampled, recently diversified, clades. One such clade is the model genus Neurospora, members of which lack recent gene duplications. Several Neurospora species are comprehensively characterized organisms apt for studying the evolution of lineage-specific genes (LSGs). Using gene synteny, we documented that 78% of Neurospora LSG clusters are located adjacent to the telomeres featuring extensive tracts of non-coding DNA and duplicated genes. Here, we report several instances of LSGs that are likely from regional rearrangements and potentially from gene rebirth. To broadly investigate the functions of LSGs, we assembled transcriptomics data from 68 experimental data points and identified co-regulatory modules using Weighted Gene Correlation Network Analysis, revealing that LSGs are widely but peripherally involved in known regulatory machinery for diverse functions. The ancestral status of the LSG mas-1, a gene with roles in cell-wall integrity and cellular sensitivity to antifungal toxins, was investigated in detail alongside its genomic neighbours, indicating that it arose from an ancient lysophospholipase precursor that is ubiquitous in lineages of the Sordariomycetes. Our discoveries illuminate a "rummage region" in the N. crassa genome that enables the formation of new genes and functions to arise via gene duplication and relocation, followed by fast mutation and recombination facilitated by sequence repeats and unconstrained non-coding sequences.

The Sordariomycetes: an expanding resource with Big Data for mining in evolutionary genomics and transcriptomics.

Advances in genomics and transcriptomics accompanying the rapid accumulation of omics data have provided new tools that have transformed and expanded the traditional concepts of model fungi. Evolutionary genomics and transcriptomics have flourished with the use of classical and newer fungal models that facilitate the study of diverse topics encompassing fungal biology and development. Technological advances have also created the opportunity to obtain and mine large datasets. One such continuously growing dataset is that of the Sordariomycetes, which exhibit a richness of species, ecological diversity, economic importance, and a profound research history on amenable models. Currently, 3,574 species of this class have been sequenced, comprising nearly one-third of the available ascomycete genomes. Among these genomes, multiple representatives of the model genera Fusarium, Neurospora, and Trichoderma are present. In this review, we examine recently published studies and data on the Sordariomycetes that have contributed novel insights to the field of fungal evolution via integrative analyses of the genetic, pathogenic, and other biological characteristics of the fungi. Some of these studies applied ancestral state analysis of gene expression among divergent lineages to infer regulatory network models, identify key genetic elements in fungal sexual development, and investigate the regulation of conidial germination and secondary metabolism. Such multispecies investigations address challenges in the study of fungal evolutionary genomics derived from studies that are often based on limited model genomes and that primarily focus on the aspects of biology driven by knowledge drawn from a few model species. Rapidly accumulating information and expanding capabilities for systems biological analysis of Big Data are setting the stage for the expansion of the concept of model systems from unitary taxonomic species/genera to inclusive clusters of well-studied models that can facilitate both the in-depth study of specific lineages and also investigation of trait diversity across lineages. The Sordariomycetes class, in particular, offers abundant omics data and a large and active global research community. As such, the Sordariomycetes can form a core omics clade, providing a blueprint for the expansion of our knowledge of evolution at the genomic scale in the exciting era of Big Data and artificial intelligence, and serving as a reference for the future analysis of different taxonomic levels within the fungal kingdom.

Differential Expression of Cell Wall Remodeling Genes Is Part of the Dynamic Phase-Specific Transcriptional Program of Conidial Germination of Trichoderma asperelloides.

The nature of saprophytic and mycoparasitic hyphal growth of Trichoderma spp. has been studied extensively, yet its initiation via conidial germination in this genus is less well understood. Using near-synchronous germinating cultures of Trichoderma asperelloides, we followed the morphological progression from dormant conidia to initial polar growth to germling formation and to evidence for first branching. We found that the stage-specific transcriptional profile of T. asperelloides is one of the most dynamic described to date: transcript abundance of over 5000 genes-comprising approximately half of the annotated genome-was unremittingly reduced in the transition from dormancy to polar growth. Conversely, after the onset of germination, the transcript abundance of approximately a quarter of the genome was unremittingly elevated during the transition from elongation to initial branching. These changes are a testimony to the substantial developmental events that accompany germination. Bayesian network analysis identified several chitinase- and glucanase-encoding genes as active transcriptional hubs during germination. Furthermore, the expression of specific members of the chitin synthase and glucan elongase families was significantly increased during germination in the presence of Rhizoctonia solani-a known host of the mycoparasite-indicating that host recognition can occur during the early stages of mycoparasite development.

