Jesús Aguirre

Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans

NADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to produce reactive oxygen species (ROS). The recent discovery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth and signalling. Here, we address the role of NADPH oxidase‐generated ROS in the biology of the filamentous fungus Aspergillus nidulans. We characterize the noxA gene and show that it encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes. Deletion of noxA specifically blocks differentiation of sexual fruit bodies (cleistothecia), without affecting hyphal growth or asexual development. Accordingly, the noxA gene is induced during sexual development, peaking at the time of cleistothecia differentiation and in parallel with the hülle cell‐associated catalase peroxidase gene cpeA. This expression pattern is not dependent on transcription factors SteA and StuA, which are essential for cleistothecia formation. In contrast, noxA‐dependent premature sexual development correlates with noxA derepression in ΔsakA null mutants, connecting stress MAPK signalling to the regulated production of ROS. Using a nitroblue tetrazolium (NBT) assay to detect superoxide, we found that hülle cells and cleistothecia initials produce superoxide in a process inhibited by NADPH oxidase inhibitor DPI and markedly reduced in ΔnoxA mutants. Furthermore, using H2DCFDA, we detected that H2O2 and possibly other ROS are generated in a NoxA‐dependent fashion, mainly in the external walls from cleistothecia initials. The essential role of NoxA‐generated ROS in A. nidulans sexual differentiation and the presence of one or two noxA homologues in all analysed filamentous fungi suggest that NADPH oxidase‐generated ROS play important roles in fungal physiology and differentiation.

SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans

In eukaryotic cells, environmental stress signals are transmitted by evolutionarily conserved MAPKs, such as Hog1 in the budding yeast Saccharomyces cerevisiae, Spc1 in the fission yeast Schizosaccharomyces pombe and p38/JNK in mammalian cells. Here, we report the identification of the Aspergillus nidulans sakA gene, which encodes a member of the stress MAPK family. The sakA gene is able to complement the S. pombe spc1− defects in both osmo‐regulation and cell cycle progression. Moreover, SakA MAPK is activated in response to osmotic and oxidative stress in both S. pombe and A. nidulans. However, in contrast to hog1 and spc1 mutants, the sakA null mutant is not sensitive to high osmolarity stress, indicating a different regulation of the osmostress response in this fungus. On the other hand, the ΔsakA mutant shows development and cell‐specific phenotypes. First, it displays premature steA‐dependent sexual development. Second, ΔsakA mutant produces asexual spores that are highly sensitive to oxidative and heat shock stress and lose viability upon storage. Indeed, SakA is transiently activated early after induction of conidiation. Our results indicate that SakA MAPK is involved in stress signal transduction and repression of sexual development, and is required for spore stress resistance and survival.

Hyperoxidant states cause microbial cell differentiation by cell isolation from dioxygen

A general theory giving an explanation of microbial cell differentiation is presented. Based on experimental results, an unstable hyperoxidant state is postulated to trigger differentiation. Simple rules, involving the reduction of dioxygen and the isolation from dioxygen by diverse mechanisms, are proposed to govern transitions between the growth state and the differentiated states. With this view, common features of microbial differentiation processes, dimorphic growth, cell differentiation in dioxygen evolving phototrophs and in anaerobes are analyzed. The theory could have implications for understanding cell differentiation in higher organisms.

NADPH Oxidases NOX-1 and NOX-2 Require the Regulatory Subunit NOR-1 To Control Cell Differentiation and Growth in Neurospora crassa†

ABSTRACT We have proposed that reactive oxygen species (ROS) play essential roles in cell differentiation. Enzymes belonging to the NADPH oxidase (NOX) family produce superoxide in a regulated manner. We have identified three distinct NOX subfamilies in the fungal kingdom and have shown that NoxA is required for sexual cell differentiation in Aspergillus nidulans. Here we show that Neurospora crassa NOX-1 elimination results in complete female sterility, decreased asexual development, and reduction of hyphal growth. The lack of NOX-2 did not affect any of these processes but led instead to the production of sexual spores that failed to germinate, even in the presence of exogenous oxidants. The elimination of NOR-1, an ortholog of the mammalian Nox2 regulatory subunit gp67phox, also caused female sterility, the production of unviable sexual spores, and a decrease in asexual development and hyphal growth. These results indicate that NOR-1 is required for NOX-1 and NOX-2 functions at different developmental stages and establish a link between NOX-generated ROS and the regulation of growth. Indeed, NOX-1 was required for the increased asexual sporulation previously observed in mutants without catalase CAT-3. We also analyzed the function of the penta-EF calcium-binding domain protein PEF-1 in N. crassa. Deletion of pef-1 resulted in increased conidiation but, in contrast to what occurs in Dictyostelium discoideum, the mutation of this peflin did not suppress the phenotypes caused by the lack of NOX-1. Our results support the role of ROS as critical cell differentiation signals and highlight a novel role for ROS in regulation of fungal growth.

