The role of ion homeostasis imbalance due to citrate accumulation in fluoroacetic acid (FAA) toxicity in Neurospora crassa.

Fluoroacetic acid (FAA) is a poison commonly used for the lethal control of invasive species in Australia and New Zealand. Despite its widespread use and long history as a pesticide, no effective treatment for accidental poisoning exists. Although it is known to inhibit the tricarboxylic acid (TCA) cycle, specific details of FAA toxicology have remained elusive, with hypocalcemia suggested to be involved in the neurological symptoms prior to death. Here, we study the effects of FAA on cell growth and mitochondrial function using the filamentous fungi Neurospora crassa as model organism. FAA toxicosis in N. crassa is characterized by an initial hyperpolarization and subsequent depolarization of the mitochondrial membranes, followed by a significant intracellular decrease in ATP and increase in Ca2+. The development of mycelium was markedly affected within 6 h, and growth impaired after 24 h of FAA exposure. Although the activity of mitochondrial complexes I, II and IV was impaired, the activity of citrate synthase was not affected. Supplementation with Ca2+ exacerbated the effects of FAA in cell growth and membrane potential. Our findings suggest that an imbalance created in the ratio of ions within the mitochondria may lead to conformational changes in ATP synthase dimers due to mitochondrial Ca2+ uptake, that ultimately result in the opening of the mitochondrial permeability transition pore (MPTP), a decrease in membrane potential, and cell death. Our findings suggest new approaches for the treatment research, as well as the possibility to use N. crassa as a high-throughput screening assay to evaluate a large number of FAA antidote candidates.

Coordinated Regulation of Membrane Homeostasis and Drug Accumulation by Novel Kinase STK-17 in Response to Antifungal Azole Treatment.

The emergence of antifungal resistance, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, makes fungal infections difficult to treat in clinics and agriculture. When exposed to azoles, fungi can make adaptive responses to alleviate azole toxicity and produce azole tolerance. However, except for azole efflux pumps and ergosterol biosynthesis genes, the role of most azole responsive genes in azole resistance is unknown. In this study, STK-17, whose transcription is upregulated by azoles, was characterized as a novel kinase that is required for azole resistance. Deletion or dysfunction of STK-17 led to azole hypersensitivity in Neurospora crassa and to other ergosterol biosynthesis inhibitors such as amorolfine, terbinafine, and amphotericin B, but not fatty acid and ceramide biosynthesis inhibitors. STK-17 was also required for oxidative stress resistance, but this was not connected to azole resistance. RNA-seq results showed that stk-17 deletion affected the basal expression and the response to ketoconazole of some membrane protein genes, indicating functional association of STK-17 with the membrane. Notably, deletion of stk-17 affected the normal response to azoles of erg genes, including the azole target-encoding gene erg11, and erg2, erg6, and erg24, and led to abnormal accumulation of sterols in the presence of azoles. HPLC-MS/MS analysis revealed increased intracellular azole accumulation in the stk-17 mutant, possibly due to enhanced azole influx and reduced azole efflux that was independent of the major efflux pump CDR4. Importantly, STK-17 was widely distributed and functionally conserved among fungi, thus providing a potential antifungal target. IMPORTANCE Antifungal resistance is increasing worldwide, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, making control of fungal infections more challenging. A lot of effort has been expended in elucidating the mechanism of azole resistance and revealing potential antifungal targets. In this study, by analyzing azole-responsive genes in Neurospora crassa, we discovered STK-17, a novel kinase, that is required for azole resistance in several types of fungi. It has a role in regulating membrane homeostasis, responses to azole by ergosterol biosynthesis genes and azole accumulation, thus, deepening our understanding on the mechanism of azole stress response. Additionally, STK-17 is conserved among fungi and plays important roles in fungal development and stress resistance. Kinase inhibitors are broadly used for treating diseases, and our study pinpoints a potential drug target for antifungal development.

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus.