Secondary Metabolism Gene Clusters Exhibit Increasingly Dynamic and Differential Expression during Asexual Growth, Conidiation, and Sexual Development in Neurospora crassa.

Secondary metabolite clusters (SMCs) encode the machinery for fungal toxin production. However, understanding their function and analyzing their products requires investigation of the developmental and environmental conditions in which they are expressed. Gene expression is often restricted to specific and unexamined stages of the life cycle. Therefore, we applied comparative genomics analyses to identify SMCs in Neurospora crassa and analyzed extensive transcriptomic data spanning nine independent experiments from diverse developmental and environmental conditions to reveal their life cycle-specific gene expression patterns. We reported 20 SMCs comprising 177 genes-a manageable set for investigation of the roles of SMCs across the life cycle of the fungal model N. crassa-as well as gene sets coordinately expressed in 18 predicted SMCs during asexual and sexual growth under three nutritional and two temperature conditions. Divergent activity of SMCs between asexual and sexual development was reported. Of 126 SMC genes that we examined for knockout phenotypes, al-2 and al-3 exhibited phenotypes in asexual growth and conidiation, whereas os-5, poi-2, and pmd-1 exhibited phenotypes in sexual development. SMCs with annotated function in mating and crossing were actively regulated during the switch between asexual and sexual growth. Our discoveries call for attention to roles that SMCs may play in the regulatory switches controlling mode of development, as well as the ecological associations of those developmental stages that may influence expression of SMCs. IMPORTANCE Secondary metabolites (SMs) are low-molecular-weight compounds that often mediate interactions between fungi and their environments. Fungi enriched with SMs are of significant research interest to agriculture and medicine, especially from the aspects of pathogen ecology and environmental epidemiology. However, SM clusters (SMCs) that have been predicted by comparative genomics alone have typically been poorly defined and insufficiently functionally annotated. Therefore, we have investigated coordinate expression in SMCs in the model system N. crassa, and our results suggest that SMCs respond to environmental signals and to stress that are associated with development. This study examined SMC regulation at the level of RNA to integrate observations and knowledge of these genes in various growth and development conditions, supporting combining comparative genomics and inclusive transcriptomics to improve computational annotation of SMCs. Our findings call for detailed study of the function of SMCs during the asexual-sexual switch, a key, often-overlooked developmental stage.

Sequencing and Analysis of the Entire Genome of the Mycoparasitic Bioeffector Fungus Trichoderma asperelloides Strain T 203 (Hypocreales).

The filamentous mycoparasitic fungus Trichoderma asperelloides (Hypocreales, Ascomycota, Dikarya) strain T 203 was isolated from soil in Israel by the Ilan Chet group in the 1980s. As it has been the subject of laboratory, greenhouse, and field experiments and has been incorporated into commercial agricultural preparations, its genome has been sequenced and analyzed.

The Culturable Mycobiome of Mesophotic Agelas oroides: Constituents and Changes Following Sponge Transplantation to Shallow Water.

Marine sponges harbor a diverse array of microorganisms and the composition of the microbial community has been suggested to be linked to holo-biont health. Most of the attention concerning sponge mycobiomes has been given to sponges present in shallow depths. Here, we describe the presence of 146 culturable mycobiome taxa isolated from mesophotic niche (100 m depth)-inhabiting samples of Agelas oroides, in the Mediterranean Sea. We identify some potential in vitro interactions between several A. oroides-associated fungi and show that sponge meso-hyl extract, but not its predominantly collagen-rich part, is sufficient to support hyphal growth. We demonstrate that changes in the diversity of culturable mycobiome constituents occur following sponge transplantation from its original mesophotic habitat to shallow (10 m) waters, where historically (60 years ago) this species was found. We conclude that among the 30 fungal genera identified as associated with A. oroides, Aspergillus, Penicillium and Trichoderma constitute the core mycobiome of A. oroides, and that they persist even when the sponge is transplanted to a suboptimal environment, indicative of the presence of constant, as well as dynamic, components of the sponge mycobiome. Other genera seemed more depth-related and appeared or disappeared upon host's transfer from 100 to 10 m.

