Filamentous Ascomycete Research Articles

Analyzing Ash Leaf-Colonizing Fungal Communities for Their Biological Control of Hymenoscyphus fraxineus.

The invasive ascomycete Hymenoscyphus fraxineus has been threatening Fraxinus excelsior populations throughout Europe for over two decades. Since the infection and first colonization by the pathogen occurs in leaves, leaf-colonizing microorganisms have been discussed as a barrier and as possible biocontrol agents against the disease. To identify fungal groups with health-supporting potential, we compared the fungal microbiota of compound leaves from susceptible and tolerant ash trees in four ash stands with high H. fraxineus exposure. The fungal communities were analyzed both culture-independently by ITS2 amplicon sequencing and by the taxonomic classification of 1,704 isolates using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) or sequencing of the entire ITS region. The fungal community structure did not show significant differences depending on the health status. However, for several OTUs and a MALDI group, a significantly higher abundance was found in tolerant ash trees. Thus, the yeast Papiliotrema flavescens was significantly increased and accounted for 12.3% of the mycobiome of tolerant ashes (OTU0003), and it had also a distinctly higher abundance among the isolates. The filamentous ascomycete Sarocladium strictum was increased 24-fold among the isolates of tolerant trees, but its abundance was comparably low. An in vitro screening for the growth inhibition of the pathogen via cocultivation resulted in 28 yeast-like isolates and 79 filamentous fungi with antagonistic activity. A statistical cocultivation test on two H. fraxineus strains confirmed six of the yeast-like isolates that suppressed H. fraxineus significantly, from 39–50%, two of them through a fungicidal effect. The highest inhibition rates among the yeasts were found for three isolates belonging to Aureobasidium pullulans and P. flavescens. The cocultivation test of the filamentous isolates revealed higher effects compared to the yeasts. Four isolates showed significant inhibition of both H. fraxineus strains with a rate of 72–100%, and five further isolates inhibited only one H. fraxineus strain significantly. The most effective isolates were members of the genus Cladosporium. During the next step, in planta tests will be necessary to verify the efficacy of the antagonistic isolates and to assess their suitability as biocontrol agents.

FgPal1 regulates morphogenesis and pathogenesis in Fusarium graminearum.

Ascospores are the primary inoculum in Fusarium graminearum, a causal agent of wheat head blight. In a previous study, FgPAL1 was found to be upregulated in the Fgama1 mutant and important for ascosporogenesis. However, the biological function of this well-conserved gene in filamentous ascomycetes is not clear. In this study, we characterized its functions in growth, differentiation and pathogenesis. The Fgpal1 mutant had severe growth defects and often displayed abnormal hyphal tips. It was defective in infectious growth in rachis tissues and spreading in wheat heads. The Fgpal1 mutant produced conidia with fewer septa and more nuclei per compartment than the wild type. In actively growing hyphal tips, FgPal1-GFP mainly localized to the subapical collar and septa. The FgPal1 and LifeAct partially co-localized at the subapical region in an interdependent manner. The Fgpal1 mutant was normal in meiosis with eight nuclei in developing asci but most asci were aborted. Taken together, our results showed that FgPal1 plays a role in maintaining polarized tip growth and coordination between nuclear division and cytokinesis, and it is also important for infectious growth and developments of ascospores by the free cell formation process.

Early endocytosis as a key to understanding mechanisms of plasma membrane tension regulation in filamentous fungi.

Two main systems regulate plasma membrane tension (PMT) and provide a close connection between the protoplast and the cell wall in fungi: turgor pressure and the actin cytoskeleton. These systems work together with the plasma membrane focal adhesion to the cell wall and their contribution to fungal cell organization and physiology has been partially studied. However, it remains controversial in model filamentous ascomycetes and oomycetes and even less investigated in filamentous basidiomycetes. Early endocytosis can be used to research the mechanisms regulating PMT since the dynamics of early endocytosis is largely dependent on this tension. This study examined the effects of actin polymerization inhibitors and hyperosmotic shock on early endocytosis and cell morphology in two filamentous basidiomycetes. The main obtained results are: (i) the depolymerisation of F-actin leads to the fast formation of endocytic pits while inhibiting of their scission from the plasma membrane and (ii) the moderate hyperosmotic shock does not affect the dynamics of early endocytosis. These and our other results have allowed suggesting a curtain model for the regulation of PMT in basidiomycetes. According to the proposed curtain model, the PMT in many non-apical cells of hyphae is more often regulated not by turgor pressure but by a system of actin driver cables that are associated with the proteins of the focal adhesion sites. The change in PMT occurs similar to the movement of a curtain along the curtain rod using the curtain drivers. This model addresses the fundamental properties of the fungal structure and physiology. It requires confirmation including the currently technically unavailable high-quality labelling of the actin cytoskeleton of the basidiomycetes.