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

Zinc Transporter Protein (tzn-1) May Also Play a Role in Conidiation Pathway of Neurospora crassa: An Insight Using Proteogenomic Approach.

BACKGROUND: Zinc transporter (tzn-1) of Neurospora crassa plays a crucial role in conidiation pathway, as its removal results in aconidiation which was reported in our earlier studies. OBJECTIVES: The main objective of this study was to analyze the role of tzn-1 in conidiation process, by comparing knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) with wild oak ridge (OR) 74 'A' strain by 'Proteo-genomic' approach. METHODS: To identify the commonly expressed protein spots in knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) by comparing with wild oak ridge (OR) 74 'A' strain. Two sets (Δtzn-1 to wild and Δacon-3 to wild) were analyzed by combining 2- Dimensional gel electrophoresis (2DE) with Matrix Associated Laser Desortion/Ionization mass spectrometry -Peptide Mass Fingerprint (MALDI-PMF). Then, the peptide sequences which were obtained by MASCOT (database software) were identified by FGSC BLASTp search analysis. Finally, to evaluate the expression of the KO mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) in comparison to wild (OR) 74 'A' type was analyzed by Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) studies. RESULTS: 2DE and MALDI-PMF has shown the nine commonly overexpressed protein spots from the two sets (Δtzn-1 to wild and Δacon-3 to wild). Peptide sequences were obtained by MASCOT (database software) analysis and peptide sequences were identified by FGSC BLASTp search. Eight sequences have shown the similarities with the genes involved during the early stages of conidial and sexual development. Our qRT-PCR analysis has shown that tzn-1 gene was upregulated in contrast to acon-3 gene in absence of iron concentration and down regulated with increase in iron concentrations in wild samples. With increase in zinc supplements, the tzn-1 gene is normally regulated and shown contrasting feature in absence of zinc and acon-3 gene is normally regulated both in presence and absence of zinc. At regular time intervals, declined growth rate was observed after 18hours of induction. CONCLUSION: Thus, we conclude that tzn-1 and acon-3 genes were actively participating in early stages of conidial process and metal ions play some crucial role in the development of the organism.

Histone Methylation by SET Domain Proteins in Fungi.

Histone-modifying enzymes are responsible for regulating transcription, recombination, DNA repair, DNA replication, chromatid cohesion, and chromosome segregation. Fungi are ideally suited for comparative chromatin biology because sequencing of numerous genomes from many clades is coupled to existing rich methodology that allows truly holistic approaches, integrating evolutionary biology with mechanistic molecular biology and ecology, promising applications in medicine or plant pathology. While genome information is rich, mechanistic studies on histone modifications are largely restricted to two yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and one filamentous fungus, Neurospora crassa-three species that arguably are not representative of this diverse kingdom. Here, histone methylation serves as a paradigm to illustrate the roles chromatin modifications may play in more complex fungal life cycles. This review summarizes recent advances in our understanding of histone H3 methylation at two sites associated with active transcription, lysine 4 and lysine 36 (H3K4, H3K36); a site associated with the formation of constitutive heterochromatin, lysine 9 (H3K9); and a site associated with the formation of facultative heterochromatin, lysine 27 (H3K27). Special attention is paid to differences in how methylation marks interact in different taxa.

The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae.

BACKGROUND: Pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize D-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp. RESULTS: In the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for D-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described D-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized D-galacturonic acid in a medium containing D-fructose as co-substrate. CONCLUSION: This work is the first demonstration of the expression of a functional heterologous pathway for D-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol.

Clerodane type diterpene as a novel antifungal agent from Polyalthia longifolia var. pendula.