Multiple Catalase Genes Are Differentially Regulated in Aspergillus nidulans

Detoxification of hydrogen peroxide is a fundamental aspect of the cellular antioxidant responses in which catalases play a major role. Two differentially regulated catalase genes, catA and catB, have been studied in Aspergillus nidulans. Here we have characterized a third catalase gene, designated catC, which predicts a 475-amino-acid polypeptide containing a peroxisome-targeting signal. With a molecular mass of 54 kDa, CatC shows high similarity to other small-subunit monofunctional catalases and is most closely related to catalases from other fungi, Archaea, and animals. In contrast, the CatA (approximately 84 kDa) and CatB (approximately 79 kDa) enzymes belong to a family of large-subunit catalases, constituting a unique fungal and bacterial group. The catC gene displayed a relatively constant pattern of expression, not being induced by oxidative or other types of stress. Targeted disruption of catC eliminated a constitutive catalase activity not detected previously in zymogram gels. However, a catalase activity detected in catA catB mutant strains during late stationary phase was still present in catC and catABC null mutants, thus demonstrating the presence of a fourth catalase, here named catalase D (CatD). Neither catC nor catABC triple mutants showed any developmental defect, and both mutants grew as well as wild-type strains in H(2)O(2)-generating substrates, such as fatty acids, and/or purines as the sole carbon and nitrogen sources, respectively. CatD activity was induced during late stationary phase by glucose starvation, high temperature, and, to a lesser extent, H(2)O(2) treatment. The existence of at least four differentially regulated catalases indicates a large and regulated capability for H(2)O(2) detoxification in filamentous fungi.

catA, a new Aspergillus nidulans gene encoding a developmentally regulated catalase

Abstract  Aspergillus nidulans asexual sporulation (conidiation) is a model system for studying gene regulation and development. The CAN5 cDNA is one of several clones isolated based on transcript induction during conidiation. Here we present the molecular characterization of its corresponding gene, demonstrating that it encodes a developmentally regulated catalase, designated catA. The catA 744-amino-acid-residue polypeptide shows significant identity to other catalases. Its similarity to prokaryotic catalases is greater than to other fungal catalases. catA mRNA is barely detectable in growing mycelia, highly induced during sporulation, and present in isolated spores. However, catA expression is not dependent on the developmental regulatory genes brlA, abaA and wetA. Direct catalase activity determination in native gels revealed the existence of two bands of activity. One of these bands represented the major activity during vegetative growth and was induced during sporulation. The second catalase activity appeared after the induction of sporulation and was the predominant activity in spores. Disruption of catA abolished the major spore catalase without eliminating the vegetative activity, indicating the existence of at least two catalase genes in A. nidulans. catA-disrupted mutants produced spores that were sensitive to H2O2, as compared to wild-type spores. The increase in the activity of the vegetative catalase and the appearance of a second catalase during asexual sporulation is consistent with the occurrence of an oxidative stress during development.

Nox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals.

The production of reactive oxygen species (ROS) in a highly regulated fashion is a hallmark of members of the NADPH oxidase (Nox) family of enzymes. Nox enzymes are present in most eukaryotic groups such as the amebozoid, fungi, algae and plants, and animals, in which they are involved in seemingly diverse biological processes. However, a comprehensive survey of Nox functions throughout biology reveals common functional themes. Noxes are often activated in response to stressful conditions such as nutrient starvation, physical damage, or pathogen attack. Although the end result varies depending on the organism and tissue, Nox-produced ROS mediate the response to the adverse stimuli, such as innate immunity responses in plants and animals or cell differentiation in Dictyostelium, fungi, and plants. These responses involve ROS-mediated signaling mechanisms occurring at intracellular or cell-to-cell levels and sometimes involve cell wall or extracellular matrix cross-linking. Indeed, Noxes are involved in local and systemic signaling from plants to fish and in cross-linking of the plant hair-cell wall, synthesis of the nematode cuticle, and formation of the sea urchin fertilization envelope. The extensive use of Nox enzymes in biology to regulate cell-to-cell signaling and morphogenesis suggests that additional functions in mammalian signaling and development remain to be discovered.

