Fungal Pathways Research Articles

Antifungal Effect of Penicillamine Due to the Selective Targeting of L-Homoserine O-Acetyltransferase.

Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway of L-methionine, an amino acid essential for humans, seems to be an attractive perspective. The MET2 gene from Candida albicans encoding L-homoserine O-acetyltransferase (CaMet2p), an enzyme catalyzing the first step in that pathway, was cloned and expressed as the native or the oligo-His-tagged fusion protein in Escherichia coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities. The purified MET2 gene product revealed the appropriate activity, catalyzed the conversion of L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH), and exhibited differential sensitivity to several L-Hom or OALH analogues, including penicillamine. Surprisingly, both penicillamine enantiomers (L- and D-Pen) displayed comparable inhibitory effects. The results of the docking of L- and D-Pen to the model of CaMet2p confirmed that both enantiomeric forms of the inhibitor are able to bind to the catalytic site of the enzyme with similar affinities and a similar binding mode. The sensitivity of some fungal cells to L-Pen, depending on the presence or absence of L-Met in the medium, clearly indicate Met2p targeting. Moreover, C. glabrata clinical strains that are resistant to fluconazole displayed a similar susceptibility to L-Pen as the wild-type strains. Our results prove the potential usefulness of Met2p as a molecular target for antifungal chemotherapy.

Child type 1 diabetes associated with mother vaginal bacteriome and mycobiome

Mother vaginal microbes contribute to microbiome of vaginally delivered neonates. Child microbiome can be associated with autoimmune diseases, such as type 1 diabetes (T1D). We collected vaginal DNA samples from 25 mothers with a vaginally delivered child diagnosed with T1D and samples from 24 control mothers who had vaginally delivered a healthy child and analyzed bacteriome and mycobiome of the samples. The total DNA of the samples was extracted, and ribosomal DNA regions (16S for bacteria, ITS2 for fungi) were amplified, followed by next-generation sequencing and machine learning. We found that alpha-diversity of bacteriome was increased (P < 0.002), whereas alpha-diversity of mycobiome was decreased (P < 0.001) in mothers with a diabetic child compared to the control mothers. Beta-diversity analysis suggested differences in mycobiomes between the mother groups (P = 0.001). Random forest models were able to effectively predict diabetes and control status of unknown samples (bacteria: 0.86 AUC, fungi: 0.96 AUC). Our data indicate several fungal genera and bacterial metabolic pathways of mother vaginal microbiome to be associated with child T1D. We suggest that early onset of T1D in a child has a relationship with altered mother vaginal microbiome and that both bacteriome and mycobiome contribute to this shift.

Identification of a D-galacturonate reductase efficiently using NADH as a cofactor

D-galacturonate reductases are catalysing the reversible reduction of D-galacturonate to L-galactonate using NAD(P)H as a cofactor. The enzymes are part of two different pathways. One pathway is the fungal pathway for the catabolism of the main compound of pectin, D-galacturonate. The other pathway is a a pathway in plants for L-ascorbic acid synthesis. The previously described naturally occurring enzymes preferably use NADPH as a cofactor. Although certain D-galacturonate reductases, such as the reductases from Aspergillus niger or Euglena gracilis also accept NADH, their activity is significantly higher with NADPH. We identified in E. gracilis a gene, called gaa1, coding for a D-galacturonate reductase with similar activities with NADH and NADPH. It is potentially useful for the metabolic engineering of microbes to make use of pectin rich biomass.

Oxidative stress response pathways in fungi.

Fungal response to any stress is intricate, specific, and multilayered, though it employs only a few evolutionarily conserved regulators. This comes with the assumption that one regulator operates more than one stress-specific response. Although the assumption holds true, the current understanding of molecular mechanisms that drive response specificity and adequacy remains rudimentary. Deciphering the response of fungi to oxidative stress may help fill those knowledge gaps since it is one of the most encountered stress types in any kind of fungal niche. Data have been accumulating on the roles of the HOG pathway and Yap1- and Skn7-related pathways in mounting distinct and robust responses in fungi upon exposure to oxidative stress. Herein, we review recent and most relevant studies reporting the contribution of each of these pathways in response to oxidative stress in pathogenic and opportunistic fungi after giving a paralleled overview in two divergent models, the budding and fission yeasts. With the concept of stress-specific response and the importance of reactive oxygen species in fungal development, we first present a preface on the expanding domain of redox biology and oxidative stress.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

Relationships between Soil Tillage Systems, Nematode Communities and Weed Seed Predation

