Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.

Glucose sensing and light regulation: A mutation in the glucose sensor RCO-3 modifies photoadaptation in Neurospora crassa.

Light regulates fungal gene transcription transiently leading to photoadaptation. In the ascomycete Neurospora crassa photoadaptation is mediated by interactions between a light-regulated transcription factor complex, the white collar complex, and the small photoreceptor VVD. Other proteins, like the RCO-1/RCM-1 repressor complex participate indirectly in photoadaptation. We show that RCO-3, a protein with high similarity to glucose transporters, is needed for photoadaptation. The mutation in rco-3 modifies the transcriptional response to light of several genes and leads to changes in photoadaptation without significantly changing the amount and regulation of WC-1. The mutation in rco-3, however, does not modify the capacity of the circadian clock to be reset by light. Our results add support to the proposal that there is a connection between glucose sensing and light regulation in Neurospora and that the fungus integrates different environmental signals to regulate transcription.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger.

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/µ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research.

Disruption of the L-arabitol dehydrogenase encoding gene in Aspergillus tubingensis results in increased xylanase production.

Fungal xylanases are of major importance to many industrial sectors, such as food and feed, paper and pulp, and biofuels. Improving their production is therefore highly relevant. We determined the molecular basis of an improved xylanase-producing strain of Aspergillus tubingensis that was generated by UV mutagenesis in an industrial strain improvement program. Using enzyme assays, gene expression, sequencing of the ladA locus in the parent and mutant, and complementation of the mutation, we were able to show that improved xylanase production was mainly caused by a chromosomal translocation that occurred between a subtilisin-like protease pepD gene and the L-arabitol dehydrogenase encoding gene (ladA), which is part of the L-arabinose catabolic pathway. This genomic rearrangement resulted in disruption of both genes and, as a consequence, the inability of the mutant to use L-arabinose as a carbon source, while growth on D-xylose was unaffected. Complementation with constitutively expressed ladA confirmed that the xylanase overproducing phenotype was mainly caused by loss of ladA function, while a knockout of xlnR in the UV mutant demonstrated that improved xylanase production was mediated by XlnR. This study demonstrates the potential of metabolic manipulation for increased production of fungal enzymes.

Strain improvement for enhanced production of cellulase in Trichoderma viride.

The filamentous fungi Trichoderma species produce extracellular cellulase. The current study was carried out to obtain an industrial strain with hyperproduction of cellulase. The wild-type strain, Trichoderma viride TL-124, was subjected to successive mutagenic treatments with UV irradiation, low-energy ion beam implantation, atmospheric pressure non-equilibrium discharge plasma (APNEDP), and N-methyl-N'-nitro-N-nitrosoguanidine to generate about 3000 mutants. Among these mutants, T. viride N879 strain exhibited the greatest relevant activity: 2.38-fold filter paper activity and 2.61-fold carboxymethyl cellulase, 2.18-fold beta-glucosidase, and 2.27-fold cellobiohydrolase activities, compared with the respective wild-type activities, under solid-state fermentation using the inexpensive raw material wheat straw as a substrate. This work represents the first application of APNEDP in eukaryotic microorganisms.

Mutation of Alternaria tenuissima FCBP-252 for hyper-active alpha-amylase.

Production of extracellular alpha-amylase enzyme by a filamentous fungus, Alternaria tenuissima was studied in solid-state fermentation (SSF) as well as submerged fermentation (SmF). The potential strain was successfully mutated by UV and ethyl methanesulfonate (EMS). High-level of alpha-amylase activity was obtained by the mutant At-Ch-5.6 (76.75 Units mL(-1)) after chemical treatment followed by UV mutant At-UV-2.8 (63.12 Units mL(-1)) which was significantly higher than parental A. tenuissima FCBP-252 (32 Units mL(-1)). These mutants with high levels of activity were genetically characterized using RAPD-PCR. Expression pattern of mutants exhibited that the mutants were isogenic variants of parent strain and out-performance of the mutants could be attributed to change in genetic make up. This work represented the first report of strain improvement in Alternaria for hyper activity of alpha-amylase enzyme and suggested that this fungus could be used to extract purified enzyme.

Genetic analysis of the Neurospora crassa RAD14 homolog mus-43 and the RAD10 homolog mus-44 reveals that they belong to the mus-38 pathway of two nucleotide excision repair systems.

