Methods for the detection of intracellular calcium in filamentous fungi.

Calcium (Ca2+), a critical secondary messenger, is also known as the molecule of life and death. The cell responds to a minute change in Ca2+ concentration and tightly maintains Ca2+ homeostasis. Therefore, determining the cell Ca2+ level is critical to understand Ca2+ distribution in the cell and various cell processes. Many techniques have been developed to measure Ca2+ in the cell. We review here different methods used to detect and measure Ca2+ in filamentous fungi. Ca2+-sensitive fluorescent chlortetracycline hydrochloride (CTC), Ca2+-selective microelectrode, Ca2+ isotopes, aequorins, and RGECOs are commonly used to measure the Ca2+ level in filamentous fungi. The use of CTC was one of the earliest methods, developed in 1988, to measure the Ca2+ gradient in the filamentous fungus Neurospora crassa. Subsequently, Ca2+-specific microelectrodes were developed later in the 1990s to identify Ca2+ ion flux variations, and to measure Ca2+ concentration. Another method for quantifying Ca2+ is by using radio-labeled Ca2+ as a tracer. The usage of 45Ca to measure Ca2+ in Saccharomyces cerevisiae was reported previously and the same methodology was also used to detect Ca2+ in N. crassa recently. Subsequently, genetically engineered Ca2+ indicators (GECIs) like aequorins and RGECOs have been developed as Ca2+ indicators to detect and visualize Ca2+ inside the cell. In this review, we summarize various methodologies used to detect and measure Ca2+ in filamentous fungi with their advantages and limitations. •Chlortetracycline (CTC) fluorescence assay is used for visualizing Ca2+ level, whereas microelectrodes technique is used to determine Ca2+ flux in the cell.•Radioactive 45Ca is useful for quantification of Ca2+ in the cellular compartments.•Genetically modified calcium indicators (GECIs) are used to study Ca2+ dynamics in the cell.

CDF family of zinc transporters ZRC-1, MSC-2, and ZRG-17 are involved in survival at high zinc conditions, vegetative development, and cellulase utilization in Neurospora crassa.

The cation diffusion facilitator (CDF) family of zinc transporters plays a crucial role in zinc homeostasis in eukaryotes, including fungi. Here, we investigated the cell functions and genetic interactions of CDF zinc transporters zrc-1 and msc-2 in Neurospora crassa. The Δzrc-1 mutant could not grow in a high-zinc environment, indicating that the zinc transporter protein ZRC-1 was essential for growth in high-zinc conditions. However, the deletion of msc-2 did not show any severe phenotypic defects. Furthermore, we studied the genetic interactions of the zinc transporters using the CDF double mutants. Previously, zrg-17 was reported to be critical, where the Δzrg-17 mutant showed defects in both vegetative development and asexual sporulation. Interestingly, the Δmsc-2;Δzrg-17 double mutant showed phenotypes similar to the wild type, and restored the phenotypic defects of the Δzrg-17 mutation. However, the Δzrc-1;Δmsc-2 and Δzrc-1;Δzrg-17 double mutants continue to display phenotypic defects like their parental single mutants. The double mutant Δzrc-1;Δzrg-17 showed severe vegetative growth defects, including slow growth, short aerial hyphae, narrowed septation, and defective asexual sporulation. In addition, aerial hyphae development of the Δzrc-1;Δmsc-2 and Δzrc-1;Δzrg-17 double mutants were reduced under endoplasmic reticulum stress. Thus, this study revealed the cell functions and genetic interactions of zrc-1, msc-2, and zrg-17 for vegetative development and tolerance to stress conditions in N. crassa.

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.

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 cmdRIP 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 cmdRIP, ∆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 cmdRIP, ∆camk-1, and ∆camk-2 mutants. The cmdRIP 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 cmdRIP, ∆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.

The cell functions of phospholipase C-1, Ca2+/H+ exchanger-1, and secretory phospholipase A2 in tolerance to stress conditions and cellulose degradation in Neurospora crassa.