The GUL-1 Protein Binds Multiple RNAs Involved in Cell Wall Remodeling and Affects the MAK-1 Pathway in Neurospora crassa.

The Neurospora crassa GUL-1 is part of the COT-1 pathway, which plays key roles in regulating polar hyphal growth and cell wall remodeling. We show that GUL-1 is a bona fide RNA-binding protein (RBP) that can associate with 828 "core" mRNA species. When cell wall integrity (CWI) is challenged, expression of over 25% of genomic RNA species are modulated (2,628 mRNAs, including the GUL-1 mRNA). GUL-1 binds mRNAs of genes related to translation, cell wall remodeling, circadian clock, endoplasmic reticulum (ER), as well as CWI and MAPK pathway components. GUL-1 interacts with over 100 different proteins, including stress-granule and P-body proteins, ER components and components of the MAPK, COT-1, and STRIPAK complexes. Several additional RBPs were also shown to physically interact with GUL-1. Under stress conditions, GUL-1 can localize to the ER and affect the CWI pathway-evident via altered phosphorylation levels of MAK-1, interaction with mak-1 transcript, and involvement in the expression level of the transcription factor adv-1. We conclude that GUL-1 functions in multiple cellular processes, including the regulation of cell wall remodeling, via a mechanism associated with the MAK-1 pathway and stress-response.

Seasonal Variations in the Culturable Mycobiome of Acropora loripes along a Depth Gradient.

Coral associated fungi are widespread, highly diverse and are part and parcel of the coral holobiont. To study how environmental conditions prevailing near the coral-host may affect fungal diversity, the culturable (isolated on potato dextrose agar) mycobiome associated with Acropora loripes colonies was seasonally sampled along a depth gradient in the Gulf of Aqaba. Fragments were sampled from both apparently healthy coral colonies as well as those exhibiting observable lesions. Based on phylogenetic analysis of 197 fungal sequences, Ascomycota were the most prevalent (91.9%). The abundance of fungi increased with increasing water depth, where corals sampled at 25 m yielded up to 70% more fungal colony forming units (CFUs) than those isolated at 6 m. Fungal diversity at 25 m was also markedly higher, with over 2-fold more fungal families represented. Diversity was also higher in lesioned coral samples, when compared to apparently healthy colonies. In winter, concurrent with water column mixing and increased levels of available nutrients, at the shallow depths, Saccharomytacea and Sporidiobolacea were more prevalent, while in spring and fall Trichocomacea (overall, the most prevalent family isolated throughout this study) were the most abundant taxa isolated at these depths as well as at deeper sampling sites. Our results highlight the dynamic nature of the culturable coral mycobiome and its sensitivity to environmental conditions and coral health.

The Third International Symposium on Fungal Stress - ISFUS.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

Growing a circular economy with fungal biotechnology: a white paper.

Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation's sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.

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.

Fungi in the Marine Environment: Open Questions and Unsolved Problems.

Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far. To date, a relatively small percentage of described species are associated with marine environments, with ∼1,100 species retrieved exclusively from the marine environment. Nevertheless, fungi have been found in nearly every marine habitat explored, from the surface of the ocean to kilometers below ocean sediments. Fungi are hypothesized to contribute to phytoplankton population cycles and the biological carbon pump and are active in the chemistry of marine sediments. Many fungi have been identified as commensals or pathogens of marine animals (e.g., corals and sponges), plants, and algae. Despite their varied roles, remarkably little is known about the diversity of this major branch of eukaryotic life in marine ecosystems or their ecological functions. This perspective emerges from a Marine Fungi Workshop held in May 2018 at the Marine Biological Laboratory in Woods Hole, MA. We present the state of knowledge as well as the multitude of open questions regarding the diversity and function of fungi in the marine biosphere and geochemical cycles.

Metabolism and Development during Conidial Germination in Response to a Carbon-Nitrogen-Rich Synthetic or a Natural Source of Nutrition in Neurospora crassa.

