Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca²⁺ Homeostasis in Aspergillus nidulans

ABSTRACT

The eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion of cchA suppresses the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans. Additionally, the hypersensitivity of the ΔcnaA strain to extracellular calcium and cell-wall-damaging agents can be suppressed by cchA deletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of either cnaA or cchA significantly decreased the [Ca²⁺]_c, but a deficiency in both cnaA and cchA switches the [Ca²⁺]_c to the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction of cchA in the ΔcnaA strain dramatically restored the expression of enaA (a major determinant for sodium detoxification), which was abolished in the ΔcnaA strain under salt stress. These results suggest that double deficiencies of cnaA and cchA could bypass the requirement of calcineurin to induce enaA expression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca²⁺ channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.

IMPORTANCE The feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One 7:e46564, 2012, http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion of cchA could suppress the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans. In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca²⁺]_c. Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca²⁺ channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca²⁺ homeostasis in response to different environments.

INTRODUCTION

To rapidly sense and respond to different environmental conditions, organisms have evolved signaling pathways to coordinate growth, proliferation, and metabolism (1–6). Among them, the calcium-mediated signaling pathway plays an important regulatory role in various physiological processes, such as cell cycle, cytoskeletal rearrangement, ion homeostasis, and stress response (7–10). Calcium homeostasis systems are highly regulated pathways used by cells to maintain cytoplasmic Ca²⁺ concentrations ([Ca²⁺]_c)within an optimal range in the cytosol and other organelles (11–14). The rise and fall of free [Ca²⁺]_c can be directly sensed, decoded, and retransmitted to cellular targets, such as the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin, which is highly conserved from yeasts to humans and mediates many important cellular processes (15, 16).

Calcineurin is a member of the serine-threonine-specific protein phosphatase (PP) family. It differs from other phosphatases in its metal ion requirements, range of substrate specificity, and cellular regulation (17). Calcineurin exists as a heterodimer of catalytic subunits (CnA) and regulatory subunits (CnB) (18, 19), and the CnA subunit contains the catalytic domain and three regulatory domains, including the CnB-binding, calmodulin-binding, and autoinhibitory domains, all located toward the carboxy terminus (20–22). Calcineurin signaling is vital to the regulation of intracellular cation homeostasis, which is crucial for the proper function of living cells (23, 24). [Ca²⁺]_c is stringently controlled by complex interactions between calcineurin and different Ca²⁺ channels, Ca²⁺ pumps, and other Ca²⁺ transporters (16). In a previous study, we implicated the calcium channel CchA and its regulatory subunit MidA, which form a high-affinity calcium influx system (HACS), in facilitating calcium influx in low-calcium-concentration environments and in stress responses in Aspergillus nidulans (25). Studies in fission yeast have shown that the addition of extracellular CaCl₂ induced an increase in [Ca²⁺]_c, and this increased Ca²⁺ activated calcineurin, which subsequently inhibited the Cch1-Mid1 calcium channel through dephosphorylation to prevent calcium toxicity (26). Studies in fungi have revealed that calcineurin has essential roles in fungal morphogenesis, cell cycle progression, cell wall integrity, and antifungal drug activity (8, 17, 27, 28). Calcineurin also plays a crucial role in osmotic adaptation. In budding yeast, osmotic pretreatment of cells enhances salt tolerance and growth in highly saline environments. However, the addition of EGTA to the osmotic pretreatment medium or deletion of cch1 reduced cellular NaCl adaptation (3). Further studies have indicated that hyperosmotic stress induces a transient increase in [Ca²⁺]_c through Cch1p-Mid1p (26, 29). However, exactly how calcineurin and CchA coordinate the [Ca²⁺]_c and the response to salt stress in fungi is not well understood.

Aspergillus species are among the most abundant fungi worldwide. Among them, Aspergillus nidulans has been used as a model to study many fungal biological processes. Our previous studies have shown that deletion of cchA in A. nidulans could restore hyphal growth defects caused by the calcineurin inhibitor FK506 under salt stress (25). In this study, the roles of calcineurin and CchA in salt stress adaption were investigated by generating calcineurin and cchA single and double deletion strains, studying calcium homeostasis under different conditions, and analyzing the transcriptional profiles of genes responsible for the salt stress response and calcium homeostasis. Our results reveal new insights into the complex relationship between calcineurin and CchA in the regulation of cell survival processes in fungi.

MATERIALS AND METHODS

Strains, media, culture conditions, and transformation.