We investigated the cell functions of the Ca2+ signaling genes phospholipase C-1 (plc-1), Ca2+/H+ exchanger (cpe-1), and secretory phospholipase A2 (splA2) for stress responses and cellulose utilization in Neurospora crassa. The Δplc-1, Δcpe-1, and ΔsplA2 mutants displayed increased sensitivity to the alkaline pH and reduced survival during induced thermotolerance. The ΔsplA2 mutant also exhibited hypersensitivity to the DTT-induced endoplasmic reticulum (ER) stress, increased microcrystalline cellulose utilization, increased protein secretion, and glucose accumulation in the culture supernatants. Moreover, the ΔsplA2 mutant could not grow on microcrystalline cellulose during ER stress. Furthermore, plc-1, cpe-1, and splA2 synthetically regulate the acquisition of thermotolerance induced by heat shock, responses to alkaline pH and ER stress, and utilization of cellulose and other alternate carbon sources in N. crassa. In addition, expression of the alkaline pH regulator, pac-3, and heat shock proteins, hsp60, and hsp80 was reduced in the Δplc-1, Δcpe-1, and ΔsplA2 single and double mutants. The expression of the unfolded protein response (UPR) markers grp-78 and pdi-1 was also significantly reduced in the mutants showing growth defect during ER stress. The increased cellulolytic activities of the ΔsplA2 and Δcpe-1; ΔsplA2 mutants were due to increased cbh-1, cbh-2, and endo-2 expression in N. crassa. Therefore, plc-1, cpe-1, and splA2 are involved in stress responses and cellulose utilization in N. crassa.

Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.2 for the single mutants lacking cpe-1, splA2, camk-1, camk-2, camk-3, camk-4, and cnb-1, suggesting that the circadian clock exhibits standard temperature compensation. However, the Q10 value for the ∆plc-1 mutant was 1.41 at 25 and 30 °C, 1.53 and 1.40 for the ∆ncs-1 mutant at 20 and 25 °C, and at 20 and 30 °C, respectively, suggesting a partial loss of temperature compensation in these two mutants. Moreover, expression of frq, a regulator of the circadian period, and the blue light receptor wc-1, were increased >2-fold in the Δplc-1, ∆plc-1; ∆cpe-1, and the ∆plc-1; ∆splA2 mutants at 20 °C. The frq mRNA level was increased >2-fold in the Δncs-1 mutant compared to the ras-1bd strain at 20 °C. Therefore, multiple Ca2+ signaling genes regulate the circadian period, by influencing expression of the frq and wc-1 genes that are critical for maintaining the normal circadian period length in N. crassa.

CRZ1 transcription factor is involved in cell survival, stress tolerance, and virulence in fungi.

The calcineurin-CRZ1 signaling pathway is conserved from yeasts to humans, and is involved in survival, tolerance to biotic and abiotic stress conditions, virulence, and drug resistance. The primary target of calcineurin is Crz1p in yeasts and nuclear factor of activated T-cells (NFAT) in mammals. Calcineurin regulates the expression of various genes involved in cell wall regeneration, ionic homeostasis, virulence, and other cellular processes. Another protein called regulator of calcineurin (RCN) also regulates calcineurin functions. This review discusses the structure, functions, and regulations of CRZ1 in fungi and related organisms. We have also discussed the role of CRZ1 in pathogenicity and virulence in fungi, plants, and animals.

Heat shock proteins and the calcineurin-crz1 signaling regulate stress responses in fungi.

The heat shock proteins (Hsps) act as a molecular chaperone to stabilize client proteins involved in various cell functions in fungi. Hsps are classified into different families such as HSP90, HSP70, HSP60, HSP40, and small HSPs (sHsps). Hsp90, a well-studied member of the Hsp family proteins, plays a role in growth, cell survival, and pathogenicity in fungi. Hsp70 and sHsps are involved in the development, tolerance to stress conditions, and drug resistance in fungi. Hsp60 is a mitochondrial chaperone, and Hsp40 regulates fungal ATPase machinery. In this review, we describe the cell functions, regulation, and the molecular link of the Hsps with the calcineurin-crz1 calcium signaling pathway for their role in cell survival, growth, virulence, and drug resistance in fungi and related organisms.