Saccharomyces cerevisiae Rad14 and Rad10 proteins are essential for nucleotide excision repair (NER). Rad14 is a UV-damaged DNA binding protein and Rad10 is a structure-specific endonuclease that functions in a complex with Rad1. In this study, we identified and characterized the RAD14 and RAD10 homolog genes in Neurospora crassa, which we named mus-43 and mus-44, respectively. Disruption of mus-43 and mus-44 conferred sensitivity to UV and 4-nitroquinoline 1-oxide, but not to methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, camptothecin, hydroxyurea, or bleomycin. The mus-44 mutant was more sensitive to UV than the mus-43 mutant. Genetic analysis indicated that mus-43 and mus-44 are epistatic to mus-38 which is a homolog of the S. cerevisiae RAD1, but not to mus-18 which belongs to a second excision repair pathway. Immunological assays demonstrated that both mus-43 and mus-44 retained the ability to excise UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts, but that excision ability was completely abolished in the mus-43 mus-18 and mus-44 mus-18 double mutants. These double mutants exhibited extremely high sensitivity to UV. In mus-43 and mus-44 mutants, the UV-induced mutation frequency increased compared to that of the wild-type. The mus-44 mutants also exhibited a partial photoreactivation defect phenotype similar to mus-38. These results suggest that both mus-43 and mus-44 function in the mus-38 NER pathway, but not in the mus-18 excision repair pathway.

Isolation and characterization of nucleotide excision repair deficient mutants of the entomopathogenic fungus, Beauveria bassiana.

To better understand DNA repair in the entomopathogenic fungus Beauveria bassiana, three ultraviolet (UV) light sensitive mutants were isolated and characterized to be deficient in nucleotide excision repair (NER). The UV sensitive mutants were scored by comparison to survival of the parental isolate, GK2016, after 36 J/m(2) UV-C irradiation. At this dose, conidial survival of GK2016 was 98% and the mutants LC75, LC194, and LC85 had survival values of 63%, 45%, and 31%, respectively. An immunological method which measured the removal of pyrimidine-(6-4)-pyrimidone photoproducts during repair confirmed the decreased ability of LC75, LC194, and LC85 to remove these UV-induced dimers by NER. The mutants were also found to be deficient in NER at swollen/ germinating conidia and blastospore life cycle stages. The germination of the moderately UV sensitive mutant, LC75, was similar to that of the parental isolate, GK2016, after UV irradiation and incubation to enhance NER. The more sensitive mutants, LC194 and LC85 were 2.1- or 2.7-fold, respectively, less likely to germinate after UV irradiation based on their ability to carry out NER. These NER deficient mutants, the first to be derived from B. bassiana, reveal the importance of NER in spore survival post-UV irradiation.

Regulation of Saccharomyces cerevisiae DNA polymerase eta transcript and protein.

RAD30-encoded DNA polymerase eta functions as a translesion polymerase that can bypass the most frequent types of UV-induced pyrimidine photoproducts in an error-free manner. Although its transcript is UV-inducible in Saccharomyces cerevisiae, Rad30 (studied as a Rad30-Myc fusion) is a stable protein whose levels do not fluctuate following UV treatment or during cell cycle progression. Rad30 protein is subject to monoubiquitination whose level is upregulated in G1 and downregulated during S-phase reentry. This downregulation is accelerated in UV-treated cells. A missense mutation (L577Q) of the ubiquitin binding domain (UBZ) confers a reduced degree of ubiquitination outside of G1 and a complete failure to stably interact with ubiquitinated substrates. This mutation confers a phenotype resembling a complete RAD30 deletion, thus attesting to the significance of the UBZ motif for polymerase eta function in vivo.

Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process.

BACKGROUND: In fungi, light is primarily known to influence general morphogenesis and both sexual and asexual sporulation. In order to expand the knowledge on the effect of light in fungi and to determine the role of the light regulatory protein ENVOY in the implementation of this effect, we performed a global screen for genes, which are specifically effected by light in the fungus Hypocrea jecorina (anamorph Trichoderma reesei) using Rapid Subtraction Hybridization (RaSH). Based on these data, we analyzed whether these genes are influenced by ENVOY and if overexpression of ENVOY in darkness would be sufficient to execute its function. RESULTS: The cellular functions of the detected light responsive genes comprised a variety of roles in transcription, translation, signal transduction, metabolism, and transport. Their response to light with respect to the involvement of ENVOY could be classified as follows: (i) ENVOY-mediated upregulation by light; (ii) ENVOY-independent upregulation by light; (iii) ENVOY-antagonized upregulation by light; ENVOY-dependent repression by light; (iv) ENVOY-independent repression by light; and (v) both positive and negative regulation by ENVOY of genes not responsive to light in the wild-type. ENVOY was found to be crucial for normal growth in light on various carbon sources and is not able to execute its regulatory function if overexpressed in the darkness. CONCLUSION: The different responses indicate that light impacts fungi like H. jecorina at several cellular processes, and that it has both positive and negative effects. The data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light.