Sporulation in Ashbya gossypii.

Ashbya gossypii is a filamentous ascomycete belonging to the yeast family of Saccharomycetaceae. At the end of its growth phase Ashbya generates abundant amounts of riboflavin and spores that form within sporangia derived from fragmented cellular compartments of hyphae. The length of spores differs within species of the genus. Needle-shaped Ashbya spores aggregate via terminal filaments. A. gossypii is a homothallic fungus which may possess a and α mating types. However, the solo-MATa type strain is self-fertile and sporulates abundantly apparently without the need of prior mating. The central components required for the regulation of sporulation, encoded by IME1, IME2, IME4, KAR4, are conserved with Saccharomyces cerevisiae. Nutrient depletion generates a strong positive signal for sporulation via the cAMP-PKA pathway and SOK2, which is also essential for sporulation. Strong inhibitors of sporulation besides mutations in the central regulatory genes are the addition of exogenous cAMP or the overexpression of the mating type gene MATα2. Sporulation has been dissected using gene-function analyses and global RNA-seq transcriptomics. This revealed a role of Msn2/4, another potential PKA-target, for spore wall formation and a key dual role of the protein A kinase Tpk2 at the onset of sporulation as well as for breaking the dormancy of spores to initiate germination. Recent work has provided an overview of ascus development, regulation of sporulation and spore maturation. This will be summarized in the current review with a focus on the central regulatory genes. Current research and open questions will also be discussed.

Early ecological succession patterns of bacterial, fungal and plant communities along a chronosequence in a recently deglaciated area of the Italian Alps.

In this study, the early ecological succession patterns of Forni Glacier (Ortles-Cevedale group, Italian Alps) forefield along an 18-year long chronosequence (with a temporal resolution of 1 year) has been reported. Bacterial and fungal community structures were inferred by high-throughput sequencing of 16S rRNA gene and ITS, respectively. In addition, the occurrence of both herbaceous and arboreous plants was also recorded at each plot. A significant decrease of alpha-diversity in more recently deglaciated areas was observed for both bacteria and plants. Time since deglaciation and pH affected the structure of both fungal and bacterial communities. Pioneer plants could be a major source of colonization for both bacterial and fungal communities. Consistently, some of the most abundant bacterial taxa and some of those significantly varying with pH along the chronosequence (Polaromonas, Granulicella, Thiobacillus, Acidiferrobacter) are known to be actively involved in rock-weathering processes due to their chemolithotrophic metabolism, thus suggesting that the early phase of the chronosequence could be mainly shaped by the biologically controlled bioavailability of metals and inorganic compounds. Fungal communities were dominated by ascomycetous filamentous fungi and basidiomycetous yeasts. Their role as cold-adapted organic matter decomposers, due to their heterotrophic metabolism, was suggested.

The Penicillium chrysogenum Q176 Antimicrobial Protein PAFC Effectively Inhibits the Growth of the Opportunistic Human Pathogen Candida albicans.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes promise treatment alternatives to licensed antifungal drugs. In this study, we characterized the Penicillium chrysogenum Q176 antifungal protein C (PAFC), which is phylogenetically distinct to the other two Penicillium antifungal proteins, PAF and PAFB, that are expressed by this biotechnologically important ascomycete. PAFC is secreted into the culture broth and is co-expressed with PAF and PAFB in the exudates of surface cultures. This observation is in line with the suggested role of AMPs in the adaptive response of the host to endogenous and/or environmental stimuli. The in silico structural model predicted five β-strands stabilized by four intramolecular disulfide bonds in PAFC. The functional characterization of recombinant PAFC provided evidence for a promising new molecule in anti-Candida therapy. The thermotolerant PAFC killed planktonic cells and reduced the metabolic activity of sessile cells in pre-established biofilms of two Candida albicans strains, one of which was a fluconazole-resistant clinical isolate showing higher PAFC sensitivity than the fluconazole-sensitive strain. Candidacidal activity was linked to severe cell morphology changes, PAFC internalization, induction of intracellular reactive oxygen species and plasma membrane disintegration. The lack of hemolytic activity further corroborates the potential applicability of PAFC in clinical therapy.

Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes.

Sexual and asexual development in filamentous ascomycetes is controlled by components of conserved signaling pathways. Here, we investigated the development of mutant strains lacking genes for kinases MAK2, MEK2, and MIK2, as well as the scaffold protein HAM5 of the pheromone response (PR) pathway. All had a defect in fruiting body development and hyphal fusion. Another phenotype was a defect in melanin-dependent ascospore germination. However, this deficiency was observed only in kinase deletion mutants, but not in strains lacking HAM5. Notably, the same developmental phenotypes were previously described for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) mutants, but the germination defect was only seen in NOX2 mutants. These data suggest a molecular link between the pheromone signaling pathway and both NOX complexes. Using data from yeast two-hybrid (Y2H) analysis, we found that the scaffolding protein HAM5 interacts with NOR1, the regulator of NOX1 and NOX2 complexes. This interaction was further confirmed using differently fluorescent-labeled proteins to demonstrate that NOR1 and HAM5 co-localize at cytoplasmic spots and tips of mature hyphae. This observation was supported by phenotypic characterization of single and double mutants. The oxidative stress response and the initiation of fruiting bodies were similar in Δham5Δnor1 and Δham5, but distinctly reduced in Δnor1, indicating that the double deletion leads to a partial suppression of the Δnor1 phenotype. We conclude that the PR and NOX1 complexes are connected by direct interaction between HAM5 and NOR1. In contrast, PR kinases are linked to the NOX2 complex without participation of HAM5.

Genetic diversity of Magnaporthe oryzae, the blast pathogen of rice in different districts of Karnataka, India determined by simple sequence repeat (SSR) markers

Blast disease caused by a filamentous heterothallic ascomycetous fungus Magnaporthe oryzae is one of the most devastating diseases of rice crop in different parts of the world. A variety of DNA markers are used for understanding the diversity and virulence spectrum of this fungus. In our current investigation, 21 simple sequence repeat markers are taken to analyse genetic diversity among 72 M. oryzae isolates obtained from different geographical locations of Karnataka, India. Results showed that there is significant variation among the isolates of M. oryzae with 92 polymorphic bands with an average of 4.38 bands for each marker. Average locus heterozygosity and polymorphic information content (PIC) of total markers were 0.73 and 0.60 respectively; Cluster analysis interestingly indicated a correlation of grouping with the geographical boundaries. We have also plotted virulence spectrum for each group and found out mixed types of reaction pattern in each group. SSR based genetic diversity of the pathogen is evolved as per the geographical boundaries and the extent of diversity might help the pathogen significantly in adapting to the changed environment. Hence during breeding program locus heterozygosity, PIC and virulence spectrum have to be considered for obtaining stable resistance cultivars.

Novel species of Huntiella from naturally-occurring forest trees in Greece and South Africa.

Huntiella species are wood-infecting, filamentous ascomycetes that occur in fresh wounds on a wide variety of tree species. These fungi are mainly known as saprobes although some have been associated with disease symptoms. Six fungal isolates with typical culture characteristics of Huntiella spp. were collected from wounds on native forest trees in Greece and South Africa. The aim of this study was to identify these isolates, using morphological characters and multigene phylogenies of the rRNA internal transcribed spacer (ITS) region, portions of the β-tubulin (BT1) and translation elongation factor 1α (TEF-1α) genes. The mating strategies of these fungi were also determined through PCR amplification of mating type genes. The study revealed two new species; one from Platanus orientalis in Greece and one from Colophospermum mopane and Senegalia nigrescens in South Africa. These novel taxa have been provided with the names, H. hellenicasp. nov. and H. krugerisp. nov., respectively. The former species was found to have a homothallic and the latter a heterothallic mating system.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

Whole Genome Sequencing Resource of the European Larch Canker Pathogen Lachnellula willkommii for Molecular Diagnostic Marker Development.