Bioactivity-guided chemical examination of methanolic extract of leaves of Polyalthia longifolia var. pendula led to the isolation of the active constituent, a diterpene 1 which was identified as 16α-hydroxycleroda-3,13(14)Z-dien-15,16-olide on the basis of its spectral data. Among the tested strains, diterpene 1 was found to exhibit antifungal activities having MIC90 values of 50.3, 100.6 and 201.2 µM against Candida albicans NCIM3557, Cryptococcus neoformans NCIM3542 (human pathogens) and Neurospora crassa NCIM870 (saprophyte), respectively. Initial, structure-activity-relationship (SAR) data generated by synthesizing some derivatives revealed that the double bond between C3-C4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. The mode of action of 1 in C. albicans is due to compromised cell membrane permeability and also probably due to disruption of cell wall structures. The red blood cell haemolysis of all the compounds (1-4) did not show any significant haemolysis and was found to be less than 15% for all the compounds when tested at highest concentration, i.e. 1200 µM. Interestingly, all the tested compounds inhibited Y-H transition in dimorphic C. albicans NCIM3557 at much lower concentration than their MIC90 values. Determination of ROS generation by diterpene 1 using DCFH-DA and DHR123 (dihydrorhodamine) staining of C. albicans NCIM3557 indicated production of intracellular ROS as a mechanism of antifungal activity.

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance.

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)-survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca(2+)](c), carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

Isolation, characterization and application of a cellulose-degrading strain Neurospora crassa S1 from oil palm empty fruit bunch.

BACKGROUND: Oil palm empty fruit bunch (EFB) is a lignocellulosic waste produced in palm oil industry. EFB mainly consists of cellulose, hemicellulose (mainly xylan) and lignin and has a great potential to be reused. Converting EFB to fermentable sugars and value-added chemicals is a much better choice than treating EFB as waste. RESULTS: A cellulase-producing strain growing on oil palm empty fruit bunch (EFB) was isolated and identified as Neurospora crassa S1, which is able to produce cellulases using EFB as the sole carbon source. The strain started to secret cellulases into the medium after 24 h of cultivation at 30°C and reached its maximal cellulase activity at 240 h. Mass spectroscopy (MS) analysis showed that more than 50 proteins were secreted into the medium when EFB was used as the sole carbon source. Among them, 7 proteins were identified as putative enzymes associated with cellulose degradation. The whole cell culture of Neurospora crassa S1 was used to hydrolyze acid-treated EFB, giving a total sugar yield of 83.2%, which is comparable with that (82.0%) using a well-known cellulase producer Trichoderma reesei RUT-C30 (ATCC56765). CONCLUSION: Neurospora crassa S1 is a commercially promising native cellulase producer for EFB hydrolysis especially when the sugars obtained are to be fermented to products that require use of non-genetically engineered strains.

Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system.

Ergothioneine is a small, sulfur-containing metabolite (229 Da) synthesized by various species of bacteria and fungi, which can accumulate to millimolar levels in tissues or cells (e.g. erythrocytes) of higher eukaryotes. It is commonly marketed as a dietary supplement due to its proposed protective and antioxidative functions. In this study we report the genes forming the two-step ergothioneine biosynthetic pathway in the fission yeast, Schizosaccharomyces pombe. We identified the first gene, egt1+ (SPBC1604.01), by sequence homology to previously published genes from Neurospora crassa and Mycobacterium smegmatis. We showed, using metabolomic analysis, that the Δegt1 deletion mutant completely lacked ergothioneine and its precursors (trimethyl histidine/hercynine and hercynylcysteine sulfoxide). Since the second step of ergothioneine biosynthesis has not been characterized in eukaryotes, we examined four putative homologs (Nfs1/SPBC21D10.11c, SPAC11D3.10, SPCC777.03c, and SPBC660.12c) of the corresponding mycobacterial enzyme EgtE. Among deletion mutants of these genes, only one (ΔSPBC660.12c, designated Δegt2) showed a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibited no phenotypic defects during vegetative growth or quiescence. To effectively study the role of ergothioneine, we constructed an egt1+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. We employed three versions of the nmt1 promoter with increasing strength of expression and confirmed corresponding accumulations of ergothioneine. We quantified the intracellular concentration of ergothioneine in S. pombe (0.3, 157.4, 41.6, and up to 1606.3 µM in vegetative, nitrogen-starved, glucose-starved, and egt1+-overexpressing cells, respectively) and described its gradual accumulation under long-term quiescence. Finally, we demonstrated that the ergothioneine pathway can also synthesize selenoneine, a selenium-containing derivative of ergothioneine, when the culture medium is supplemented with selenium. We further found that selenoneine biosynthesis involves a novel intermediate compound, hercynylselenocysteine.