Light represses conidiation in koji mold Aspergillus oryzae.

In the filamentous fungus Aspergillus oryzae, there has been no report on photoreaction. Here we investigated the effect of light in A. oryzae and found that conidiation was repressed by white light. This reaction is contrary to that of other Aspergilli, which show abundant conidiation under light. Moreover, red light also caused reduced conidiation. Genome sequencing of A. oryzae indicated the existence of homologs of some light-related genes in other filamentous fungi. To approach the molecular mechanism of this photoresponse, the effect of red light on the expression level of several genes putatively responsible for conidiation or photoperception, i.e., brlA, a gene known to be required for conidiation, AofphA, the putative homolog of the A. nidulans phytochrome gene fphA, and AoveA, the putative homolog of the negative regulator gene in conidiation in A. nidulans, was examined. These three genes showed no significant response to red light at the transcriptional level. The results indicate that A. oryzae perceives and responds to red light in a manner independent of the transcriptional regulation of these genes.

DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

Roles of Saccharomyces cerevisiae DNA polymerases Poleta and Polzeta in response to irradiation by simulated sunlight.

Sunlight causes lesions in DNA that if unrepaired and inaccurately replicated by DNA polymerases yield mutations that result in skin cancer in humans. Two enzymes involved in translesion synthesis (TLS) of UV-induced photolesions are DNA polymerase eta (Poleta) and polymerase zeta (Polzeta), encoded by the RAD30A and REV3 genes, respectively. Previous studies have investigated the TLS roles of these polymerases in human and yeast cells irradiated with monochromatic, short wavelength UVC radiation (254 nm). However, less is known about cellular responses to solar radiation, which is of higher and mixed wavelengths (310-1100 nm) and produces a different spectrum of DNA lesions, including Dewar photoproducts and oxidative lesions. Here we report on the comparative cytotoxic and mutagenic effects of simulated sunlight (SSL) and UVC radiation on yeast wild-type, rad30Delta, rev3Delta and rev3Delta rad30Delta strains. The results with SSL support several previous interpretations on the roles of these two polymerases in TLS of photodimers and (6-4) photoproducts derived from studies with UVC. They further suggest that Poleta participates in the non-mutagenic bypass of SSL-dependent cytosine-containing Dewar photoproducts and 8-oxoguanine, while Polzeta is mainly responsible for the mutagenic bypass of all types of Dewar photoproducts. They also suggest that in the absence of Polzeta, Poleta contributes to UVC- and SSL-induced mutagenesis, possibly by the bypass of photodimers containing deaminated cytosine.

Histone deacetylase Hda1 acts as repressor of the Ustilago maydis biotrophic marker gene mig1.

The Ustilago maydis mig1 gene is extensively up-regulated during growth within its host plant. A genetic approach was set up to identify mutants expressing mig1 during axenic growth. Five independent mutants were identified that not only displayed increased transcript levels of mig1 but also of egl1, an endoglucanase expressed in dikaryotic filaments. egl1 has recently been shown to be repressed by Hda1, a putative histone deacetylase [Reichmann et al., submitted]. The identified UV mutants shared other phenotypes with hda1 deletion mutants like enhanced pigmentation and the inability to produce teliospores in maize tumours. Complementation and sequence analysis demonstrated that all five UV mutants contained point mutations in the hda1 gene. Despite a common repression mechanism, expression levels of mig1 and egl1 were significantly different during axenic and biotrophic growth, providing evidence for additional regulatory inputs from the respective growth stage. Furthermore, while egl1 is subject to repression by the U. maydis regulator Rum1, this was not the case for mig1. U. maydis strains deleted in either hda1 or rum1 were not affected in mig1 expression in the tumour stage. Transcript levels conferred by mig1 promoters deleted in negatively cis-acting sequences exceeded those in hda1 mutants, suggesting additional negative factors governing mig1 expression.

RAD9, RAD24, RAD16 and RAD26 are required for the inducible nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers from the transcribed and non-transcribed regions of the Saccharomyces cerevisiae MFA2 gene.