The filamentous ascomycete fungus Lachnellula willkommii is the causal agent of European larch canker (ELC), one of the most destructive diseases of larch in Europe and a regulated plant pathogen of quarantine significance in Canada and the United States. L. willkommii was first detected in Massachusetts, North America in 1927 on a larch plantation cultivated with nursery stock imported from Great Britain. Despite the decades of practices aimed at eliminating the pathogen, it has reappeared in coastal areas of Canada and the United States. There is concern ELC could spread throughout the range of eastern larch, a transcontinental species typical of the Boreal forest that spans the North American landscape. There is geographic range overlap between several nonpathogenic indigenous Lachnellula species and the reported distribution of L. willkommii in North America. Morphological and biological methods to distinguish L. willkommii are often inadequate as the fungus does not always produce the phenotypic structures that distinguish it from these other saprophytic Lachnellula species. Whole genome sequencing technologies were used to obtain the draft genome sequences of L. willkommii and six other Lachnellula species. Molecular markers identified from the genomic data may be used to discriminate L. willkommii from its nonpathogenic relatives.

Biofungicidal Potential of Neosartorya (Aspergillus) Fischeri Antifungal Protein NFAP and Novel Synthetic γ-Core Peptides.

Because of enormous crop losses worldwide due to pesticide-resistant plant pathogenic fungi, there is an increasing demand for the development of novel antifungal strategies in agriculture. Antifungal proteins (APs) and peptides are considered potential biofungicides; however, several factors limit their direct agricultural application, such as the high cost of production, narrow antifungal spectrum, and detrimental effects to plant development and human/animal health. This study evaluated the safety of the application of APs and peptides from the ascomycete Neosartorya fischeri as crop preservatives. The full-length N. fischeri AP (NFAP) and novel rationally designed γ-core peptide derivatives (PDs) γNFAP-opt and γNFAP-optGZ exhibited efficacy by inhibiting the growth of the agriculturally relevant filamentous ascomycetes in vitro. A high positive net charge, however, neither the hydrophilicity nor the primary structure supported the antifungal efficacy of these PDs. Further testing demonstrated that the antifungal activity did not require a conformational change of the β-pleated NFAP or the canonically ordered conformation of the synthetic PDs. Neither hemolysis nor cytotoxicity was observed when the NFAP and γNFAP-opt were applied at antifungally effective concentrations in human cell lines. Similarly, the Medicago truncatula plants that served as toxicity model and were grown from seedlings that were treated with NFAP, γNFAP-opt, or γNFAP-optGZ failed to exhibit morphological aberrations, reduction in primary root length, or the number of lateral roots. Crop protection experiments demonstrated that NFAP and associated antifungal active γ-core PDs were able to protect tomato fruits against the postharvest fungal pathogen Cladosporium herbarum.

Comparative analysis of genome-wide DNA methylation in Neurospora

ABSTRACT DNA methylation is an epigenetic mark that plays an important role in genetic regulation in eukaryotes. Major progress has been made in dissecting the molecular pathways that regulate DNA methylation. Yet, little is known about DNA methylation variation over evolutionary time. Here we present an investigation of the variation of DNA methylation and transposable element (TE) content in species of the filamentous ascomycetes Neurospora. We generated genome-wide DNA methylation data at single-base resolution, together with genomic TE content and gene expression data, of 10 individuals representing five closely related Neurospora species. We found that the methylation levels were low (ranging from 1.3% to 2.5%) and varied among the genomes in a species-specific way. Furthermore, we found that the TEs over 400 bp long were targeted by DNA methylation, and in all genomes, high methylation correlated with low GC, confirming a conserved link between DNA methylation and Repeat Induced Point (RIP) mutations in this group of fungi. Both TE content and DNA methylation pattern showed phylogenetic signal, and the species with the highest TE load (N. crassa) also exhibited the highest methylation level per TE. Our results suggest that DNA methylation is an evolvable trait and indicate that the genomes of Neurospora are shaped by an evolutionary arms race between TEs and host defence.

Anion Inhibition Studies of the β-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora.

CAS3 is a newly cloned cytosolic β-class carbonic anhydrase (CA, EC 4.2.1.1) from the filamentous ascomycete Sordaria macrospora. This enzyme has a high catalytic activity for the physiological CO2 hydration reaction and herein, we report the inhibition profile of CAS3 with anions and small molecules. The most effective CAS3 anions/small molecule inhibitors were diethyl-dithiocarbamate, sulfamide, sulfamate, phenyl boronic and phenyl arsonic acids, with KIs in the range of 0.89 mM–97 µM. Anions such as iodide, the pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrogensulfide, stannate, selenate, tellurate, tetraborate, perrhenate, perruthenate, selenocyanide and trithiocarbonate were low millimolar CAS3 inhibitors. The light halides, sulfate, hydrogensulfite, peroxydisulfate, diphosphate, divanadate, perchlorate, tetrafluoroborate, fluorosulfonate and iminodisulfonate did not significantly inhibit this enzyme. These data may be useful for developing antifungals based on CA inhibition, considering the fact that many of the inhibitors reported here may be used as lead molecules and, by incorporating the appropriate organic scaffolds, potent nanomolar inhibitors could be developed.