Response Regulators SrrA and SskA Are Central Components of a Phosphorelay System Involved in Stress Signal Transduction and Asexual Sporulation in Aspergillus nidulans

ABSTRACT Among eukaryotes, only slime molds, fungi, and plants contain signal transduction phosphorelay systems. In filamentous fungi, multiple sensor kinases appear to use a single histidine-containing phosphotransfer (HPt) protein to relay signals to two response regulators (RR). In Aspergillus nidulans, the RR SskA mediates activation of the mitogen-activated protein kinase SakA in response to osmotic and oxidative stress, whereas the functions of the RR SrrA were unknown. We used a genetic approach to characterize the srrA gene as a new member of the skn7/prr1 family and to analyze the roles of SrrA in the phosphorelay system composed of the RR SskA, the HPt protein YpdA, and the sensor kinase NikA. While mutants lacking the HPt protein YpdA are unviable, mutants lacking SskA (ΔsskA), SrrA (ΔsrrA), or both RR (ΔsrrA ΔsskA) are viable and differentially affected in osmotic and oxidative stress responses. Both RR are involved in osmostress resistance, but ΔsskA mutants are more sensitive to this stress, and only SrrA is required for H2O2 resistance and H2O2-mediated induction of catalase CatB. In contrast, both RR are individually required for fungicide sensitivity and calcofluor resistance and for normal sporulation and conidiospore viability. The ΔsrrA and ΔsskA sporulation defects appear to be related to decreased mRNA levels of the key sporulation gene brlA. In contrast, conidiospore viability defects do not correlate with the activity of the spore-specific catalase CatA. Our results support a model in which NikA acts upstream of SrrA and SskA to transmit fungicide signals and to regulate asexual sporulation and conidiospore viability. In contrast, NikA appears dispensable for osmotic and oxidative stress signaling. These results highlight important differences in stress signal transmission among fungi and define a phosphorelay system involved in oxidative and osmotic stress, cell wall maintenance, fungicide sensitivity, asexual reproduction, and spore viability.

Aspergillus nidulans transcription factor AtfA interacts with the MAPK SakA to regulate general stress responses, development and spore functions.

Fungi utilize a phosphorelay system coupled to a MAP kinase module for sensing and processing environmental signals. In Aspergillus nidulans, response regulator SskA transmits osmotic and oxidative stress signals to the stress MAPK (SAPK) SakA. Using a genetic approach together with GFP tagging and molecular bifluorescence we show that SakA and ATF/CREB transcription factor AtfA define a general stress‐signalling pathway that plays differential roles in oxidative stress responses during growth and development. AtfA is permanently localized in the nucleus, while SakA accumulates in the nucleus in response to oxidative or osmotic stress signals or during normal spore development, where it physically interacts with AtfA. AtfA is required for expression of several genes, the conidial accumulation of SakA and the viability of conidia. Furthermore, SakA is active (phosphorylated) in asexual spores, remaining phosphorylated in dormant conidia and becoming dephosphorylated during germination. SakA phosphorylation in spores depends on certain (SskA) but not other (SrrA and NikA) components of the phosphorelay system. Constitutive phosphorylation of SakA induced by the fungicide fludioxonil prevents both, germ tube formation and nuclear division. Similarly, Neurospora crassa SakA orthologue OS‐2 is phosphorylated in intact conidia and gets dephosphorylated during germination. We propose that SakA–AtfA interaction regulates gene expression during stress and conidiophore development and that SAPK phosphorylation is a conserved mechanism to regulate transitions between non‐growing (spore) and growing (mycelia) states.

Efficient Transformation of Aspergillus nidulans by Electroporation of Germinated Conidia

We report the transformation of swollen A. nidulans conidia by electroporation. With this method, transformation frequencies were similar to those obtained by using protoplast fusion. The methodology employed is simple, requiring no enzymes nor osmotic stabilizers. The effects of conidial age, DNA topology/ concentration and electric field strength are presented Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol43/iss1/21 Efficient Transformation of Aspergillus nidulans by Electroporation of Germinated Conidia Olivia Sánchez and Jesús Aguirre Instituto de Fisiología Celular. Uiversidad Nacional Autónoma de Mexico. Apdo. Postal 70-242, 04510 México, D. F., Mexico. We report the transformation of swollen A. nidulans conidia by electroporation. With this method, transformation frequencies were similar to those obtained by using protoplast fusion. The methodology employed is simple, requiring no enzymes nor osmotic stabilizers. The effects of conidial age, DNA topology/concentration and electric field strength are presented. A. nidulans transformation by protoplast fusion is highly dependent on a good enzyme-mediated protoplast preparation (Ballance et al. 1983 Biochem. Biophys. Res. Commun. 112:284-289, Tilburn et al. 1983 Gene 26:205-221, Yelton et al. 1984 Proc. Nat. Acad. Sci. USA 81:14701474). During this process, there is a serious compromise between protoplast yield and viability, which are greatly affected by variations in different enzyme lots, mycelial age and osmotic medium composition. Electroporation as an alternative transformation method has proven successful for different types of fungi (Sánchez et al. 1993 Appl. Environ. Microbiol. 59:2087-2092, Xoconostle-Cázares et al. 1996 Microbiology 142:377-387, Vann, C., 1995 Fungal Genet. Newsl. 42A:53). However, the only method reported for A. nidulans (Richey et al. 1989 Phytopathology 79:844-847) still required protoplast production. We have adapted a previously reported electroporation method (Sánchez et al. 1993 Appl. Environ. Microbiol. 59:2087-2092), to transform germinated conidia at frequencies similar to those obtained by protoplast fusion. Conidia from strain RMS011 (pabaA1, yA2, argB::trpC B, veA1, trpC801; M. Stringer), washed 5 times with 10 ml of distilled water were used to inoculate 400 ml of Kafers minimal-nitrate medium plus supplements, at a density of 1 X 10 conidia/ml and incubated at 37 C in a rotary shaker (300 rpm) for 0, 2 or 7 h. Conidia recovered by centrifugation were resuspended in 400 ml of ice-cold sterile water, centrifuged again, resuspended in 25 ml of ice-cold pretreating buffer YED (1% yeast extract, 1% glucose) plus 12.5 mM DTT and 20 mM HEPES (adjusted to pH 8.0 with 100 mM Tris) and incubated for 1 h at 30 C in a rotary shaker at 100 rpm. Although the experiments reported here included DTT, we later found that it had no effect on transformation frequency. After this 60 min incubation, conidia were centrifuged and resuspended in 2.5 ml (about 1.6 X 10 conidia/ml final) of ice-cold electroporation buffer (10 mM Tris-HCl [pH 7.5], 270 mM sucrose, 1 mM lithium acetate) and kept on ice. For electroporation, 1 ug of dialyzed DNA was added to 50 ul of the ice cold conidial supension. The final volume was adjusted to 60 ul with distilled water, the mixture was incubated on ice for 15 min and then transferred to a 0.2-cm cuvette. Electroporation was performed using the Bio-Rad Gene Pulser and Pulse Controller Apparatus. Voltage was adjusted to 1,000 V, capacitance to 25 uF and resistance was 400 Ohms (pulse length varied between 5.1 and 5.8 ms). Under these conditions, about 35 % of the conidia were killed. Published by New Prairie Press, 2017 Following electroporation, 1 ml of ice-cold YED was added to the cuvette and the cell suspension was transferred to a sterile 10 ml tube, kept on ice for 15 min and incubated at 30 C for 90 min in a rotary shaker at 100 rpm. Conidia subjected to electroporation were spread on supplemented minimal plates lacking arginine (250 ul/plate) and incubated at 37 C. Most of the plated conidia germinated on selective medium but failed to grow further; the actual transformants being evident after 48 h. Transformant stability was tested by velvet-replica plating in selective medium. Only healthy growing, well sporulated colonies were counted as transformants. Table 1. Effect of conidial germination time and freezing-thawing on transformation frequency by electroporation. Number of Transformants / ug DNA _______________________________________________ Plasmid 0 h 2 h 7 h 2h thawed * _________________________________________________________ None 0 0 0 0 pDHG25 30 551 97 372 _________________________________________________________ Conidia were germinated for the indicated times and electroporated, using 1 ug of the autonomous replicating plasmid pDHG25. * 250 ul aliquots of 2 h germinated electrocompetent conidia were transferred to -70 C, stored for several days, thawed by incubating on ice and electroporated. Salt traces in DNA were removed by spin-filtration trough water-equilibrated Sephadex-G25-80 minicolumns. Except for 0 h, numbers represent mean values from two independent experiments, with a maximum variation of 10 % about the mean. Table 2. Effect of DNA topology and replication type on transformation frequency by electroporation . Type of DNA No. of Transformants/ ug DNA _______________________________________________________________________ pDHG25, circular 623 pDHG25, linear (BamHI) 1,189 pREN2, circular 11 pREN2, linear (KpnI) 19 _______________________________________________________________________ a DNAs digested with the indicated enzymes were filtered through Sephadex and stored at 4 C (Navarro et al. 1996 Curr. Genet. 29:352-359). For pDHG25, numbers represent mean values http://newprairiepress.org/fgr/vol43/iss1/21 DOI: 10.4148/1941-4765.1317 from four independent experiments. For pREN2, numbers are mean values from duplicates. Maximum variations were 9 and 26 % about the mean, respectively. Using this protocol, and the autonomous replicating plasmid pDHG25, which carries the argB gene as a selective marker (Gems et al. 1991 Gene 98:61-67), we evaluated the effect of germination time on transformation frequency. Results in Table 1 show that in the 2 h germination time the number of transformants per microgram of DNA was about 18 and 5 times more than those obtained after 0 and 7 h germination times, respectively. Only 2 h germinated conidia were used in all further experiments. The number of transformants obtained with pDHG25 using the 2 h germinated conidia is slightly higher than what we routinely obtain using protoplast fusion; 400-500 transformants/ ug DNA. When frozen electrocompetent conidia were electroporated with pDHG25, transformation frequency was about 30 % lower than with unfrozen conidia (Table 1). The convenience of having frozen stocks from different strains ready to be transformed could compensate for this reduction. We used frozen conidia to test the effects of plasmid concentration and field strength parameters on transformation efficiency. Within the DNA range tested (100 to 2,000 ng) we observed a non-linear response and a plateau in the number of transformants after 500 ng of DNA (Figure 1). With Regards to the effect of field strength, we found that 5 kV/cm (1000 Volts/0.2 cm) resulted in the highest number of transformants (Figure 2). Figure 1. Effect of plasmid concentration on transformation efficiency. Frozen electrocompetent conidia were thawed, incubated with the indicated amounts of circular pDHG25 and electroporated. The results are means from duplicates with a maximum variation of 4 % about the mean. Published by New Prairie Press, 2017 Figure 2. Effect of Field Strength on Transformation Frequency.50 ul aliquots from a frozen conidial pool were incubated with 1 ug of circular pDHG25 and electroporated using the indicated kV/cm. DNAs with different topology and replication types were also tested for electroporation efficiency. Results in Table 2 show that transformation frequency for the integrative plasmid pREN2 was much lower than for the self replicating plasmid pDHG25. However, the number of transformants obtained with pREN2 is also similar to those obtained by us and others when using protoplast fusion (5-10 transformants/ ug; Upshall, A. 1986 Curr. Genet. 10:593-599). Using linear forms of both types of plasmids increased transformation frequency about 2 times, perhaps reflecting an increased frequency of DNA entrance to the cells. Southern blot analysis of several transformants using argB and catA as probes showed that 8 out of 8 PDHG25-derived transformants contained argB sequences as part of the self replicating plasmid, whereas from 9 pREN2-derived transformants, 1 contained pREN2 sequences integrated at catA , 7 contained a single copy of pREN2 integrated at other genomic regions and 1 contained multiple integrations. Although we have not tested other electroporation apparatus, conditions described here are not too different from those reported for yeast (Becker and Guarente 1991 Methods in Enzymology 194:182-187). Therefore, yeast protocols for other electroporators should be a good starting point, provided the use of germinated conidia and a self replicating plasmid to optimize conditions.