Calmodulin, Calcium/Calmodulin-Dependent Kinases-1 and 2 Regulate Expression of the Heat Shock Proteins for Heat Shock Tolerance and Pheromone Signaling Genes for Sexual Development in Neurospora crassa

Abstract

Calmodulin (CaM) is a primary Ca²⁺ sensor that binds and activates numerous target proteins and regulates several cellular processes in eukaryotes. CaM is essential in Neurospora crassa; therefore, we generated a CaM mutant using repeat-induced point (RIP) mutation and investigated the cmd^RIP mutant phenotypes. We also studied knockout mutants of four Ca²⁺/CaM kinases (camk-1, 2, 3, and 4) for their role during stress conditions and sexual development. The cmd^RIP, ∆camk-1, and ∆camk-2 mutants showed reduced survival and growth rates under heat stress, oxidative stress, pH, and ER stress conditions. In addition, under the heat stress conditions, expression of the heat shock protein genes hsp70 and hsp80 was reduced in the cmd^RIP, ∆camk-1, and ∆camk-2 mutants. The cmd^RIP mutant was also defective in cell fusion, its vegetative hyphae could not support the fertilized wild type perithecia graft, and female sterile. Furthermore, the expression of pheromone signaling genes pre-1, pre-2, ccg-4, mfa-1, and fmf-1 was reduced in the cmd^RIP, ∆camk-1, and ∆camk-2 mutants. Therefore, CaM, Ca²⁺/CaMK-1 and 2 are involved in the tolerance to heat stress conditions and sexual development by regulating the heat shock and pheromone response pathways, respectively, in N. crassa.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-023-01091-8.

Introduction

The cell pathways for sensing and adaption to environmental changes are essential for the survival of an organism. A change in the environmental conditions may trigger stress response pathways to cope with stress in all organisms, including fungi [1]. In the heterothallic filamentous fungus N. crassa, calcium (Ca²⁺) signaling responds and transduces various external stimuli and regulates numerous cellular processes required for normal asexual and sexual developments [2, 3]. The cell maintains a cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) level of ~ 100 nM that is maintained by pumps, antiporters, and Ca²⁺ binding proteins [2, 3]. In response to extracellular stimuli, the influx of extracellular Ca²⁺ or release of Ca²⁺ from the intracellular stores transiently increases in the [Ca²⁺]_c levels to 1 µM or more, activating Ca²⁺ sensing proteins for inducing an intracellular signal [4, 5].

Calmodulin (CaM), the principal ubiquitous Ca²⁺ sensing protein conserved in eukaryotes, binds to Ca²⁺ with high affinity and specificity. CaM binds to four Ca²⁺ with the help of EF-hand motifs, where each EF-hand domain consists of 30 amino acid residues with two helices flanking a loop characterized by 12 residues, where the 1(+ X), 3(+ Y), 5(+ Z), 7(− Y), 9(− X), 12(− Z) residues provide the oxygen ligands to form the co-ordination with Ca²⁺ in a pentagonal bipyramidal geometry [6, 7]. The binding of Ca²⁺ to CaM is crucial for CaM function and activation of over 300 target proteins [8].

In N. crassa, the NCU04120 gene encodes a CaM protein of 149 amino acid residues consisting of four conserved EF-hand domains [5]. The N. crassa CaM shows only one amino acid difference to A. nidulans calmodulin, 59% and 85% homology to the calmodulin from yeast and human, respectively. N. crassa CaM contains an unusually high amount of serine, resembles that of plant CaM except for the absence of trimethyllysine, and is slightly less acidic than the vertebrate counterpart [5, 9, 10]. CaM is essential for viability in N. crassa; therefore, CaM was studied by using either CaM antagonists or viable CaM mutants generated via repeat-induced point mutation (RIP) [9, 11]. The previous studies using the CaM antagonist trifluoperazine (TFP), chlorpromazine (CPZ), and cmd^RIP mutant showed that CaM is essential for the growth, hyphal development, circadian rhythm, activation of chitin synthase required for cell wall formation, carotenoid accumulation, ultraviolet survival, and sexual development [5, 9].