Fungal spores germinate and undergo vegetative growth, leading to either asexual or sexual reproductive dispersal. Previous research has indicated that among developmental regulatory genes, expression is conserved across nutritional environments, whereas pathways for carbon and nitrogen metabolism appear highly responsive-perhaps to accommodate differential nutritive processing. To comprehensively investigate conidial germination and the adaptive life history decision-making underlying these two modes of reproduction, we profiled transcription of Neurospora crassa germinating on two media: synthetic Bird medium, designed to promote asexual reproduction; and a natural maple sap medium, on which both asexual reproduction and sexual reproduction manifest. A later start to germination but faster development was observed on synthetic medium. Metabolic genes exhibited altered expression in response to nutrients-at least 34% of the genes in the genome were significantly downregulated during the first two stages of conidial germination on synthetic medium. Knockouts of genes exhibiting differential expression across development altered germination and growth rates, as well as in one case causing abnormal germination. A consensus Bayesian network of these genes indicated especially tight integration of environmental sensing, asexual and sexual development, and nitrogen metabolism on a natural medium, suggesting that in natural environments, a more dynamic and tentative balance of asexual and sexual development may be typical of N. crassa colonies.IMPORTANCE One of the most remarkable successes of life is its ability to flourish in response to temporally and spatially varying environments. Fungi occupy diverse ecosystems, and their sensitivity to these environmental changes often drives major fungal life history decisions, including the major switch from vegetative growth to asexual or sexual reproduction. Spore germination comprises the first and simplest stage of vegetative growth. We examined the dependence of this early life history on the nutritional environment using genome-wide transcriptomics. We demonstrated that for developmental regulatory genes, expression was generally conserved across nutritional environments, whereas metabolic gene expression was highly labile. The level of activation of developmental genes did depend on current nutrient conditions, as did the modularity of metabolic and developmental response network interactions. This knowledge is critical to the development of future technologies that could manipulate fungal growth for medical, agricultural, or industrial purposes.

Transcriptional profiling and localization of GUL-1, a COT-1 pathway component, in Neurospora crassa.

Impairment of theNeurospora crassaCOT-1 kinase results in defects in hyphal polarity. Some of these effects are partially suppressed by inactivation of gul-1 (encoding an mRNA-binding protein involved in translational regulation). Here, we report on the transcriptional profiling of cot-1 inactivation and demonstrate that gul-1 affects transcript abundance of multiple genes in the COT-1 pathway, including processes such as cell wall remodeling, nitrogen and amino acid metabolism. The GUL-1 protein itself was found to be distributed within the entire hyphal cell, along with a clear presence of aggregates that traffic within the cytoplasm. Live imaging of GUL-1-GFP demonstrated that GUL-1 transport is microtubule-dependent. Cellular stress, as imposed by the presence of the cell wall biosynthesis inhibitor Nikkomycin Z or by nitrogen limitation, resulted in a 2-3-fold increase of GUL-1 aggregate association with nuclei. Taken together, this study demonstrates that GUL-1 affects multiple processes, its function is stress-related and linked with cellular traffic and nuclear association.

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.

The diversity of Trichoderma species from soil in South Africa, with five new additions.

Fourteen Trichoderma (Hypocreales) species were identified during a survey of the genus in South Africa. These include T. afroharzianum, T. asperelloides, T. asperellum, T. atrobrunneum, T. atroviride, T. camerunense, T. gamsii, T. hamatum, T. koningii, T. koningiopsis, T. saturnisporum, T. spirale, T. virens, and T. viride. Ten of these species were not known to occur in South Africa prior to this investigation. Five additional species were novel and are described here as T. beinartii, T. caeruleimontis, T. chetii, T. restrictum, and T. undulatum. These novel Trichoderma species display morphological traits that are typical of the genus. Based on molecular identification using calmodulin, endochitinase, nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2), RNA polymerase II subunit B, and translation elongation factor 1-α gene sequence data, T. beinartii, T. caeruleimontis, and T. chetii were found to belong to the Longibrachiatum clade, whereas T. restrictum is a member of the Hamatum clade. Trichoderma undulatum occupies a distinct lineage distantly related to other Trichoderma species. Strains of T. beinartii and T. chetii were isolated previously in Hawaii and Israel; however, T. caeruleimontis, T. restrictum, and T. undulatum are so far known only from South Africa.