Soil tillage is generally recognized as having a negative effect on soil health and weed seed predators. Recent advancements in conservation tillage practices allow for further comparison of how different levels of soil disturbances could influence soil food web communities. Field trials were conducted in 2017 and 2018 at two different sites to measure the effects of four cover crop termination treatments: conventional till (CT), no-till (NT), strip-till following roller-crimping (ST-RC), and strip-till with a living mulch between crop rows (ST-LM) on soil health using nematode community indices as soil health bioindicators. Following cover crop termination, the soil was monitored in the subsequent bell pepper (Capsicum annuum) crop over three sampling dates (June, August, and October). In the ST-RC treatment plots, soil nutrients were enriched (increased Enrichment Index, EI) and the soil food web structure was improved (higher Structure Index, SI) by the mid-season of the 2017 trial. In the 2018 trial, the ST-RC treatment enhanced fungal decomposition pathways (fungivore/fungivore + bacterivore ratio) throughout the bell pepper crop cycle and enhanced bacterial decomposition (abundance of bacterivorous nematodes) by the end of the cropping cycle compared to the no-till treatment, suggesting that the ST-RC treatment can further improve soil health conditions achieved by the NT treatment. Scatter plots of sampling points revealed that the treatments CT and NT had very distinct nematode-weed community assemblages in both trials, with the ST-RC and ST-LM treatments distributed closely with the NT treatment. Multivariate analysis among soil health bioindicators, weed pressure and weed seed predation explained 76.4 and 55.7% of the variance in the 2017 and 2018 trials, respectively. Weed pressure was consistently negatively related to (1) the SI, indicating soil disturbance would lead to more weed pressure; (2) the EI, indicating soil with higher weed pressure was linked to poor soil nutrient cycling, (3) cover crop residues left in the field from conservation tillage, or (4) how well the living mulch of red clover covered the ground. This study did not show that weed seed predation would lead to lower weed pressure but weed seed predation increased as weed biomass increased.

Genes and evolutionary fates of the amanitin biosynthesis pathway in poisonous mushrooms

The deadly toxin α-amanitin is a bicyclic octapeptide biosynthesized on ribosomes. A phylogenetically disjunct group of mushrooms in Agaricales (Amanita, Lepiota, and Galerina) synthesizes α-amanitin. This distribution of the toxin biosynthetic pathway is possibly related to the horizontal transfer of metabolic gene clusters among taxonomically unrelated mushrooms with overlapping habitats. Here, our work confirms that two biosynthetic genes, P450-29 and FMO1, are oxygenases important for amanitin biosynthesis. Phylogenetic and genetic analyses of these genes strongly support their origin through horizontal transfer, as is the case for the previously characterized biosynthetic genes MSDIN and POPB. Our analysis of multiple genomes showed that the evolution of the α-amanitin biosynthetic pathways in the poisonous agarics in the Amanita, Lepiota, and Galerina clades entailed distinct evolutionary pathways including gene family expansion, biosynthetic genes, and genomic rearrangements. Unrelated poisonous fungi produce the same deadly amanitin toxins using variations of the same pathway. Furthermore, the evolution of the amanitin biosynthetic pathway(s) in Amanita species generates a much wider range of toxic cyclic peptides. The results reported here expand our understanding of the genetics, diversity, and evolution of the toxin biosynthetic pathway in fungi.

Toward more efficient ergothioneine production using the fungal ergothioneine biosynthetic pathway

BackgroundErgothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy.ResultsHere, we showed that Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes (tregt1 and tregt2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions.ConclusionsOur results showed that the overproduction of ERG in E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.

Fungal Melanin Biosynthesis Pathway as Source for Fungal Toxins.

ABSTRACTContamination of food and feed with toxin-producing fungi is a major threat in agriculture and for human health. The filamentous fungus Alternaria alternata is one of the most widespread postharvest contaminants and a weak plant pathogen. It produces a large variety of secondary metabolites with alternariol and its derivatives as characteristic mycotoxin. Other important phyto- and mycotoxins are perylene quinones (PQs), some of which have anticancer properties. Here, we discovered that the PQ altertoxin (ATX) biosynthesis shares most enzymes with the 1,8-dihydroxynaphthalene (1,8-DHN) melanin pathway. However, melanin was formed in aerial hyphae and spores, and ATXs were synthesized in substrate hyphae. This spatial separation is achieved through the promiscuity of a polyketide synthase, presumably producing a pentaketide (T4HN), a hexaketide (AT4HN), and a heptaketide (YWA1) as products. T4HN directly enters the altertoxin and DHN melanin pathway, whereas AT4HN and YWA1 can be converted only in aerial hyphae, which probably leads to a higher T4HN concentration, favoring 1,8-DHN melanin formation. Whereas the production of ATXs was strictly dependent on the CmrA transcription factor, melanin could still be produced in the absence of CmrA to some extent. This suggests that different cues regulate melanin and toxin formation. Since DHN melanin is produced by many fungi, PQs or related compounds may be produced in many more fungi than so far assumed.

Microbial Involvement in the Bioremediation of Total Petroleum Hydrocarbon Polluted Soils: Challenges and Perspectives

Nowadays, soil contamination by total petroleum hydrocarbons is still one of the most widespread forms of contamination. Intervention technologies are consolidated; however, full-scale interventions turn out to be not sustainable. Sustainability is essential not only in terms of costs, but also in terms of restoration of the soil resilience. Bioremediation has the possibility to fill the gap of sustainability with proper knowledge. Bioremediation should be optimized by the exploitation of the recent “omic” approaches to the study of hydrocarburoclastic microbiomes. To reach the goal, an extensive and deep knowledge in the study of bacterial and fungal degradative pathways, their interactions within microbiomes and of microbiomes with the soil matrix has to be gained. “Omic” approaches permits to study both the culturable and the unculturable soil microbial communities active in degradation processes, offering the instruments to identify the key organisms responsible for soil contaminant depletion and restoration of soil resilience. Tools for the investigation of both microbial communities, their degradation pathways and their interaction, will be discussed, describing the dedicated genomic and metagenomic approaches, as well as the interpretative tools of the deriving data, that are exploitable for both optimizing bio-based approaches for the treatment of total petroleum hydrocarbon contaminated soils and for the correct scaling up of the technologies at the industrial scale.

Impact of Bis-O-dihydroferuloyl-1,4-butanediol Content on the Chemical, Enzymatic and Fungal Degradation Processes of Poly(3-hydroxybutyrate).

Poly-β-hydroxybutyrate (PHB) is a very common bio-based and biocompatible polymer obtained from the fermentation of soil bacteria. Due to its important crystallinity, PHB is extremely brittle in nature, which results in poor mechanical properties with low extension at the break. To overcome these issues, the crystallinity of PHB can be reduced by blending with plasticizers such as ferulic acid derivatives, e.g., bis-O-dihydroferuloyl-1,4-butanediol (BDF). The degradation potential of polymer blends of PHB containing various percentages (0, 5, 10, 20, and 40 w%) of BDF was investigated through chemical, enzymatic and fungal pathways. Chemical degradation revealed that, in 0.25 M NaOH solution, the presence of BDF in the blend was necessary to carry out the degradation, which increased as the BDF percentage increased. Whereas no enzymatic degradation could be achieved in the tested conditions. Fungal degradation was achieved with a strain isolated from the soil and monitored through imagery processing. Similar to the chemical degradation, higher BDF content resulted in higher degradation by the fungus.

Long-term cattle grazing shifts the ecological state of forest soils.

Cattle grazing profoundly affects abiotic and biotic characteristics of ecosystems. While most research has been performed on grasslands, the effect of large managed ungulates on forest ecosystems has largely been neglected. Compared to a baseline seminatural state, we investigated how long‐term cattle grazing of birch forest patches affected the abiotic state and the ecological community (microbes and invertebrates) of the soil subsystem. Grazing strongly modified the soil abiotic environment by increasing phosphorus content, pH, and bulk density, while reducing the C:N ratio. The reduced C:N ratio was strongly associated with a lower microbial biomass, mainly caused by a reduction of fungal biomass. This was linked to a decrease in fungivorous nematode abundance and the nematode channel index, indicating a relative uplift in the importance of the bacterial energy‐channel in the nematode assemblages. Cattle grazing highly modified invertebrate community composition producing distinct assemblages from the seminatural situation. Richness and abundance of microarthropods was consistently reduced by grazing (excepting collembolan richness) and grazing‐associated changes in soil pH, Olsen P, and reduced soil pore volume (bulk density) limiting niche space and refuge from physical disturbance. Anecic earthworm species predominated in grazed patches, but were absent from ungrazed forest, and may benefit from manure inputs, while their deep vertical burrowing behavior protects them from physical disturbance. Perturbation of birch forest habitat by long‐term ungulate grazing profoundly modified soil biodiversity, either directly through increased physical disturbance and manure input or indirectly by modifying soil abiotic conditions. Comparative analyses revealed the ecosystem engineering potential of large ungulate grazers in forest systems through major shifts in the composition and structure of microbial and invertebrate assemblages, including the potential for reduced energy flow through the fungal decomposition pathway. The precise consequences for species trophic interactions and biodiversity–ecosystem function relationships remain to be established, however.

Capturing an Early Gene Induction Event during Wood Decay by the Brown Rot Fungus Rhodonia placenta.

ABSTRACTBrown rot fungi dominate wood decomposition in coniferous forests, and their carbohydrate-selective mechanisms are of commercial interest. Brown rot was recently described as a two-step, sequential mechanism orchestrated by fungi using differentially expressed genes (DEGs) and consisting of oxidation via reactive oxygen species (ROS) followed by enzymatic saccharification. There have been indications, however, that the initial oxidation step itself might require induction. To capture this early gene regulation event, here, we integrated fine-scale cryosectioning with whole-transcriptome sequencing to dissect gene expression at the single-hyphal-cell scale (tens of micrometers). This improved the spatial resolution 50-fold, relative to previous work, and we were able to capture the activity of the first 100 μm of hyphal front growth by Rhodonia placenta in aspen wood. This early decay period was dominated by delayed gene expression patterns as the fungus ramped up its mechanism. These delayed DEGs included many genes implicated in ROS pathways (lignocellulose oxidation [LOX]) that were previously and incorrectly assumed to be constitutively expressed. These delayed DEGs, which include those with and without predicted functions, also create a focused subset of target genes for functional genomics. However, this delayed pattern was not universal, with a few genes being upregulated immediately at the hyphal front. Most notably, this included a gene commonly implicated in hydroquinone and iron redox cycling: benzoquinone reductase.IMPORTANCE Earth’s aboveground terrestrial biomass is primarily wood, and fungi dominate wood decomposition. Here, we studied these fungal pathways in a common “brown rot”-type fungus, Rhodonia placenta, that selectively extracts sugars from carbohydrates embedded within wood lignin. Using a space-for-time design to map fungal gene expression at the extreme hyphal front in wood, we made two discoveries. First, we found that many genes long assumed to be “on” (constitutively expressed) from the very beginning of decay were instead “off” before being upregulated, when mapped (via transcriptome sequencing [RNA-seq]) at a high resolution. Second, we found that the gene encoding benzoquinone reductase was “on” in incipient decay and quickly downregulated, implying a key role in “kick-starting” brown rot.

The integration of nematode communities into the soil biological health framework by factor analysis

A critique of the soil health framework is that biological indicators currently lag behind chemical and physical indicators of soil health. Incorporating nematode communities into the soil health framework could help to better reflect key aspects of soil food web structure and function and significantly contribute to ecosystem processes. However, little is understood regarding how nematode communities relate to soil biological health indicators such as permanganate oxidizable carbon (POXC), soil protein, mineralizable C, and enzyme activities in agroecosystems. Here, we use an exploratory factor analysis (EFA) to quantitatively explore which set of soil health indicators best explain a given factor (i.e. soil health trait) using data from two long term experimental trials and 44 farm fields across Ohio. Specifically, this paper aims to 1) integrate our understanding of nematode structure and function with other soil biological health indicators to describe soil health traits; and 2) determine how management practices alter soil health traits. We found that soil biological health indicators represented four underlying soil health traits: fungal organic matter processing pathway, the rate of nutrient cycling, trophic complexity, and cumulative disturbance. Results indicated that soil biological health indicators, such as enzyme activities, POXC, mineralizable C and soil protein were more integrated with nematode feeding groups than with nematode indices. Additionally, tillage intensity had a significant effect on the fungal organic matter processing pathway and the rate of nutrient cycling. This study indicates that nematode feeding groups can be readily incorporated into the soil health framework and future soil health assessments.

Effect of photodegradation on fungal colonization on wood during initial stage of brown-rot decay

Abstract Photodegradation and decay may happen simultaneously on wood under many exposure conditions. The aim of this study was to explore the effect of photodegradation on fungal colonization during initial stage of brown-rot decay of wood. For this purpose, southern pine (Pinus spp.) wood was exposed to accelerated UV weathering for different durations, and then decayed by a brown-rot fungus (Gloeophyllum trabeum). The changes in wood microstructure, chemical composition, surface color, crystallinity, fungal colonization pathways, and photodegradation products after weathering and decay were evaluated and characterized. The results showed that both weathering and decay led to obvious color change on wood surface. The photodegradation of wood resulted in the formation of cracking in cell wall, thus creating new voids. The photodegradation products, which mainly consisted of phenols, diterpenoid acids, and other lignin derived compounds, appeared in the cell lumen. During the initial stage of brown-rot decay, fungal hyphae invaded into wood through inherent voids such as ray cells, axial tracheids, and pits, and completed the initial colonization after 20 days. After photodegradation, hyphae could also invade wood from the new voids, and the acidic photodegradation products promoted the non-enzymatic degradation process of brown-rot. However, lignin derived compounds seemed to inhibit the further activity of decay fungi.