In response to Ca²⁺ stimulus, CaM interacts with the enzyme family called Ca²⁺/CaM dependent kinases (Ca²⁺/CaMKs). Ca²⁺/CaMKs are Ser/Thr kinases, and their domain organization comprises an N-terminal catalytic domain, Ca²⁺ /CaM-binding regions, and a C-terminal regulatory domain consisting of the overlapping autoinhibitory [12]. In N. crassa, there are four Ca²⁺/CaMKs, where CaMK-1 and CaMK-2 are essential for full fertility and involved in the tolerance to stress conditions [13, 14].

To investigate the cell functions and mechanism of CaM and Ca²⁺/CaMKs during different growth conditions and development phases, we studied the phenotypes of the cmd^RIP and ∆camks mutants under heat, oxidative, pH, and endoplasmic reticulum (ER) stress conditions. We found that cmd, camk-1, and camk-2 genes regulate the heat shock response pathway under stress. In addition, the cmd^RIP mutant showed defects in cell fusion, an inability to support the development of fertilized perithecia, and a female sterile phenotype. Moreover, the cmd, camk-1, and camk-2 genes regulate the pheromone response pathway during sexual development.

Materials and Methods

Strains, Media, and Culture Conditions

The N. crassa wild type strains 74-OR23-IVA (FGSC 2489) and ORS-SL6a (FGSC 4200), and the knockout mutant strains of Ca²⁺ signaling genes ΔNCU09123:: hph mat A (FGSC 12547), ΔNCU09123:: hph mat a (FGSC 12548), ΔNCU02283:: hph mat A (FGSC 12449), ΔNCU02283:: hph mat A (FGSC 12448), ΔNCU06177:: hph mat A (FGSC 11536), ΔNCU06177:: hph mat a (FGSC 11537) and ΔNCU09212:: hph mat a (FGSC 11545) were obtained from the Fungal Genetic Stock Center (FGSC), University of Missouri, Kansas City, MO 64110 [15]. The Δpan-2::bar::P_tcu-1::cmd::V5::gfp; mat a (850) and Δpan-2::bar::P_tcu-1::cmd^RIP::V5::gfp; mat A (26) strains were generated in our laboratory [9, Table S1]. For routine culture, growth, and maintenance, strains were grown on 1X Vogel’s [16] glucose minimal medium (VGM) containing 1.5% of glucose and 1.5% Bacto agar (Himedia, India) for solid medium. For sexual development, strains of opposite mating types were crossed on 1X Synthetic Crossing Medium (SCM) [17]. Ascospore germination was performed on a medium containing 0.05% fructose, 0.05% glucose, 2% sorbose (FGS), and 2% Bacto agar. Calcium-D pantothenic acid (0.01 mg/ml) and bathocuproinedisulfonic acid (BCS; 250 µM) were supplemented in the mediums as indicated.

Temperature Sensitivity and Thermotolerance Assay

For temperature sensitivity assay, the strains were first grown in VGM agar at 30 °C in the dark for 2–3 days and then at room temperature under light for additional 2 days. Freshly harvested conidial cell suspension of ~ 7 µl of 2 × 10⁷ conidia/ml was inoculated in the center of 90-mm-Petri dishes containing VGM and incubated at low (20 °C), optimum (30 °C), and elevated (40 °C) temperatures. The colony diameter was measured at regular intervals of 3 h, starting at 12 h till 24 h. The radial growth rate was calculated as cm h⁻¹.

For thermotolerance assay, fresh conidial suspension of ~ 1 × 10⁶ conidia/ml was inoculated in liquid VGM and incubated for 2 h in the dark at 30 °C, shaking at 180 rpm. The germlings were then divided equally into three tubes, which were held at 30 °C (control), 30 °C (uninduced), and 44 °C (induced). Both the 30 °C (uninduced) and 44 °C (induced) cultures were then given a lethal heat shock at 52 °C for 20 min. An aliquot of the germlings was plated on the FGS agar medium, followed by incubation at 30 °C for 24 h [18]. The germlings were then observed under a stereomicroscope, and viable colonies were counted for each condition. The percent survival rate was determined by dividing the number of viable colonies in the uninduced and induced treated plates by the number of viable colonies in the control plate and multiplying by 100.