A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations.

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.

Polygalacturonases from Moniliophthora perniciosa are regulated by fermentable carbon sources and possible post-translational modifications.

We report the first molecular and in silico analysis of Monilophthora perniciosa polygalacturonases (PGs). Three MpPG genes (MpPG1, MpPG2 and MpPG3) were identified and analyzed at transcriptional level, by RT-qPCR, in dikaryotic M. perniciosa mycelium grown on solid-bran based medium and on liquid medium supplemented with different fermentable and non-fermentable carbon sources. The MpPG genes presented different expression patterns suggesting different individual regulation. However, all are mainly regulated by fermentable carbon sources (galactose and mannose). The integrated analysis of PG gene expression and systems biology (using MpG1 and MpG2 orthologs in Neurospora crassa, named NCU06961 and NCU02369, respectively) allowed identifying some possible mechanism of protein regulation during the necrotrophic fungal phase. MpPG1-NCU06961 and MpPG2-NCU02369 directly or indirectly interacted with central and highly connected proteins involved in protein synthesis and protein regulation associated to post-translational modifications, in cell wall metabolism, and in cellular metabolism related to energy production. This analysis also allowed the identification of key proteins for further studies of M. perniciosa development and/or for disease management, such as MpPG2, a pectin methylesterase, an acetolactate synthase and the small ubiquitin-like modifier SMT3-like.

Microbial transformations of isophorone by Alternaria alternata and Neurospora crassa.

Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one), a monoterpene, and the structurally related 1,8-cineole and camphor, have demonstrated a protective effect against cancer, biological activity against a variety of microorganisms, and anti-oxidant properties. The derivatization of isophorone is, therefore, an important field of xenobiochemistry, pharmacology and toxicology. The aim of this study was to obtain derivatives of isophorone through microbial biotransformation and evaluate the biotransformation metabolites as potential antimicrobial agents. Incubation of isophorone with the fungi Alternaria alternata and Neurospora crassa afforded 4a-hydroxy- and 7-hydroxy-isophorone as transformation metabolites. The antimicrobial activities of isophorone and the metabolites were evaluated in vitro both by using agar dilution and microdilution methods. However, no significant antibacterial activity was observed when compared with those of standard substances.

The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa.

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.

The Neurospora crassa chr-1 gene is up-regulated by chromate and its encoded CHR-1 protein causes chromate sensitivity and chromium accumulation.

The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate resistance by exporting chromate ions from the cell's cytoplasm. The Neurospora crassa strain 74-A chr-1 gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the chr-1 gene was up-regulated by chromate exposure of N. crassa cultures. Introduction in N. crassa of sense and antisense fragments of the chr-1 gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in N crassa strains deleted in the genomic chr-1 gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from N. crassa chr-1 gene (Ncchr-1) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast Saccharomyces cerevisiae. Galactose-induced S. cerevisiae transformants expressing Ncchr-1 were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect S. cerevisiae chr-1 transformants from chromate toxicity. These data indicate that the N. crassa CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein.

Transgenic Nicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper.

The diets of two-thirds of the world's population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T(0) and T(1)) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd(2+) and Zn(2+) did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper.

Expression of fungal diacylglycerol acyltransferase2 genes to increase kernel oil in maize.