Investigating Fungal Biosynthetic Pathways Using Pichia pastoris as a Heterologous Host.

Fungal natural products have extensive biological activities, and thus have been largely commercialized in the pharmaceutical, agricultural, and food industries. Recently, heterologous expression has become an irreplaceable technique to functionalize fungal biosynthetic gene clusters and synthesize fungal natural products in various chassis organisms. This chapter describes the general method of using Pichia pastoris as a chassis host to investigate fungal biosynthetic pathways.

Cell death induction in Aspergillus fumigatus: accentuating drug toxicity through inhibition of the unfolded protein response (UPR).

One of the most potent opportunistic fungal pathogens of humans is Aspergillus fumigatus, an environmental mold that causes a life-threatening pneumonia with a high rate of morbidity and mortality. Despite advances in therapy, issues of drug toxicity and antifungal resistance remain an obstacle to effective therapy. This underscores the need for more information on fungal pathways that could be pharmacologically manipulated to either reduce the viability of the fungus during infection, or to unleash the fungicidal potential of current antifungal drugs. In this review, we summarize the emerging evidence that the ability of A. fumigatus to sustain viability during stress relies heavily on an adaptive signaling pathway known as the unfolded protein response (UPR), thereby exposing a vulnerability in this fungus that has strong potential for future therapeutic intervention.

Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus oryzae as a Heterologous Host.

A suite of molecular techniques have been developed in recent decades, which allow gene clusters coding for the biosynthesis of fungal natural products to be investigated and characterized in great detail. Many of these involve the manipulation of the native producer, for example, to increase yields of natural products or investigate the biosynthetic pathway through gene disruptions. However, an alternative and powerful means of investigating biosynthetic pathways, which does not rely on a cooperative native host, is the refactoring and heterologous expression of pathways in a suitable host strain. This protocol aims to walk the reader through the various steps required for the heterologous expression of a fungal biosynthetic gene cluster, specifically using Aspergillus oryzae strain NSAR1 and the pTYGS series of expression vectors. Briefly, this process involves the design and construction of up to four multigene expression vectors using yeast recombination, PEG-mediation transformation of A. oryzae protoplasts, and chemical extraction of the resulting transformants to screen for the presence of metabolites.

Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus nidulans as a Heterologous Host.

Fungal natural products encompass an important source of pharmaceutically relevant molecules. Heterologous expression of biosynthetic pathways in chassis strains enables the discovery of new secondary metabolites and characterization of pathway enzymes. In our laboratory, biosynthetic genes in a clustered pathway have been refactored in engineered heterologous hosts such as Aspergillus nidulans. Here we describe the assembly of heterologous expression vectors, transformation into A. nidulans, and detection of new compounds in the transformant strains.

Drafting Penicillium oxalicum calcineurin-CrzA pathway by combining the analysis of phenotype, transcriptome, and endogenous protein–protein interactions

Fungi sense environmental signals and coordinate growth, development, and metabolism accordingly. Calcium-calmodulin-calcineurin signaling is a conserved cascade pathway in fungi. One of the most important downstream targets of this pathway is the transcription factor Crz1/CrzA, which plays an essential role in various cellular processes. The putative collaborators of Penicillium oxalicum CrzA (PoCrzA) were found, through tandem affinity purification followed by mass spectrometric analysis (TAP-MS). A total of 50 protein–protein interaction collaborators of PoCrzA were observed. Among them, some collaborators, such as the catalytic subunit of calcineurin (Cna1, calcineurin A), the regulatory catalytic subunit of calcineurin (Cnb1, calcineurin B), and a 14-3-3 protein Bmh1, which were previously reported in yeast, were identified. Some putative collaborators, including two karyopherins (exportin Los1 and importin Srp1), two kinases (Fus3 and Slt2p), and a general transcriptional corepressor (Cyc8), were also found. The CrzA deletion mutant ΔPocrzA exhibited slow hyphal growth, impaired conidiogenesis, and reduced extracellular cellulase synthesis. Phenotype and transcriptome analysis showed that PoCrzA regulated fungal development in a Flbs-BrlA-dependent manner and participated in cellulase synthesis by modulating cellulolytic gene expression. On the basis of the results of TAP-MS, transcriptome, and phenotypic analysis in P. oxalicum, our study was the first to draft the calcineurin-CrzA pathway in cellulolytic fungi.