RNA Isolation and Quantitative Real-Time PCR Analysis

To study the expression of genes involved in the acquisition of thermotolerance, strains were grown in VG agar medium at 30 °C in the dark for 2–3 days, then ~ 1 × 10⁶ conidia/ml were inoculated in liquid VG medium and incubated at 28 °C in the dark with constant shaking at 180 rpm for 14 h and then exposed to heat shock at 48 °C for 1 h as describes previously [19]. For the expression of genes related to sexual development under nitrogen-starved conditions, ~ 1 × 10⁶ conidia/ml were inoculated in liquid SCM and incubated at 22 °C under constant light with shaking at 180 rpm for 18 h [20]. The mycelium from these cultures was harvested by filtration and then pulverized into a fine powder using mortar-pestle and liquid nitrogen. RNA was isolated from the powdered mycelia using TRIzol reagent (Life Technologies, USA), and cDNA was synthesized using the Verso™ cDNA Synthesis Kit (Thermo Fisher Scientific, USA) as per the manufacturer's protocol. Quantitative Real-Time PCR (qRT PCR) analysis was performed using specific primer pairs (Table S2), the SYBR Select Master Mix (Life, City, State, USA), and the 7500 Real-Time PCR System (Applied Biosystems, City, State, USA) or the Quant Studio 5 Real-Time PCR system (ThermoScientific, USA) as per the manufacturer’s protocol. The fold change in the relative expression of the target genes was calculated using the 2^−ΔΔC_T method [21] with β-tubulin gene expression level as the reference control [22].

Results

The cmd^RIP (26), ∆camk-1, and ∆camk-2 Mutants Showed Reduced Growth and Survival Rates Under the Heat, Oxidative, and ER Stress Conditions

The cmd^RIP (26), ∆camk-1, and ∆camk-2 mutants showed decreased colony growth rate at lower and elevated temperatures in comparison to the wild type (Fig. 1). To confirm that the growth defects were due to RIP-induced mutations in the cmd and deletion of camk-1 and camk-2 genes, we studied the temperature sensitivity of the parental cmd (850) strain [9], and the camk-1 (19) and camk-2 (8) homokaryotic strains (Table S1) that were generated by inserting the camk-1 and camk-2 genes from the wild type into the pan-2 locus for complementation analysis (described in the Supplementary Materials and Methods). The complemented strains restored the wild type phenotype, which confirmed that the growth defects were due to RIP-induced mutations of the cmd and deletion of the camk-1 and camk-2 genes (Fig. 1). Furthermore, we investigated the role of CaM and Ca²⁺/CaMKs in growth and survival under heat (Fig. S1), oxidative (Figs. S2, S3), alkaline pH (Fig. S4), and ER stress conditions (Fig. S5). These results suggested that CaM, CaMK-1, and CaMK-2 are necessary for survival under various stress conditions.

Fig. 1.

Temperature sensitivity of the cmd^RIP (26), ∆camk-1, and ∆camk-2 mutants. The sensitivity of the wild type, cmd^RIP (26), cmd (850), ∆camk-1, camk-1 (19), ∆camk-2, camk-2 (8), ∆camk-3, and ∆camk-4 strains (Table S1) was evaluated by growing the strains grown on VGM and incubating at three different temperature conditions of 20 °C (low), 30 °C (optimum), and 40 °C (high) for 24 h. A Colony growth on Petri dish. B Average colony growth rate (cm h⁻¹). Error bars indicate the standard deviations calculated from the data for three independent experiments (n = 3) and the significance given by P values < 0.05 (*), < 0.01 (**), and < 0.001 (***) as measured by the one-way ANOVA test

The cmd Gene is Necessary for Female Fertility, Normal Perithecia Development, and Cell Fusion

Previous studies showed that the cmd gene is required for sexual development [9, 11]. To understand the role and mechanism of CaM during sexual development, we performed the male–female fertility assay. The cmd^RIP (26) mutant was sterile as a female parent, suggesting that cmd is necessary for the female sexual structure development in N. crassa (Fig. S7). Furthermore, the perithecia grafting assay showed that cmd is necessary for perithecium development (Fig. S8). In addition, the cell fusion assay suggested that cmd is also required for hyphal fusion during vegetative growth (Fig. S9).

The cmd, camk-1, and camk-2 genes are Necessary for the Normal Expression of the Heat Shock Response and Pheromone Signaling Genes

The expression levels for cmd, camk-1, and camk-2 were increased under heat stress conditions, suggesting a role for these genes in the acquisition of thermotolerance (Fig. S10). To further understand how CaM and Ca²⁺/CaMKs mediate heat stress tolerance, we studied the expression of three genes encoding the heat shock proteins Hsp60, Hsp 70, and Hsp80 in the wild type, cmd^RIP, ∆camk-1, and ∆camk-2 mutant strains. We found that the expression of hsp70 and hsp80 was decreased in the cmd^RIP, ∆camk-1, and ∆camk-2 mutants, while only a minor reduction in the expression of hsp60 was observed in the mutants (Fig. 2A). Therefore, CaM and CaMKs are involved in the regulation of Hsp70 and Hsp80 under heat stress for the acquisition of thermotolerance.

Fig. 2.

Expression analysis of the heat shock response and pheromone signaling genes. A Expression of heat shock response genes hsp60, hsp70, and hsp80 in the wild type, cmd^RIP (26), ∆camk-1,and ∆camk-2 mutant strains. RNA was isolated from the 14 h cultures of N. crassa wild type and mutant strains treated with heat shock conditions for 1 h at 48 °C. Then, the expression of the heat shock response genes hsp60, hsp70, and hsp80 was studied by qRT-PCR and normalized to the expression of the β-tubulin gene. Standard deviations are indicated by error bars calculated from three independent experiments (n = 3), and the significance given by P values < 0.05 (*), < 0.01 (**), and < 0.001 (***) as measured by the one-way ANOVA test. B Expression of the pheromone signaling genes in wild type, cmd^RIP (26), ∆camk-1, and ∆camk-2 mutants. RNA was isolated from the N. crassa wild type and mutant strains cultured in the SCM, and the expression of pheromone signaling genes pre-1, pre-2, ccg-4, mfa-1, and fmf-1 was determined using qRT-PCR and normalized to the expression of the β-tubulin gene. Standard deviations are indicated by error bars calculated from three independent experiments (n = 3), and the significance given by P values < 0.05 (*), < 0.01 (**), and < 0.001 (***) as measured by the one-way ANOVA test

Pheromones and their receptors determine the chemotropic attraction, recognition, and fusion between mating cells during sexual development for proper perithecial development and ascospore production [23]. Therefore, to examine if the pheromone signaling pathway is affected in these mutants, we studied the expression genes encoding pheromone receptors pre-1 and pre-2, the pheromone precursor genes clock-controlled gene-4 (ccg-4), mating factor a-1 (mfa-1), and the female male fertility-1 (fmf-1) gene involved in pheromone signaling under nitrogen starved condition. The expression of the pre-1, pre-2, ccg-4, mfa-1, and fmf-1 genes was reduced in the mutants compared to the wild type (Fig. 2B), indicating that pheromone signaling has been affected in these strains. Therefore, this result suggested that CaM, CaMK-1, and CaMK-2 regulate the pheromone response during sexual development in N. crassa.