Dominant mutants of the calcineurin catalytic subunit (CNA-1) showed developmental defects, increased sensitivity to stress conditions, and CNA-1 interacts with CaM and CRZ-1 in Neurospora crassa.

We studied a dominant mutant of the Neurospora crassa calcineurin catalytic (cna-1) subunit generated using the repeat-induced point mutation (RIP). The Cna-1RIP mutants showed defects in morphology, aerial hyphae development, carotenoid accumulation, and fertility. The Cna-1RIP mutants also showed sensitivity to osmotic stress and a reduction in acquisition of thermotolerance on exposure to lethal heat shock temperature. The Cna-1RIP allele was dominant over the wild type cna-1 allele, suggesting that the CNA-1RIP mutant protein acts in a dominant-negative manner. In addition, we studied calcineurin regulatory subunit (cnb-1) mutants, which were previously generated using RIP. The cnb-1RIP mutants showed sensitivity to calcium (Ca2+) and heat-shock stress conditions. Thus, calcineurin plays an essential role for vegetative and sexual developments, and tolerance to stress conditions in N. crassa. Moreover, CNA-1 directly interacts with the calmodulin (CaM) and calcineurin-responsive zinc finger-1 (CRZ-1) proteins under high Ca2+ condition in N. crassa.

Calcium signaling is involved in diverse cellular processes in fungi.

Calcium (Ca2+) is a universal signalling molecule of life. The Ca2+ signalling is an evolutionarily conserved process from prokaryotes to eukaryotes. Ca2+ at high concentration is deleterious to the cell; therefore, cell maintains a low resting level of intracellular free Ca2+ concentration ([Ca2+]c). The resting [Ca2+]c is tightly regulated, and a transient increase of the [Ca2+]c initiates a signalling cascade in the cell. Ca2+ signalling plays an essential role in various processes, including growth, development, reproduction, tolerance to stress conditions, and virulence in fungi. In this review, we describe the evolutionary aspects of Ca2+ signalling and cell functions of major Ca2+ signalling proteins in different fungi.

Calcineurin responsive zinc-finger-1 binds to a unique promoter sequence to upregulate neuronal calcium sensor-1, whose interaction with MID-1 increases tolerance to calcium stress in Neurospora crassa.

We studied the molecular mechanism of neuronal calcium sensor-1 (NCS-1) signaling pathway for tolerance to Ca2+ stress in Neurospora crassa. Increasing concentration of Ca2+ increased the expression of ncs-1; however, the calcineurin inhibitor FK506 severely reduced ncs-1 mRNA transcript levels. Chromatin immunoprecipitation (ChIP) studies revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and CRZ-1 binding upregulated ncs-1 expression under high Ca2+ concentrations. These results suggested the regulation of NCS-1 function through calcineurin- CRZ-1 signaling pathway. Furthermore, the electrophoretic mobility shift assay (EMSA) revealed that the CRZ-1 binds specifically to an 8 bp sequence 5'-CCTTCACA-3' in the ncs-1 promoter 216 bp upstream of the ATG start codon. We also showed that NCS-1 binds to the Ca2+ permeable channel MID-1 for tolerance to Ca2+ stress. Therefore, CRZ-1 binds to a unique sequence in the ncs-1 promoter, causing upregulation of NCS-1 that binds to MID-1 for tolerance to Ca2+ stress.

Phospholipases play multiple cellular roles including growth, stress tolerance, sexual development, and virulence in fungi.