A list of the A. nidulans strains used in this study is given in Table 1. TN02A7, a strain with deletion of a gene required for nonhomologous end joining in double-strand break repair (30), was used in all transformation experiments as a reference strain. The following media were used: YAG (2% glucose, 0.5% yeast extract, with trace elements as needed), YUU (YAG supplemented with 5 mM uridine and 10 mM uracil), YAGK (YAG with 0.6 M KCl); YUUK (YUU with 0.6 M KCl), MMPDRUU (minimal medium with 2% glucose, nitrate salts, trace elements, 0.5 mg/liter pyridoxine, 2.5 mg/liter riboflavin, 5 mM uridine, 10 mM uracil, pH 6.5, with trace elements and nitrate salts added to the medium as described previously [31, 32]), and MMPGRUU (same as MMPDRUU, except with 2% glucose replaced with 1% vol/vol glycerol). For solid media, 2% agar was added. Growth conditions and genetic crosses were performed as previously described (33). Standard DNA transformation procedures were done according to a method described for A. nidulans (34, 35). For growth rate analysis, 1 × 10⁵ spores were cultured on either MMPDRUU or MMPDRUU with 800 mM NaCl, and the change in colony diameter was monitored over 48 h. To quantify the production of conidia, 1 × 10⁵ conidia of relevant strains were spotted on MMPDRUU. The inoculations were cultured for 2.5 days at 37°C, and then the number of conidia was counted. The determination of total biomass was performed by inoculating a total number of 1 × 10⁸ conidia into 100 ml of MMPDRUU liquid medium. After 18 h of growth at 37°C with shaking at 220 rpm, each sample was dried before a final dry weight was recorded. All experiments were performed in triplicate.

TABLE 1.

List of the A. nidulans strains used in this study

Strain	Genotypea	Reference or source
TN02A7	pyrG89 riboB2 pyroA4 nkuA::argB2 veA1	30
CNA1	pyrG89 ΔcnaA::pyroA pyroA4 wA3	37
CJA01	pyrG89 ΔcnaA::pyroA pyroA4 alcA(p)::YFP-CchA::pyr-4	7
HHA02	pyrG89 riboB2 pyroA4 nkuA::argB2 alcA(p)::YFP-CchA::pyr-4 veA1	7
WSA05	pyrG89 riboB2 pyroA4 nkuA::argB2 ΔcchA::pyrG veA1	25
WSA08	pyrG89 ΔcnaB::pyrG riboB2 pyroA4 nkuA::argB2 veA1	This study
LXA04	pyrG89 riboB2 ΔcnaA::pyroA pyroA4 nkuA::argB2 ΔcchA::pyrG veA1	This study
LXA05	pyrG89 ΔcnaB::pyrG nkuA::argB2 ΔcchA::pyrG veA1	This study
ZHA01	pyrG89 riboB2 pyroA4 nkuA::argB2 ΔyvcA::pyrG veA1	This study
ZSA01	pyrG89 riboB2 ΔcnaA::pyroA pyroA4 nkuA::argB2 ΔyvcA::pyrG veA1	This study

^aYFP, yellow fluorescent protein.

Genetic mutant strain construction.

The Aspergillus fumigatus pyrG gene, used as a selectable nutritional marker for transformation, was amplified from the plasmid pXDRFP4 using the primers pyrG5′ and pyrG3′. To generate constructs for the ΔcnaB strain WSA08, the double-joint PCR method was used as previously described (36). In brief, a 1,093-bp 5′-flank DNA fragment and a 1,089-bp 3′-flank DNA fragment were amplified using the primers cnaB-p1 and cnaB-p3 and cnaB-p4 and cnaB-p6, respectively, from genomic DNA (gDNA) of A. nidulans reference strain TN02A7. The linearized DNA fragment, including a 5′ flank of cnaB, pyrG, and a 3′ flank of cnaB, was amplified with primers cnaB-p2 and cnaB-p5. The final fusion PCR product was purified and transformed into TN02A7. A diagnostic PCR assay was performed to detect cnaB replaced by A. fumigatus pyrG (AfpyrG) at the original cnaB locus using primers cnaB-p1 and Diag-pyrG. The same strategy was used to construct the yvcA deletion strain ZSA01. The primers for the genetic mutant strain construction are listed in Table 2.

TABLE 2.

List of primers used in this study

To construct the ΔcnaA cchA^re conditional mutant strain, the ΔcnaA strain CNA1 (37) was crossed with the cchA^re strain HHA02 (7), resulting in ΔcnaA cchA^re strain CJA01. In the conditional mutant strain CJA01, cchA was under the control of alc(p), which was repressed (“re” in “cchA^re”) by glucose on MMPDRUU or not repressed by glycerol on MMPGRUU (25). To construct the calcineurin and cchA double deletion strain, the ΔcnaA strain CNA1 and the ΔcnaB strain WSA08 were crossed with the ΔcchA strain WSA05 (25), respectively, resulting in the ΔcnaA ΔcchA strain LXA04 and ΔcnaB ΔcchA strain LXA05. To construct the yvcA and cnaA double deletion strain, the ΔyvcA strain ZSA01 was crossed with the ΔcnaA strain CNA1. All progeny were screened according to a standard protocol (38).

For the mutants expressing the codon-optimized aequorin, the plasmid pAEQS1-15 containing codon-optimized aequorin (39) and the plasmid pQa-pyroA or pJH37 containing selective marker genes pyroA or riboB, respectively, were cotransformed into the indicated mutants. Transformants were screened for aequorin expression using methods described previously (34, 35), and a high-aequorin-expression strain was selected after homokaryon purification.