Maize (Zea mays) oil has high value but is only about 4% of the grain by weight. To increase kernel oil content, fungal diacylglycerol acyltransferase2 (DGAT2) genes from Umbelopsis (formerly Mortierella) ramanniana and Neurospora crassa were introduced into maize using an embryo-enhanced promoter. The protein encoded by the N. crassa gene was longer than that of U. ramanniana. It included 353 amino acids that aligned to the U. ramanniana DGAT2A protein and a 243-amino acid sequence at the amino terminus that was unique to the N. crassa DGAT2 protein. Two forms of N. crassa DGAT2 were tested: the predicted full-length protein (L-NcDGAT2) and a shorter form (S-NcDGAT2) that encoded just the sequences that share homology with the U. ramanniana protein. Expression of all three transgenes in maize resulted in small but statistically significant increases in kernel oil. S-NcDGAT2 had the biggest impact on kernel oil, with a 26% (relative) increase in oil in kernels of the best events (inbred). Increases in kernel oil were also obtained in both conventional and high-oil hybrids, and grain yield was not affected by expression of these fungal DGAT2 transgenes.

The antifungal activity of the Penicillium chrysogenum protein PAF disrupts calcium homeostasis in Neurospora crassa.

The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca(2+) signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca(2+) concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca(2+) ([Ca(2+)](c)). The Ca(2+) signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid], a Ca(2+) selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca(2+) homeostasis. These results indicate that extracellular Ca(2+) was the major source of these PAF-induced effects. The L-type Ca(2+) channel blocker diltiazem disrupted Ca(2+) homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca(2+) signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca(2+)](c) resting level. However, experiments with an aequorin-expressing Deltacch-1 deletion strain of N. crassa indicated that the L-type Ca(2+) channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca(2+)](c) resting level. This study is the first demonstration of the perturbation of fungal Ca(2+) homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.

Biotransformation of alpha-cedrol and caryophyllene oxide by the fungus Neurospora crassa.

Incubation of alpha-cedrol and caryophyllene oxide with Neurospora crassa afforded 12beta-hydroxy cedrol, 10alpha-hydroxy cedrol, and 3beta-hydroxy cedrol, and 12beta-hydroxy caryophyllene oxide as major metabolites, respectively. The antibacterial and radical scavenging activities of the metabolites were evaluated in vitro using broth microdilution and bioauthographic techniques. However, no significant antibacterial and antioxidant activities were observed when compared with those of standard substances.

Functional characterization of tzn1 and tzn2-zinc transporter genes in Neurospora crassa.

Previous work from our laboratory involved the description of the Neurospora metal transportome, which included seven hypothetical zinc transporters belonging to the ZIP family. The aim of the present study was to make a comparative functional evaluation of two hypothetical zinc transporters named tzn1 (NCU07621.3) and tzn2 (NCU11414.3). Phenotypic analysis of tzn1 and tzn2 mutants and a double mutant (tzn1tzn2) revealed that the deletion of tzn1 causes aconidiation and a greater defect in growth than the single deletion of tzn2. Supplementation with zinc restores growth but not conidiation in tzn1 and tzn1tzn2. TZN1 complemented a zinc-uptake-deficient Saccharomyces cerevisiae mutant (zrt1zrt2) in zinc-deficient conditions, while tzn2 restored growth upon supplementation with zinc (0.05 mM). Furthermore, the Deltatzn1 mutant was found to have severely reduced zinc content indicating that tzn1 functions as a key regulator of intracellular zinc levels in Neurospora crassa. Zinc uptake studies indicate tzn1 is a specific transporter of zinc, while tzn2 transports both zinc and cadmium. Quantitative RT-PCR showed up-regulation of tzn1 (128-fold) under zinc-depleted conditions and down-regulation (>1,000-fold) in zinc-replete conditions. The present study indicates that the zinc transport proteins encoded by tzn1 and tzn2 are members of the zinc uptake system regulated by zinc status in N. crassa.