Discussion

In this study, we found that the cmd^RIP(26), Δcamk-1, and Δcamk-2 mutants showed an increased sensitivity to temperature, oxidative, pH, and ER stress conditions. We also observed increased expression of cmd, camk-1, and camk-2 genes in the wild type in response to heat shock treatment, indicating their role in tolerance to thermal stress. In N. crassa, Hsp70 and Hsp80 (a Hsp90 family member), the major heat-inducible cytosolic member of eukaryotic Hsp70 and 90 families, were shown rapidly expressed in response to heat stress [24–26]. Hsps belonging to the chaperonin Hsp60, Hsp70, and Hsp90 families are induced rapidly in response to heat shock and play a critical role under stress conditions [24]. Hsps protect the target protein from denaturation mediated by misfolding and aggregation caused by heat shock conditions [27]. Previous work showed that CaM is required for normal growth, carotenoid production, and ultraviolet (UV) survival during sexual development in N. crassa [9, 11]. In addition, CaM directly interacts with the calcineurin catalytic subunit (CNA-1), which is required for growth and proper morphology, carotenoid accumulation, stress tolerance, and sexual development in N. crassa [28]. CaM responds to cellular stress conditions mainly by activating Ca²⁺/CaM-dependent kinases and Ca²⁺/CaM-dependent protein phosphatase calcineurin in S. cerevisiae [29]. The Ca²⁺ and CaM also regulate HSP activity in response to heat shock in maize [30]. In sorghum, Ca²⁺/CaM modulates the in vivo expression of the hsp90 family members (Hsp87 and Hsp85) under normal and heat stress conditions [31]. CaMK II-δB in mammals protects the heart from multiple death-inducing stimuli by phosphorylating and activating the HSF1, which subsequently induces the expression of HSP70 family proteins [32]. The silencing of sscmk1 gene using RNAi technology resulted in a decreased tolerance to heat stress, and the interaction of SSCMK1 with HSP90 was observed using the yeast two-hybrid assay in Sporothrix schenkii [33]. Under heat and other stress conditions, CaM might respond by directly binding and activating the HSF1 transcription factor or by its downstream targets to activate the expression and production HSPs. However, further work is needed to fully understand the mechanism of CaM-mediated regulation of the heat shock response pathway in N. crassa.

In N. crassa, the sexual phase is initiated when the cultures are subjected to light, low temperature, and nitrogen-starved conditions [34, 35]. Perithecia grafting and cell fusion assay showed that the vegetative hyphae were unable to support the development of the fertilized wild type perithecia and were defective during cell fusion, suggesting that CaM is involved in facilitating the pathways required during the formation of sexual structures in N. crassa (Figs. S8, 9). Furthermore, expression of the pheromone receptor genes pre-1 and pre-2, precursor genes ccg-4 and mfa-1, and the ste11 homolog fmf-1 gene was decreased under nitrogen-starved conditions in the cmd^RIP(26), Δcamk-1, and Δcamk-2 mutant strains (Fig. 2B). The regulatory sequences related to pheromone response elements and other important putative regulators were also found in the promoter region of cmd, camk-1, and camk-2 genes [11, 14]. In N. crassa, two pheromone receptors, PRE-1 and PRE-2 were identified, which show high sequence similarities to the a-factor receptor Ste3p and α-factor receptor Ste2p belonging to the family of G-protein coupled receptors (GPCRs) in S. cerevisiae [36]. The pheromone receptors bind to their respective pheromones, where PRE-1 and PRE-2 bind to the MFA-1 and CCG-4, respectively [23]. In addition, the Schizosaccharomyces pombe Ste11p homolog FMF-1, is required for mating pheromone signaling in both the male and female parents in N. crassa [37]. Thus, this study showed that cmd, camk-1, and camk-2 are involved in the regulations of the genes involved in pheromone signaling during the sexual phase in N. crassa.

Conclusion

The cmd, camk-1, and camk-2 genes are necessary for growth and survival under heat, oxidative, and ER stress conditions. Furthermore, cmd is also essential for female fertility, perithecia development, and cell fusion during the sexual phase in N. crassa. In addition, cmd, camk-1, and camk-2 are involved in regulating the genes involved in pheromone signaling during the sexual phase in N. crassa. Future work will reveal the detailed mechanism of the interactions of CaM, CaMK-1, and CaMK-2 with the target proteins in response to various stress conditions and during the asexual and sexual developments in N. crassa.

Supplementary Information

Below is the link to the electronic supplementary material.