Plate assays.

Unless indicated elsewhere, MMPDRUU or MMPGRUU, in which pH was adjusted to 6.5, was used as the plate assay. For each test, at least three replicates were prepared for each strain. The influence of osmotic stress or ionic stress was tested by adding 800 mM NaCl, 600 mM KCl, or 1 M sorbitol into MMPDRUU or MMPGRUU, respectively. To assess the role of the absence of extracellular calcium under salt stress, MMPDRUU was supplemented with 800 mM NaCl and 3 mM EGTA. Two microliters of 1 × 10⁷/ml conidia from the indicated strains was spotted onto relevant media and cultured at 37°C for 2.5 days. For the cell wall integrity test, aliquots of 2 μl from a series of 10-fold dilutions derived from a starting suspension of 10⁸/ml conidia as indicated were spotted onto solid MMPDRUU supplemented with 30 μg/ml calcofluor white or 100 μg/ml Congo red after sterile filtration as indicated in the text. After being cultured for 2.5 days at 37°C, the colonies were observed and imaged unless stated otherwise.

Real-time monitoring of the cytoplasmic Ca²⁺ level.

The cytoplasmic Ca²⁺ concentrations were determined using a previously described method with minor modifications (39). In brief, the vector pAEQS1-15 harboring the codon-optimized aequorin gene was transformed into the reference strain and relevant mutant strains, respectively. A total of 1 × 10⁶ spores were cultured on either MMPDRUU or MMPDRUU with 800 mM NaCl in a 96-well microtiter plate (Thermo Fischer, United Kingdom) and then incubated at 37°C for 18 h. The medium was then removed, and the mycelia were rinsed twice with PGM: 20 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)] (pH 6.7), 50 mM glucose, and 1 mM MgCl₂. Aequorin was reconstituted by incubation of mycelia in 100 μl PGM containing 2.5 μM coelenterazine for 4 h at 4°C in the dark. After reconstitution, mycelia were washed two times with 100 μl PGM and allowed to recover to room temperature for 1 h (40). Following recovery, luminescence was measured for 180 s after 15 mM CaCl₂ addition within 20 s. At the end of each experiment, the active aequorin was completely discharged by permeabilizing the cells with 20% (vol/vol) ethanol in the presence of excess Ca²⁺ (3 M CaCl₂). Luminescence was measured with an LB 96P Microlumat luminometer (Berthold Technologies, Germany). The data from relative light unit (RLU) values detected were converted into [Ca²⁺]_c by using the empirically derived calibration formula pCa = 0.332588 (−log k) + 5.5593, where k is luminescence (RLU) s⁻¹/total luminescence (RLU) (39).

Microscopic observation and image processing.

For hyphal growth microscopic observations, conidia of the relevant strains were inoculated onto precleaned glass slides overlaid with MMPDRUU or MMPDRUU amended with 800 mM NaCl. Strains were grown on slides at 37°C for 16 h, and hyphae dispersing at the edge of colony of each strain were observed under a microscope. The branching frequency was measured by assessing the percentage of subapical hyphal compartments that had one or more branches or lateral buds (41). More than 400 individual hyphal filaments per strain were measured for analysis. To assess the influence of extracellular calcium on fungal growth, strains were inoculated onto precleaned glass coverslips within a petri dish overlaid with liquid MMPDRUU in the presence or absence of 15 mM CaCl₂, 15 mM CaCl₂ plus 15% polyethylene glycol (PEG), or 15 mM CaCl₂ plus 1 M sorbitol and cultured for 12 h at 37°C. Differential interference contrast (DIC) images of the cells were collected with a Zeiss Axio imager A1 microscope (Zeiss, Jena, Germany). These images were then collected and analyzed by a SensiCamQE cooled digital camera system (Cooke Corporation, Germany), and the results were assembled in Adobe Photoshop (Adobe, San Jose, CA).

RNA isolation and quantitative RT-PCR assays.

For RNA isolation, briefly, a total of 1 × 10⁸ conidia of the relevant strains were incubated in 100 ml MMPDRUU at 37°C with shaking at 220 rpm for 18 h, and then the cultures underwent an additional incubation supplemented with or without NaCl (800 mM final concentration) at 37°C for 30 min. The samples were harvested by filtration and ground to a fine powder in the presence of liquid nitrogen. Total RNA was isolated using TRIzol (Invitrogen catalog no. 15596-025) following the manufacturer's instruction. Reverse transcription-PCR (RT-PCR) was carried out using HiScript Q RT SuperMix (Vazyme catalog no. R123-01), and then cDNA was used for the real-time analysis, which was performed using an ABI one-step fast thermocycler (Applied Biosystems), and the reaction products were detected with SYBR Premix Ex Taq (TaKaRa catalog no. DRR041A). Primer information is provided in Table 2. Independent assays were performed with three biological replicates, and transcript levels were calculated by the comparative threshold cycle (ΔC_T) method and normalized against the expression of actin mRNA level in A. nidulans. The 2^−ΔΔCT method was used to determine the change in expression (42).

RESULTS

Hyphal growth defects caused by the loss of calcineurin are suppressed by the absence of cchA under salt stress conditions.

Calcineurin plays a central role in the regulation of fungal morphogenesis, cell wall integrity, cation homeostasis, and stress adaptation (43–45). Interestingly, our previously published data showed that the absence of the high-affinity calcium channels CchA and MidA could suppress hyphal growth defects caused by the addition of the calcineurin inhibitor FK506 under salt stress conditions (25). To gain insights into the relevance of calcineurin and the calcium channel CchA in the regulation of fungal hyphal growth under salt stress and confirm the accuracy of our previous findings, both the ΔcnaA cchA^re and ΔcnaA ΔcchA strains were generated. The relevant strains were analyzed for hyphal growth on MMPDRUU in the presence or absence of salt stress (800 mM NaCl or 600 mM KCl) and osmotic stress (1 M sorbitol). Consistent with our previous report, the ΔcnaA strain displayed compact colony morphology with severe defects on hyphal radial growth, which amounted to a decrease to 29.2% ± 1.4% compared with the reference strain (Fig. 1A and B). Furthermore, the percentage of subapical hyphal compartments that had one or more branches was measured by microscopic observation. As shown in Fig. 1C, the reference strain showed organized, parallel, and defined hyphal filaments under both normal and salt stress conditions, while both the ΔcnaA and the ΔcnaA cchA^re strains showed dramatically increased hyphal branching frequencies of 93.33% ± 1.53% and 91.8% ± 1.31%, respectively, in comparison to 7.53% ± 0.5% in the reference strain on MMPDRUU (Fig. 1D). Moreover, the presence of NaCl, KCl, or sorbitol did not restore the hyphal growth defects in the ΔcnaA strain. However, in contrast to the ΔcnaA strain, the presence of NaCl restored the hyphal growth defects in the ΔcnaA cchA^re and ΔcnaA ΔcchA strains on MMPDRUU. As shown in Fig. 1B and D, the hyphal radial growth in the ΔcnaA cchA^re strain increased from 32.7% on MMPDRUU alone to 78.1% on MMPDRUU with 800 mM NaCl compared to the reference strain, and the hyphal branching frequency decreased from 91.8% on MMPDRUU alone to 3.97% on MMPDRUU with 800 mM NaCl. What needs illustration is that the ΔcnaA cchA^re strain showed an identical phenotype to the ΔcnaA strain under the nonrepression medium MMPGRUU, with which the expression of cchA was turned on by glycerol (see Fig. S1 in the supplemental material).

FIG 1.

The hyphal growth defects of the ΔcnaA or ΔcnaB strains could be suppressed by the dysfunction of CchA under salt stress. (A) Colony morphology of the ΔcnaA, ΔcnaA cchA^re, ΔcnaB, and ΔcnaB ΔcchA strains and the reference strain. The conidia were spotted on solid MMPDRUU and MMPDRUU supplemented with 800 mM NaCl, 600 mM KCl, or 1 M sorbitol, respectively, at 37°C for 2.5 days. (B) Graphic representation of radial growth rates of the ΔcnaA and ΔcnaA cchA^re strains and the reference strain. The values are means ± standard deviations (SD) from three independent experiments. (C) Differential interference contrast images of hyphae grown on MMPDRUU in the presence or absence of salt stress at 37°C for 16 h. Bar, 10 μm. (D) Analysis of branching frequencies in hyphal filaments of the ΔcnaA and ΔcnaA cchA^re strains and the reference strain. The values are means ± SD from three independent experiments.

With the aim of understanding the functions of cnaB in the regulation of hyphal growth in A. nidulans, the cnaB deletion strain WSA08 was generated (see Fig. S2 in the supplemental material). In a similar fashion to the ΔcnaA strain, the ΔcnaB strain displayed small densely packed colonies with severe defects in hyphal radial growth and increased hyphal branching. The conidial quantification assay for the whole colony showed that loss of either cnaA or cnaB caused a sharp reduction in conidial production. The percentages of conidial production were 1.44% ± 0.17% and 0.06% ± 0.01% in the ΔcnaA and ΔcnaB strains, respectively, compared with that of the reference strain (100%). Interestingly, when either the ΔcnaA or ΔcnaB strain was exposed to salt stress, the compact colony turned green compared to the control. The conidia produced by the ΔcnaA and ΔcnaB strains under salt stress were approximately increased 13-fold and 100-fold, respectively, compared to those on MMPDRUU alone, suggesting that salt stress was able to induce conidiation in calcineurin-null strains.

Since our aforementioned data verified that the deletion of cchA could suppress hyphal growth defects in the ΔcnaA strain under salt stress conditions, this prompted us to explore whether deletion of cchA could restore the hyphal growth defect in the ΔcnaB strain under salt stress. As expected, the hyphal growth defects in the ΔcnaB ΔcchA strain were reversed by growth under salt stress, which was indistinguishable from the phenomenon identified in the ΔcnaA cchA^re strain on MMPDRUU (Fig. 1A and C). Since cchA is thought to encode an alpha-subunit of the high-affinity Ca²⁺ channel, the most likely reason for the lack of cchA-based suppression of hyphal defects in the ΔcnaA strain would be due to reduced calcium uptake via CchA. To test this hypothesis, we inoculated the relevant strains on MMPDRUU solid medium supplemented with the Ca²⁺ chelator EGTA or EGTA plus NaCl. As expected, the presence of EGTA under salt stress alleviated the growth defects of the ΔcnaA or ΔcnaB strains in comparison with the control (see Fig. S3 in the supplemental material). Taken together, the above results indicated the loss of either cnaA or cnaB resulted in identical hyphal growth defects. Moreover, the absence of cchA suppressed the hyphal growth defects caused by the loss of either cnaA or cnaB under salt stress.

Hypersensitivity to extracellular calcium in calcineurin-null mutants is suppressed by cchA deletion.

To address how calcineurin and CchA coordinately regulate biological processes in the presence of exogenous Ca²⁺, the phenotypic response to calcium tolerance was determined. As shown in Fig. 2A, when strains were exposed to 15 mM CaCl₂, unlike the reference strain, which contained fully extended hyphae, the ΔcnaA strain exhibited drumstick-shaped hyphal tips, in which the protoplasm was bulging out. However, in comparison, the ΔcnaA cchA^re and ΔcnaB ΔcchA strains both displayed increased-calcium-tolerance phenotypes compared to the ΔcnaA or ΔcnaB strains, resulting in relatively long hyphal growth under the same conditions (Fig. 2A; see Fig. S4 in the supplemental material). Considering that the hypersensitivity to extracellular calcium in the cnaA- and cnaB-null mutants may be associated with cell wall defects, we tested whether osmotic stabilizers could alleviate this growth defect in the presence of Ca²⁺. As expected, the hyphal growth of both the ΔcnaA and ΔcnaA cchA^re strains was improved by adding PEG or sorbitol under the exogenous calcium condition. The same recovery phenotype occurred in the ΔcnaB and ΔcnaB ΔcchA strains (Fig. 2A). Consistently, the biomasses of the ΔcnaA and ΔcnaB strains were reduced by 80% ± 4% and 90% ± 1.6%, respectively, when grown with 15 mM CaCl₂ compared with growth in MMPDRUU alone. However, the biomasses of the ΔcnaA cchA^re and ΔcnaB ΔcchA strains decreased by only 50% ± 5% and 35% ± 4%, respectively, in the same calcium-enriched environment (Fig. 2B). Collectively, the above results suggested that calcineurin inhibition most likely perturbed cell wall synthesis and the absence of CchA could enhance the tolerance of the calcineurin-null strain to calcium toxicity.

FIG 2.

Calcium tolerance assay. (A) Phenotypic comparison of the ΔcnaA and ΔcnaA cchA^re strains and the reference strain cultured on liquid medium in the presence or absence of 15 mM CaCl₂ or 15 mM CaCl₂ plus 15% PEG or 1 M sorbitol at 37°C for 12 h. Bars, 10 μm. (B) Biomass assay of the indicated strains. The error bars indicate the standard deviations of results from three independent replicates. Significance was set at P < 0.05 (*) and P < 0.01 (**) between calcium pretreatment and no calcium pretreatment.

The loss of CchA increases the resistance to cell-wall-damaging agents in calcineurin-null mutants.

To detect whether calcineurin and CchA cooperatively contribute to the modulation of cell wall biosynthesis, the growth of the relevant strains in the presence of the cell-wall-damaging stressors calcofluor white and Congo red was tested. In comparison to the ΔcnaA and ΔcnaB strains, the ΔcnaA cchA^re and ΔcnaB ΔcchA strains were moderately sensitive to calcofluor white and Congo red. To more clearly define the subtle growth differences between the relevant strains, different inocula of conidia were spotted onto MMPDRUU supplemented with calcofluor white and Congo red. As shown in Fig. 3, the ΔcnaA cchA^re and ΔcnaB ΔcchA strains grew faster than their corresponding single deletion strains in response to those wall-damaging stressors. These data suggested that the ΔcnaA cchA^re and ΔcnaB ΔcchA strains were more resistant to cell-wall-damaging agents than the ΔcnaA and ΔcnaB strains.

FIG 3.

The loss of CchA increases the resistance to cell-wall-damaging agents in calcineurin-null mutants. A series of 2-μl 10-fold dilutions derived from a starting suspension of 1 × 10⁸/ml conidia as indicated were spotted onto solid MMPDRUU supplemented with 40 μg/ml calcofluor white and 100 μg/ml Congo red, respectively. All plates were incubated at 37°C for 2.5 days.

Calcineurin negatively regulates CchA upon calcium uptake in response to extracellular calcium.

Our data shown above have demonstrated that the absence of cchA in calcineurin deletion strains enhances tolerance to extracellular calcium. To further explore the roles of calcineurin and CchA in the regulation of [Ca²⁺]_c, real-time monitoring of [Ca²⁺]_c in living hyphal cells was carried out. As shown in Fig. 4, upon addition of 15 mM CaCl₂, the level of [Ca²⁺]_c increased immediately and reached a peak level in all of the tested strains. As expected, deletion of cchA caused a reduction of 21.9% in transient [Ca²⁺]_c compared with that of the reference strain (0.66 ± 0.17 μM for the reference strain versus 0.51 ± 0.09 μM for the ΔcchA strain). In contrast, the resulting transient [Ca²⁺]_c was increased approximately 5-fold in the ΔcnaA strain compared with that in the reference strain (0.66 ± 0.17 μM for the reference strain versus 3.06 ± 0.18 μM for the ΔcnaA strain). This suggests that the deletion of cnaA leads to calcium hyperaccumulation in the presence of extracellular calcium, confirming the important function of calcineurin as a key regulator of cellular calcium homeostasis. However, the deletion of cchA abolished the abnormal calcium hyperaccumulation in the ΔcnaA strain. Collectively, the above data show that calcineurin negatively regulates CchA upon calcium uptake, and the [Ca²⁺]_c increase in the ΔcnaA strain is mediated by the CchA channel in response to external calcium.

FIG 4.

Real-time monitoring of [Ca²⁺]_c level in response to extracellular calcium. The bar graph shows the peak [Ca²⁺]_c of the indicated strains after treatment with 15 mM CaCl₂. *, P < 0.05; **, P < 0.01. The basal [Ca²⁺]_c level is indicated by the horizontal line at the bottom (approximately 0.09 μM). In each experiment, values represent averages from six wells, and error bars represent SD (n = 6).

CnaA and CchA act synergistically to coordinate calcium influx in salt-stress-pretreated cells.

To gain further insights into the roles of calcineurin and CchA in the regulation of calcium homeostasis under salt stress, the [Ca²⁺]_c levels were monitored after salt stress pretreatment. As described in Materials and Methods, the spores of the TN02A7, ΔcnaA, ΔcchA, and ΔcnaA cchA^re strains were first cultured on MMPDRUU supplemented with 800 mM NaCl for 18 h at 37°C, and then the [Ca²⁺]_c was measured for 3 min after the addition of 15 mM CaCl₂. Interestingly, compared to cells cultured normally (Fig. 4), the NaCl-pretreated cells had an approximately 5-fold increase in transient [Ca²⁺]_c at the peak of the burst in the reference strain (3.26 ± 0.03 μM for salt-stress-pretreated cells versus 0.66 ± 0.17 μM for normally cultured cells) in response to the addition of calcium. Not surprisingly, transient [Ca²⁺]_c in the cchA deletion strain decreased by 24.2% compared to the reference strain, indicating that increased transient [Ca²⁺]_c under salt stress is partly mediated by the CchA channel. Unexpectedly, the cnaA deletion strain also exhibited decreased transient [Ca²⁺]_c amplitudes under the same stimulating conditions. Therefore, the loss of either cnaA or cchA in salt-stress-pretreated cells was able to decrease the transient [Ca²⁺]_c, suggesting that calcineurin and CchA were required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. However, the deficiency of cchA in the cnaA deletion background unexpectedly switched the transient [Ca²⁺]_c amplitude to 3.0 ± 0.16 μM, a nearly normal level compared with the reference strain when pretreated with salt stress (Fig. 5). These results indicated that the restoration of calcium homeostasis in the ΔcnaA cchA^re strain under salt stress is probably due to a bypass of the calcineurin-CchA pathway.

FIG 5.

Real-time monitoring of [Ca²⁺]_c in salt-stress-pretreated cells. The bar graph shows the peak [Ca²⁺]_c of the indicated strains after treatment with 15 mM CaCl₂. *, P < 0.05; **, P < 0.01. The basal [Ca²⁺]_c level is indicated by the horizontal line at the bottom (approximately 0.09 μM). In each experiment, values represent averages from six wells, and error bars represent SD (n = 6).

Comparison of transcriptional responses of salt-stress-induced genes and Ca²⁺ signaling-related genes.

To validate the regulatory roles of calcineurin and CchA in the maintenance of [Ca²⁺]_c homeostasis under salt stress, the expression levels of genes encoding calcium pumps, exchangers, and transporters were measured by real-time qPCR in relevant strains either pretreated with 800 mM NaCl or not pretreated. As shown in Fig. 6A, vcxA, a vacuolar Ca²⁺/H⁺ exchanger responsible for Ca²⁺ uptake into vacuoles in vivo (46, 47) was upregulated approximately 3- and 6-fold in the ΔcnaA and ΔcnaA cchA^re strains, respectively, when these strains were treated with 800 mM NaCl, in comparison to results in the absence of salt stress. Furthermore, there were no detectable changes in the reference strain pretreated with salt stress. This result suggested that more calcium may be taken up into vacuoles in the ΔcnaA strain and ΔcnaA cchA^re strain than in the reference strain under salt stress. Meanwhile, expression of yvcA, a transient receptor potential channel (TRPC) family Ca²⁺ channel gene that mediates Ca²⁺ release from vacuoles into the cytosol (48), was increased 5-fold in both the reference strain and the ΔcnaA strain when stimulated by salt stress, while no changes were observed in the ΔcnaA cchA^re strain. This suggested that more calcium was probably released from vacuoles into the cytosol in the reference strain and the ΔcnaA strain compared with the ΔcnaA cchA^re strain under salt stress. Taken together, these results indicated that vacuolar Ca²⁺ exchangers/channels are not likely to be involved in the increased [Ca²⁺]_c in the ΔcnaA cchA^re strain under salt stress. Additionally, there were no significant differences (>3-fold) in the mRNA levels of pmcA (plasma membrane calcium-ATPase) and pmrA (plasma membrane ATPase related) between the ΔcnaA and ΔcnaA cchA^re strains.

FIG 6.

Expression analysis of Ca²⁺-signaling-related and salt-stress-induced genes in response to salt stress by quantitative PCR. (A) Fold changes in mRNA levels, including vcxA, yvcA, pmrA, and pmcA, after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. (B) Fold changes in mRNA levels, including enaA, nhaA, and trkA, after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. Data representing the indicated strains' mRNA levels from salt stress pretreatment were normalized to the non-salt-pretreatment condition. The error bars indicate the standard deviations from three independent replicates.

Furthermore, the expression of genes involved in ion transport was determined. As shown in Fig. 6B, enaA, a putative P-type ATPase sodium pump, was upregulated 70-fold when the reference strain was treated with 800 mM NaCl compared to growth in MMPDRUU alone. However, no obvious increase was observed in the ΔcnaA strain under the same treatment conditions, consistent with reports that the expression of the enaA gene is mediated by calcineurin (29, 49). Most interestingly, the loss of cchA in the ΔcnaA strain dramatically restored the expression of enaA under salt stress. Expression of enaA was upregulated approximately 31-fold in the ΔcnaA cchA^re strain after salt stress treatment compared with the non-salt-treated strain. However, the expression of two other sodium transporter genes, nhnA and trkA (50–52), showed no significant differences among the tested strains when treated with 800 mM NaCl. These results suggested that increased expression of enaA may bypass the requirement for calcineurin in the ΔcnaA cchA^re strain under salt stress.

YvcA compensates for calcineurin-CchA in fungal salt stress adaption.

In budding yeast, hypertonic shock induces the release of calcium from internal stores through Yvc1p (53). To further explore the roles of YvcA in the calcineurin-CchA pathway in fungal salt stress adaption, we constructed the ΔyvcA and ΔyvcA ΔcnaA strains. As shown in Fig. 7, the deletion of yvcA does not affect the hyphal radial growth and conidiation of strains grown on MMPDRUU in the presence or absence of the Ca²⁺ chelator EGTA or EGTA plus NaCl. As mentioned in Fig. S3 in the supplemental material, the addition of EGTA to the ΔcnaA strain culture remarkably alleviated the growth defects of the ΔcnaA strain under salt stress, which mimics the phenotype of the ΔcnaA ΔcchA strain under salt stress. However, the addition of EGTA was unable to suppress the growth defects of the ΔyvcA ΔcnaA strain under salt stress. These results indicated that YvcA compensates for calcineurin-CchA in fungal salt stress adaption.

FIG 7.

Colony morphologies of the ΔyvcA, ΔcnaA, and ΔcnaA ΔyvcA strains grown on MMPDRUU in the presence or absence of 800 mM NaCl or 800 mM NaCl plus 3 mM EGTA at 37°C for 2.5 days.

DISCUSSION

Calcium signaling has been implicated in a broad spectrum of developmental processes in a variety of biological systems. Calcineurin, as the central regulator of calcium homeostasis, has been shown to play a role in morphogenesis and stress response in both yeasts and filamentous fungi (17, 44, 54, 55). Moreover, it has been shown that the calcium channel MidA-CchA complex is involved in regulating hyphal polarity and salt stress adaption (25). However, exactly how MidA-CchA and calcineurin coordinate the regulation of morphogenesis and the response to salt stress remains to be fully elucidated. In A. fumigatus, the presence of sorbitol improved the growth of the ΔcnaA strain but not the ΔcnaB strain, suggesting that CnaA and CnaB might play different roles in the regulation of cell wall biosynthesis (56). In contrast to the results from A. fumigatus, the addition of sorbitol could not alleviate the growth defects in either the ΔcnaA or ΔcnaB strain of A. nidulans. Furthermore, both the ΔcnaA and ΔcnaB strains were sensitive to external calcium, especially in liquid culture, where the protoplasm was extruded from drumstick-shaped hyphal tips. Moreover, the calcium toxicity phenotype could be alleviated by adding the osmotic stabilizer PEG or sorbitol, suggesting that calcineurin contributes to cell wall organization. These data are consistent with the function of calcineurin in cell wall biosynthesis, where calcineurin acts as a positive regulator of the expression of the FKS2 gene, which encodes a component of the β-1,3-glucan synthase complex necessary for cell wall integrity (56, 57).

Since the major roles of calcineurin and CchA are in calcium regulation, real-time monitoring of the [Ca²⁺]_c in living hyphal cells exposed to external calcium was carried out in both normally cultured cells and salt-stress-pretreated cells. In normally cultured cells, the deletion of cnaA led to calcium overaccumulation in the presence of external calcium in A. nidulans. Moreover, the abnormal increase in [Ca²⁺]_c in theΔcnaA strain was dependent on the calcium CchA channel, and the deletion of cchA completely abolished the overaccumulation of [Ca²⁺]_c seen in the ΔcnaA strain. However, fungi may use different mechanisms to adapt to salt stress. As shown in Fig. 5, significantly higher [Ca²⁺]_c was triggered in salt-stress-pretreated cells than in normally cultured cells in response to the same external calcium stimulus. It appears that more calcium was needed for fungi to better survive under salt stress than under the normal condition. Unexpectedly, the cnaA deletion strain showed lower transient [Ca²⁺]_c amplitude than the reference strain under the same salt-stress-pretreated culture conditions. It should be noted that the decrease in transient [Ca²⁺]_c in the ΔcnaA strain in the salt-stress-pretreated cells was completely opposite to the results seen in normally cultured cells in response to CaCl₂ stimulation, where the ΔcnaA strain showed a 5-fold increase in [Ca²⁺]_c over the reference strain. Therefore, we conclude that CnaA and CchA synergistically coordinate the calcium influx in salt-stress-pretreated cells. Interestingly, the loss of both cnaA and cchA unexpectedly switched [Ca²⁺]_c amplitude to a nearly normal level in salt-stress-pretreated cells, indicating that the [Ca²⁺]_c was restored to a normal level, possibly by bypassing the calcineurin-CchA pathway in the ΔcnaA cchA^re strain under salt stress.

The origin of increased [Ca²⁺]_c upon salt stress in the ΔcnaA cchA^re strain may be due to Ca²⁺ release from internal stores. In the budding yeast Saccharomyces cerevisiae, hypertonic shock induces the release of calcium from internal stores through the vacuolar membrane-localized transient receptor potential (TRP) channel-like protein, Yvc1p (53). However, in A. nidulans, yvcA was increased up to 5-fold by salt stimulation in both the reference strain and the ΔcnaA strain but not in the ΔcnaA cchA^re strain. Also, vcxA, a vacuolar Ca²⁺/H⁺ exchanger responsible for Ca²⁺ uptake into the vacuole (58), was upregulated approximately 3- and 6-fold in the ΔcnaA and ΔcnaA cchA^re strains, respectively, but not in the reference strain. Thus, it seems that vacuoles may not contribute to increased [Ca²⁺]_c upon salt stress in the ΔcnaA cchA^re strain of A. nidulans. However, it is probably not accurate to qualify the activities of calcium channels using transcriptional expression. Therefore, to more fully characterize the relationship among YvcA, CchA and calcineurin in fungal salt stress adaption, a ΔyvcA ΔcnaA strain was constructed. The results showed that salt stress was unable to suppress the hyphal growth defects in the presence of EGTA in the ΔyvcA ΔcnaA strain, indicating that YvcA compensates for calcineurin-CchA in fungal salt stress adaption.

To maintain a low intracellular sodium level, cells need to extrude excess sodium cations. The ENA system is the major determinant of sodium detoxification (29). The link between calcineurin signaling and ENA regulation has been reported previously (18, 59, 60). Calcineurin activation of ENA is mediated by the dephosphorylation of Crz1. Two Crz1 binding regions have been identified in the ENA1 promoter (29, 49). Consistent with previous reports, enaA was significantly upregulated (70-fold) in the reference strain grown under salt stress compared with that grown in MMPDRUU alone. However, no obvious increase was observed in the absence of cnaA under the same treatment. Most interestingly, the expression of enaA was upregulated approximately 31-fold in the ΔcnaA cchA^re strain under the same conditions. Thus, the increased expression of enaA that occurred in the ΔcnaA cchA^re strain may lead to hyphal growth remediation in response to salt stress. Considering the loss of cnaA and increased [Ca²⁺]_c in the ΔcnaA cchA^re strain under salt stress, it is possible that calcium-dependent upstream regulators of enaA other than calcineurin exist. Collectively, the findings in this study provide insights into the complex regulatory links between calcineurin and CchA. Calcineurin may negatively regulate or synergistically coordinate with CchA to maintain the cytoplasmic Ca²⁺ homeostasis in response to different environments.