The effect of atmospheric plasma jet on Fusarium species producing mycotoxins T2 and DON: An approach for physical and chemical investigation

Fusarium genus is a filamentous ascomycete fungi with the ability to produce toxins. Sampling was carried out between 2015-2016 in three tea courses from Mazandaran and Gilan provinces. At intervals of 3, 7 and 15 days, the plates were examined and the colonies grown in each plate were identified and their macroscopic characteristics were recorded. Finally, 150 colonies were selected. Samples cultured in a plate were cultured in liquid medium sB + ME and sB + yE. The environments were examined for successive days and slides were prepared and stained with lacto phenol. This is to ensure the inactivation of fungi exposed to plasma or the removal of spores from the sample surface. There is a significant relationship between the DON produced in the potato medium containing yeast extract and DON contained in the sample taken from the same medium that was exposed to plasma jet for 60 seconds This feature was statistically significant in comparison between changes in the toxicity of T2 and toxin after 60 seconds of treatment with jet plasma and T2 reducibility severity to DON. Plasma treatment results showed a significant reduction in time-dependent toxin concentrations. These results indicate that the plasma system has a high potential not only to destroy mycotoxins, but also toxins produced from fungi, which can be greatly reduced and can be used effectively in the food industry.

Sulfonamide Inhibition Studies of the β-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora.

A new β-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: kcat of (7.9 ± 0.2) × 105 s−1, and kcat/Km of (9.5 ± 0.12) × 107 M−1∙s−1. An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with KIs in the range of 54–95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.

A novel Pezizomycotina‐specific protein with gelsolin domains regulates contractile actin ring assembly and constriction in perforated septum formation

Septum formation in fungi is equivalent to cytokinesis. It differs mechanistically in filamentous ascomycetes (Pezizomycotina) from that of ascomycete yeasts by the retention of a central septal pore in the former group. However, septum formation in both groups is accomplished by contractile actin ring (CAR) assembly and constriction. The specific components regulating septal pore organization during septum formation are poorly understood. In this study, a novel Pezizomycotina-specific actin regulatory protein GlpA containing gelsolin domains was identified using bioinformatics. A glpA deletion mutant exhibited increased distances between septa, abnormal septum morphology and defective regulation of septal pore closure. In glpA deletion mutant hyphae, overaccumulation of actin filament (F-actin) was observed, and the CAR was abnormal with improper assembly and failure in constriction. In wild-type cells, GlpA was found at the septum formation site similarly to the CAR. The N-terminal 329 residues of GlpA are required for its localization to the septum formation site and essential for proper septum formation, while its C-terminal gelsolin domains are required for the regular CAR dynamics during septum formation. Finally, in this study we elucidated a novel Pezizomycotina-specific actin modulating component, which participates in septum formation by regulating the CAR dynamics.

Chrysoviruses Inhabited Symbiotic Fungi of Lichens.

A lichen body is formed most often from green alga cells trapped in a net of ascomycetous fungi and accompanied by endolichenic or parasitic fungi, other algae, and symbiotic or free-living bacteria. The lichen’s microcosmos is inhabited by mites, insects, and other animals for which the lichen is a source of food or a place to live. Novel, four-segmented dsRNA viruses were detected in saxicolous Chrysothrix chlorina and Lepraria incana lichens. Comparison of encoded genome proteins revealed classification of the viruses to the genus Alphachrysovirus and a relationship to chrysoviruses from filamentous ascomycetous fungi. We propose the names Chrysothrix chrysovirus 1 (CcCV1) and Lepraria chrysovirus 1 (LiCV1) as acronyms for these viruses. Surprisingly, observation of Chrysothrix chlorina hybridization with fluorescent-labelled virus probe by confocal microscope revealed that the CcCV1 virus is not present in the lichen body-forming fungus but in accompanying endolichenic Penicillium citreosulfuratum fungus. These are the first descriptions of mycoviruses from a lichen environment.

CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer

Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.

Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum.

Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum.