Olivia Sánchez

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.

Starvation stress modulates the expression of the Aspergillus nidulans brlA regulatory gene.

Expression of the Aspergillus nidulans brlA gene plays a fundamental role in the switch from vegetative growth to asexual reproduction. Using a media-shifting protocol to induce submerged sporulation and brlA-lacZ as an expression marker, it was shown that carbon and nitrogen starvation stress induced brlA transcription to different degrees. Glucose starvation induced briA rapidly to high levels and resulted in spore formation on reduced conidiophores, whereas nitrogen starvation induced brlA gradually to lower levels and sporulation occurred to a lesser extent but from more complex conidiophores. beta-Galactosidase activity paralleled brlA alpha and brlA beta mRNA. No clear qualitative differences between the two brlA transcripts were found in these starvation conditions, suggesting that the different patterns of sporulation could be explained by quantitative expression differences. Since brlA mRNA did not accumulate in the presence of a high glucose concentration, we investigated the role of other carbon sources on brlA expression. Non-repressing carbon sources such as glycerol, acetate and arabinose were as effective as glucose in preventing brlA mRNA accumulation, suggesting that the glucose effects on brlA expression could be explained as a response to nutrient starvation, rather than by carbon catabolite repression. Despite similar low levels of brlA transcripts being detected during growth in glucose or non-repressing carbon sources, conidiophores were formed only in medium containing glycerol, acetate or arabinose. When mycelia were not shifted to starvation conditions, sporulation was not observed in standard minimal medium even after glucose was exhausted, unless the medium was buffered.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

TmpA, a member of a novel family of putative membrane flavoproteins, regulates asexual development in Aspergillus nidulans.

Asexual reproduction (conidiation) in Aspergillus nidulans is induced by environmental signals like exposure to air or nutrient starvation, and depends on brlA gene activation. The study of ‘fluffy’ mutants showing delayed asexual development and reduced brlA expression has defined the fluG pathway, involved in regulation of this differentiation process. Genetic characterization of a ‘fluffy’ mutant identified tmpA as a new gene involved in regulation of conidiation. TmpA defines a new family of putative transmembrane proteins of unknown function, widespread in filamentous fungi and plants, with homologues showing similarity to non‐ribosomal peptide synthetases. The deletion of tmpA resulted in decreased brlA expression and conidiation in air‐exposed colonies. This defect was suppressed when ΔtmpA mutants were grown next to wild‐type or ΔfluG mutant colonies, even without direct contact between hyphae. In liquid culture, tmpA was essential for conidiation induced by nitrogen but not by carbon starvation, whereas the overexpression of different tmpA tagged alleles resulted in conidiation. The overexpression of fluG‐induced conidiation independently of tmpA and ΔtmpAΔfluG double mutants showed an additive ‘fluffy’ phenotype, indicating that tmpA and fluG regulate asexual sporulation through different pathways. TmpA and its homologues appear to have diverged from the ferric reductase family, retaining overall transmembrane architecture, NAD(P), flavin adenine dinucleotide (FAD) and possibly haem‐binding domains. Based on our results, we propose that TmpA is a membrane oxidoreductase involved in the synthesis of a developmental signal.

FlbD, a Myb Transcription Factor of Aspergillus nidulans, Is Uniquely Involved in both Asexual and Sexual Differentiation

ABSTRACT In the fungus Aspergillus nidulans, inactivation of the flbA to -E, fluG, fluF, and tmpA genes results in similar phenotypes, characterized by a delay in conidiophore and asexual spore production. flbB to -D encode transcription factors needed for proper expression of the brlA gene, which is essential for asexual development. However, recent evidence indicates that FlbB and FlbE also have nontranscriptional functions. Here we show that fluF1 is an allele of flbD which results in an R47P substitution. Amino acids C46 and R47 are highly conserved in FlbD and many other Myb proteins, and C46 has been proposed to mediate redox regulation. Comparison of ΔflbD and flbDR47P mutants uncovered a new and specific role for flbD during sexual development. While flbDR47P mutants retain partial function during conidiation, both ΔflbD and flbDR47P mutants are unable to develop the peridium, a specialized external tissue that differentiates during fruiting body formation and ends up surrounding the sexual spores. This function, unique among other fluffy genes, does not affect the viability of the naked ascospores produced by mutant strains. Notably, ascospore development in these mutants is still dependent on the NADPH oxidase NoxA. We generated R47K, C46D, C46S, and C46A mutant alleles and evaluated their effects on asexual and sexual development. Conidiation defects were most severe in ΔflbD mutants and stronger in R47P, C46D, and C46S strains than in R47K strains. In contrast, mutants carrying the flbDC46A allele exhibited conidiation defects in liquid culture only under nitrogen starvation conditions. The R47K, R47P, C46D, and C46S mutants failed to develop any peridial tissue, while the flbDC46A strain showed normal peridium development and increased cleistothecium formation. Our results show that FlbD regulates both asexual and sexual differentiation, suggesting that both processes require FlbD DNA binding activity and that FlbD is involved in the response to nitrogen starvation.

NapA Mediates a Redox Regulation of the Antioxidant Response, Carbon Utilization and Development in Aspergillus nidulans

The redox-regulated transcription factors (TFs) of the bZIP AP1 family, such as yeast Yap1 and fission yeast Pap1, are activated by peroxiredoxin proteins (Prxs) to regulate the antioxidant response. Previously, Aspergillus nidulans mutants lacking the Yap1 ortholog NapA have been characterized as sensitive to H2O2 and menadione. Here we study NapA roles in relation to TFs SrrA and AtfA, also involved in oxidant detoxification, showing that these TFs play different roles in oxidative stress resistance, catalase gene regulation and development, during A. nidulans life cycle. We also uncover novel NapA roles in repression of sexual development, normal conidiation, conidial mRNA accumulation, and carbon utilization. The phenotypic characterization of ΔgpxA, ΔtpxA, and ΔtpxB single, double and triple peroxiredoxin mutants in wild type or ΔnapA backgrounds shows that none of these Prxs is required for NapA function in H2O2 and menadione resistance. However, these Prxs participate in a minor NapA-independent H2O2 resistance pathway and NapA and TpxA appear to regulate conidiation along the same route. Using transcriptomic analysis we show that during conidial development NapA-dependent gene expression pattern is different from canonical oxidative stress patterns. In the course of conidiation, NapA is required for regulation of at least 214 genes, including ethanol utilization genes alcR, alcA and aldA, and large sets of genes encoding proteins involved in transcriptional regulation, drug detoxification, carbohydrate utilization and secondary metabolism, comprising multiple oxidoreductases, membrane transporters and hydrolases. In agreement with this, ΔnapA mutants fail to grow or grow very poorly in ethanol, arabinose or fructose as sole carbon sources. Moreover, we show that NapA nuclear localization is induced not only by oxidative stress but also by growth in ethanol and by carbon starvation. Together with our previous work, these results show that SakA-AtfA, SrrA and NapA oxidative stress-sensing pathways regulate essential aspects of spore physiology (i.e., cell cycle arrest, dormancy, drug production and detoxification, and carbohydrate utilization).

The Adenylate-Forming Enzymes AfeA and TmpB Are Involved in Aspergillus nidulans Self-Communication during Asexual Development.

Aspergillus nidulans asexual sporulation (conidiation) is triggered by different environmental signals and involves the differentiation of specialized structures called conidiophores. The elimination of genes flbA-E, fluG, and tmpA results in a fluffy phenotype characterized by delayed conidiophore development and decreased expression of the conidiation essential gene brlA. While flbA-E encode regulatory proteins, fluG and tmpA encode enzymes involved in the biosynthesis of independent signals needed for normal conidiation. Here we identify afeA and tmpB as new genes encoding members the adenylate-forming enzyme superfamily, whose inactivation cause different fluffy phenotypes and decreased conidiation and brlA expression. AfeA is most similar to unknown function coumarate ligase-like (4CL-Lk) enzymes and consistent with this, a K544N active site modification eliminates AfeA function. TmpB, identified previously as a larger homolog of the oxidoreductase TmpA, contains a NRPS-type adenylation domain. A high degree of synteny in the afeA-tmpA and tmpB regions in the Aspergilli suggests that these genes are part of conserved gene clusters. afeA, tmpA, and tmpB double and triple mutant analysis as well as afeA overexpression experiments indicate that TmpA and AfeA act in the same conidiation pathway, with TmpB acting in a different pathway. Fluorescent protein tagging shows that functional versions of AfeA are localized in lipid bodies and the plasma membrane, while TmpA and TmpB are localized at the plasma membrane. We propose that AfeA participates in the biosynthesis of an acylated compound, either a p-cuomaryl type or a fatty acid compound, which might be oxidized by TmpA and/or TmpB, while TmpB adenylation domain would be involved in the activation of a hydrophobic amino acid, which in turn would be oxidized by the TmpB oxidoreductase domain. Both, AfeA-TmpA and TmpB signals are involved in self-communication and reproduction in A. nidulans.

SakA and MpkC Stress MAPKs Show Opposite and Common Functions During Stress Responses and Development in Aspergillus nidulans

Stress activated MAP kinases (SAPKs) of the Hog1/Sty1/p38 family are specialized in transducing stress signals. In contrast to what is seen in animal cells, very few fungal species contain more than one SAPK. Aspergillus nidulans and other Aspergilli contain two SAPKs called SakA/HogA and MpkC. We have shown that SakA is essential for conidia to maintain their viability and to survive high H2O2 concentrations. H2O2 induces SakA nuclear accumulation and its interaction with transcription factor AtfA. Although SakA and MpkC show physical interaction, little is known about MpkC functions. Here we show that ΔmpkC mutants are not sensitive to oxidative stress but in fact MpkC inactivation partially restores the oxidative stress resistance of ΔsakA mutants. ΔmpkC mutants display about twofold increase in the production of fully viable conidia. The inactivation of the SakA upstream MAPKK PbsB or the simultaneous elimination of sakA and mpkC result in virtually identical phenotypes, including decreased radial growth, a drastic reduction of conidiation and a sharp, progressive loss of conidial viability. SakA and to a minor extent MpkC also regulate cell-wall integrity. Given the roles of MpkC in conidiation and oxidative stress sensitivity, we used a functional MpkC::GFP fusion to determine MpkC nuclear localization as an in vivo indicator of MpkC activation during asexual development and stress. MpkC is mostly localized in the cytoplasm of intact conidia, accumulates in nuclei during the first 2 h of germination and then becomes progressively excluded from nuclei in growing hyphae. In the conidiophore, MpkC nuclear accumulation increases in vesicles, metulae and phialides and decreases in older conidia. Oxidative and osmotic stresses induce MpkC nuclear accumulation in both germinating conidia and hyphae. In all these cases, MpkC nuclear accumulation is largely dependent on the MAPKK PbsB. Our results indicate that SakA and MpkC play major, distinct and sometimes opposing roles in conidiation and conidiospore physiology, as well as common roles in response to stress. We propose that two SAPKs are necessary to delay (MpkC) or fully stop (SakA) mitosis during conidiogenesis and the terminal differentiation of conidia, in the highly prolific phialoconidiation process characteristic of the Aspergilli.