Phospholipases are ubiquitous enzymes that hydrolyze phospholipids. Based on the cleavage site of the ester linkage in the substrate phospholipids, phospholipases are classified into four major types, phospholipase A (PLA), phospholipase B (PLB), phospholipase C (PLC), and phospholipase D (PLD), which are further classified into various subtypes. Phospholipases hydrolyze phospholipids into various signaling products including phosphatidic acid (PA), diacylglycerol (DAG), free fatty acids (FFAs), and lyso-phospholipids (LPLs). These signaling products regulate numerous processes such as cytoskeletal dynamics, growth, homeostasis, membrane remodeling, nutrient acquisition, secretion, signal transduction, stress tolerance, sexual development, and virulence in various organisms including fungi. Due to these key cellular roles, phospholipases are also promising targets in diagnostic and therapeutic applications. In this review, we discuss current knowledge about the cellular roles of different classes of phospholipases in fungi.

The NcZrg-17 gene of Neurospora crassa encodes a cation diffusion facilitator transporter required for vegetative development, tolerance to endoplasmic reticulum stress and cellulose degradation under low zinc conditions.

The Neurospora crassa gene NcZrg-17 encodes a membrane protein with homology to the cation diffusion facilitator (CDF) family of transporters. We analyzed the phenotypic and functional characteristics of ΔNcZrg-17 and the implications of these characteristics in vivo. The ΔNcZrg-17 mutant showed several phenotypes that are zinc suppressible such as reduced growth rate, short aerial hyphae, increased hyphal branching, early and enhanced conidiation and delayed conidial germination. Furthermore, the NcZrg-17 gene was found to be crucial for survival in the presence of endoplasmic reticulum (ER) stress inducing chemical agents. In addition, we found that ΔNcZrg-17 mutant is defective in protein secretion on cellulose media under low zinc conditions, pointing towards a physiological role for NcZrg-17 in N. crassa. A gradual and delayed transcriptional upregulation (~ threefold) of NcZrg-17 on exposure to low zinc suggests its role in adaptation to low zinc rather than zinc homeostasis. Together our findings support a function of NcZrg-17 in normal vegetative growth, tolerance to ER stress and degradation of cellulose under low zinc conditions in N. crassa.

The pleiotropic vegetative and sexual development phenotypes of Neurospora crassa arise from double mutants of the calcium signaling genes plc-1, splA2, and cpe-1.

We investigated phenotypes of the double mutants of the calcium (Ca2+) signaling genes plc-1, splA2, and cpe-1 encoding for a phospholipase C1 (PLC-1), a secretory phospholipase A2 (sPLA2), and a Ca2+/H+ exchanger (CPE-1), respectively, to understand the cell functions regulated by their genetic interactions. Mutants lacking plc-1 and either splA2 or cpe-1 exhibited numerous defects including reduced colonial growth, stunted aerial hyphae, premature conidiation on plates with delayed germination, inappropriate conidiation in submerged culture, and lesser mycelial pigmentation. Moreover, the ∆plc-1; ∆splA2 and ∆plc-1; ∆cpe-1 double mutants were female-sterile when crossed with wild type as the male parent. In addition, ∆plc-1, ∆splA2, and ∆cpe-1 single mutants displayed higher carotenoid accumulation and an increased level of intracellular reactive oxygen species (ROS). Therefore, the pleiotropic phenotype of the double mutants of plc-1, splA2, and cpe-1 suggested that the genetic interaction of these genes plays a critical role for normal vegetative and sexual development in N. crassa.

The calmodulin gene in Neurospora crassa is required for normal vegetative growth, ultraviolet survival, and sexual development.

We isolated a Neurospora crassa mutant of the calmodulin (cmd) gene using repeat-induced point mutation and studied its phenotypes. The cmd RIP mutant showed a defect in growth, reduced aerial hyphae, decreased carotenoid accumulation, a severe reduction in viability upon ultraviolet (UV) irradiation, and a fertility defect. Moreover, meiotic silencing of the cmd gene resulted in a barren phenotype. In addition, we also performed site-directed mutational analysis of the calcium/calmodulin-dependent kinase-2 (Ca2+/CaMK-2), a target of the CaM protein encoded by the cmd gene. The camk-2 S247A and the camk-2 T267A mutants in a homozygous cross, or in a cross with a Δcamk-2 mutant, displayed an intermediate phenotype, suggesting that serine 247 and threonine 267 phosphorylation sites of the Ca2+/CaMK-2 are essential for full fertility in N. crassa. Therefore, CaM in N. crassa is required for normal vegetative growth, UV survival, and sexual development. Additionally, serine 247 and threonine 267 phosphorylation sites are important for the Ca2+/CaMK-2 function.

Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

Calcineurin Subunits A and B Interact to Regulate Growth and Asexual and Sexual Development in Neurospora crassa.

Calcineurin is a calcium/calmodulin dependent protein phosphatase in eukaryotes that consists of a catalytic subunit A and a regulatory subunit B. Previous studies in the filamentous fungus Neurospora crassa had suggested that the catalytic subunit of calcineurin might be an essential protein. We generated N. crassa strains expressing the A (cna-1) and B (cnb-1) subunit genes under the regulation of Ptcu-1, a copper-responsive promoter. In these strains, addition of bathocuproinedisulfonic acid (BCS), a copper chelator, results in induction of cna-1 and cnb-1, while excess Cu2+ represses gene expression. Through analysis of these strains under repressing and inducing conditions, we found that the calcineurin is required for normal growth, asexual development and female fertility in N. crassa. Moreover, we isolated and analyzed cnb-1 mutant alleles generated by repeat-induced point mutation (RIP), with the results further supporting roles for calcineurin in growth and fertility in N. crassa. We demonstrated a direct interaction between the CNA-1 and CNB-1 proteins using an assay system developed to study protein-protein interactions in N. crassa.

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

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

Calcium/calmodulin-dependent kinases are involved in growth, thermotolerance, oxidative stress survival, and fertility in Neurospora crassa.

Calcium/calmodulin-dependent kinases (Ca(2+)/CaMKs) are Ser/Thr protein kinases that respond to change in cytosolic free Ca(2+) ([Ca(2+)]c) and play multiple cellular roles in organisms ranging from fungi to humans. In the filamentous fungus Neurospora crassa, four Ca(2+)/CaM-dependent kinases, Ca(2+)/CaMK-1 to 4, are encoded by the genes NCU09123, NCU02283, NCU06177, and NCU09212, respectively. We found that camk-1 and camk-2 are essential for full fertility in N. crassa. The survival of ∆camk-2 mutant was increased in induced thermotolerance and oxidative stress conditions. In addition, the ∆camk-1 ∆camk-2, ∆camk-4 ∆camk-2, and ∆camk-3 ∆camk-2 double mutants display slow growth phenotype, reduced aerial hyphae, decreased thermotolerance, and increased sensitivity to oxidative stress, revealing the genetic interactions among these kinases. Therefore, Ca(2+)/CaMKs are involved in growth, thermotolerance, oxidative stress tolerance, and fertility in N. crassa.

Neurospora crassa homologue of Neuronal Calcium Sensor-1 has a role in growth, calcium stress tolerance, and ultraviolet survival.

NCU04379 gene encodes a conserved Ca(2+) and/or calmodulin binding protein that possesses a consensus signal for N-terminal myristoylation and four EF-hands, characteristics of Neuronal Calcium Sensor-1proteins. The NCU04379.2 knockout mutant shows slow growth rate, increased sensitivity to calcium and ultraviolet (UV) irradiation, and a wild-type fragment carrying NCU04379 gene complements the mutant. Therefore, NCU04379 gene has a role in growth, calcium stress tolerance, and UV survival. Crosses homozygous for ΔNCU04379.2 mutant strains were fully fertile; however, we found evidence for involvement of Ca(2+)/calmodulin-dependent protein kinase encoding genes NCU02283 and NCU09123 in sexual development.