In this study, the effect of a prior UV irradiation on the removal of cyclobutane pyrimidine dimers (CPDs) from the transcribed strand (TS) and non-transcribed strand (NTS) of the MFA2 gene in haploid Saccharomyces cerevisiae (S. cerevisiae) cells was investigated. In NER competent cells, the pre-irradiation with a dose of 20J/m2 enhances the removal of CPDs induced by a second UV dose of 100J/m2 in the TS and the NTS of MFA2 gene except for the CPDs in the region +258 to +298 in the NTS, where the enhanced repair was absent. No inducible repair was observed in rad9, rad24, rad16 and rad26 cells, indicating two checkpoint genes RAD9 and RAD24, the global repair gene RAD16 and the transcription coupled repair gene RAD26 are essential for inducible NER.

The proteasome regulates the UV-induced activation of the AP-1-like transcription factor Gcn4.

The proteasome is well known for its regulation of the cell cycle and degradation of mis-folded proteins, yet many of its functions are still unknown. We show that RPN11, a gene encoding a subunit of the regulatory cap of the proteasome, is required for UV-stimulated activation of Gcn4p target genes, but is dispensable for their activation by the general control pathway. We provide evidence that RPN11 functions downstream of RAS2, and show that mutation of two additional proteasome subunits results in identical phenotypes. Our analysis defines a novel function of the proteasome: regulation of the RAS- and AP-1 transcription factor-dependent UV resistance pathway.

A Neurospora double-strand-break repair gene, mus-11, encodes a RAD52 homologue and is inducible by mutagens.

We isolated a Neurospora crassa cDNA that encodes a Rad52 homologue (ncRAD52) by PCR, using degenerate primers. RFLP mapping demonstrated that the cloned gene is located close to the ro-4 locus on the right arm of linkage group V (LGVR). In a second experiment, we used sib selection to identify a cosmid clone containing the mus-11 gene in a N. crassa genomic library. Fine-scale mapping of the mus-11 mutant showed the gene order on LGVR near ro-4 to be: ad-7 - (9.5 mu) - pab-2 (7.8 mu) - mus-11 - (3.7 mu) - inv. The nucleotide sequence of the mus-11 gene matched that of the ncRAD52 cDNA. Thus, the mus-11 gene encodes the Rad52 homologue. The deduced amino acid sequence of the MUS11 protein shows 32.0% and 27.5% overall identity to the Schizosaccharomyces pombe Rad22 protein and the human hRad52 protein, respectively, and a higher level of identity (55-66%) within the conserved N-terminal region (141 residues). The MUS11 protein shows homology to Rad52 from budding yeast only within the N-terminal region (53.2% identity over 141 amino acids) which is conserved among Rad52 homologues. Yeast two-hybrid analysis reveals that the MUS11 protein binds to both the MEI-3 protein, a Rad51 homologue, and to itself in vivo. An ncRAD52 mutant obtained by the RIPping procedure showed the same sensitivity as the original mus-11 mutant to the following mutagens and chemicals: UV light, 4NQO (4-nitroquinoline 1-oxide), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), TBHP (tert-butyl hydroperoxide), HU (hydroxyurea) and histidine. Unlike the RAD52 transcript in Saccharomyces cerevisiae, the mus-11 transcript could not be detected in mycelium under normal growth conditions, but expression of the gene was induced by UV irradiation or treatment with MMS.

Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells.

Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.

Characterization of a Neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.

We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.

Spontaneous mutation, oxidative DNA damage, and the roles of base and nucleotide excision repair in the yeast Saccharomyces cerevisiae.

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG). When compared to wild-type, ogg1 mutants show an increase in the frequency of GC to TA transversions, indicating a role for Ogg1 in the repair of 8-OxoG. Here we report an increased frequency of forward mutation to canavanine resistance in mutants defective in the nucleotide excision repair (NER) gene RAD14. This was not increased further in strains additionally defective in OGG1. However, when compared to strains solely defective in OGG1, ogg1radl4 mutants displayed an increase in spontaneous GC to TA transversions. Intriguingly, reversion of the lys1-1 ochre allele was not increased in rad14 mutants, suggesting that oxidative base damage may only represent a substrate for NER in certain regions of the genome. We also examined repair of oxidative DNA damage by transforming mutant strains with plasmid DNA treated with methylene blue plus visible light. Mutants defective in OGG1 showed no significant reduction in transformation efficiency compared with wild-type strains. In contrast, disruption of RAD14 reduced the efficiency of transformation, yet there was no further decrease in an ogg1rad14 mutant. This strongly supports a role for NER in the repair of oxidative base damage in yeast, and differs from similar experiments carried out in E. coli, where transformation efficiency is only reduced in mutants defective in both fpg and uvrA. Finally, the repair of Fpg-sensitive sites was examined at the MATalpha and HMLalpha mating type loci, and NER was found to play a role in their removal.