In fungi the PHO regulon includes genes that respond to phosphate starvation or sufficiency; initially it was reported that the PHO regulon in S. cerevisiae is formed by 22 genes, nine of which are regulatory genes (Ogawa et al., 2000). Similarly, nine genes homologous to those of the yeasts PHO regulon were identified later in the genome of Aspergillus fumigatus (Serra-Cardona et al., 2014), however, recent studies have reported a larger number of genes controlled by phosphate in the genome of some fungi (Tomar and Sinha, 2014). A study of Fusarium graminearum phosphatome identified up to 82 phosphatase genes (Yun et al., 2015). Eleven of these genes were found to be essential but the remaining 71 phosphatase genes have also impact in fungal development. These phosphatases were shown to be involved in fungal growth and differentiation, in the biosynthesis of secondary metabolites, pathogenesis and virulence.

The proteins encoded by the PHO regulon genes are located in the plasma membrane, the periplasmic space, vacuoles, mitochondria or the cytosol. Several genes were found to encode proteins for scavenging phosphate from the extracellular medium, for polyphosphate hydrolysis and transport across vacuole or mitochondria membrane systems (Persson et al., 2003). Phosphate transport and metabolism are important factors in fungal virulence and pathogenesis. Mutants of Cryptococcus neoformans defective in phosphate acquisition and storage showed altered virulence and pathogenicity associated with the production of bioactive metabolites (Kretschmer et al., 2014).

Particularly relevant was the finding in fungi of genes encoding proteins for the transport of inorganic phosphate and regulatory genes that control inorganic phosphate homeostasis. Two of the PHO regulon proteins, encoded by pho4 and pho2, are master regulators that control expression of many pho genes. The S. cerevisiae Pho4 is a 312 amino acids transcriptional regulator that belongs to the basic helix-loop-helix (bHlH) family (Persson et al., 2003). Pho4 is a dimer that works in collaboration with Pho2 controlling the transcription of the PHO regulon genes and its action is modulated by phosphorylation at serine-threonine residues, that is catalysed by Pho85/Pho80 cyclin-kinase complex (see below) (Komeili, 1999; Secco et al., 2012a; Tomar and Sinha, 2014). Initial studies on the control of gene expression by this transcriptional regulator was made by studying the expression of pho5, a gene that encodes the yeast secreted acid phosphatase, an easy to measure reporter of the Pho4-mediated regulon genes. Two activating sequences Uasp1 and Uasp2 that bind Pho4 were found in the region upstream of the pho5 promoter (Rudolph and Hinnen, 1987; Ogawa et al., 2000). The binding sequences of the Pho4 protein are CACGTG or CACGTT, the Uasp2 sequence is protected by the nucleosome structure and therefore only the Uasp1 sequence is accessible unless the chromatin is remodelled by different protein modifications including acetylation/deacetylation or phosphorylation (Steger et al., 2003). Pho4 binding sequences have been found in almost all the promoters of the PHO regulon genes (Ogawa et al., 2000). Pho4 plays also an important role in the control of pathogenicity and virulence of pathogenic fungi. Studies using a C. neoformans mutant defective in Pho4 showed that this regulatory protein is required for phosphate uptake, growth and C. neoformans dissemination into the brain (Lev et al., 2017; Bhallan et al., 2022).

The mechanism by which fungal cells sense inorganic phosphate is poorly characterized. Inositol pyrophosphates are a family of metabolic messengers characterized by the presence of one or more pyrophosphate (PPi) groups attached to a mio-inositol ring (Wera et al., 2001). The presence of energy rich phosphoanhydre bonds in these molecules is one of the characteristic features that explain their important role in regulation of phosphate metabolism. The intracellular messenger IP3 (1,4,5 inositol-triphosphate) is formed by phospholipase C that cleaves phosphatidyl-inositol-4,5 diphosphate yielding IP3 and diacylglycerol. In yeasts, higher members of this family e.g. mio-inositolheptakisphosphate (IP7) are formed by sequential phosphorylation of IP3 by pyrophosphate kinases and higher members that contain more phosphate groups than carbon atoms reflects the energetic and phosphorylation status of the cell. Particularly, the ATP concentration required for the conversion of mio-inositol hexakisphosphate (IP6) to IP7 since the IP6 kinase has a Km for ATP in the millimolar range (Azevedo and Saiardi, 2017).

In this respect an important finding was the observation that the intracellular concentration of inositol pyrophosphate changes in response to the phosphate concentration in the medium. Proteins involved in cellular reactions of phosphate metabolism, e.g. phosphate transporters, phosphate signaling proteins or polyphosphate polymerases contain a so called SPX domain that interact with inositol pyrophosphates signaling molecules (Secco et al., 2012b). These domains consist of 135–380 amino acid residues and are located in the amino terminal region of the corresponding protein. Initial studies showed that the SPX domain serves as substrate for the interaction of inositol pyrophosphate signaling molecules in plants and animals. In S. cerevisiae the pho90 and pho87 phosphate transporters interact with the SPL2 protein at its SPX domain and this decreases the phosphate uptake (Wykoff et al., 2007; Hürliman et al., 2009). Three SPX domains of different organisms, including S. cerevisiae (Hothorn et al., 2009) were crystalized, providing evidence of the SPX surface that interacts with inositol pyrophosphate (Samyn et al., 2012; Wild et al., 2016). Furthermore, IP7 and higher inositol pyrophosphates act as donors of orthophosphate in protein modifications catalysed by pyrophosphorylases that result in the introduction of an additional phosphate group in the serine phosphate residues in proteins to form serine pyrophosphate units. Therefore, increments in the ATP levels of the cell raises the intracellular level of IP7. In summary, inositol pyrophosphate molecules communicate the inorganic phosphate extracellular level to the SPX domain of the phosphate transporters and other proteins involved in phosphate metabolism, regulating their activity.

Phosphate availability is extremely important to fungi for growth. Inorganic phosphate is taken up in yeasts and filamentous fungi by the high affinity transporters systems encoded by pho84 and pho89 (Bun-ya et al., 1991; Martínez and Persson, 1998; Persson et al., 1999) and low affinity transporters encoded by pho87 and pho90 (Bun-ya et al., 1996; Ghillebert et al., 2011; Wykoff and O´Shea, 2001). When the phosphate concentration in the medium approaches the Km of the low affinity system then the cells trigger the formation of high affinity transporters. This provides a time lapse for the cell to survive using the phosphate low affinity transport system until the high affinity transport is fully induced. The importance of phosphate availability in fungal nutrition is highlighted by the presence of five phosphate transport genes pho84, pho87, pho89, pho90 and pho91 in S. cerevisiae; only quintuple mutants defective in the five transporters are unable to grow, indicating that all of them are active phosphate transporters (Wykoff and O’Shea, 2001).

Analysis of the hydrophobicity of the encoded proteins in yeast showed that both Pho84 and Pho89, belong to the multiple facilitator superfamily (MFS transporters) and have 587 and 574 amino acids respectively, have 12 transmembrane spanning domains with an intervening hydrophilic loop of 74 amino acids between the transmembrane domains TMS6 and TMS7 in Pho84, and a loop of 110 amino acids located between TMS7 and TMS8 in Pho89 (Bottger and Petterson, 2002; Persson et al., 2003). The MFS proteins include many transporters for several primary or secondary metabolites that are involved in communication between cells in pure cultures and/or between different species (Martín et al., 2005). Pho84 and Pho89 are symporters that introduce inorganic phosphate and either protons or Na+, respectively. The S. cerevisiae Pho89 high affinity symporter is functionally similar to other sodium transporters in mammals and Neurospora crassa (Bottger and Petersson, 2002). The similarity between Pho84 and Pho89 sequences is low (15% amino acids identity) and there are important differences between them. Pho84 has an optimal pH of 4.5 while the optimal pH for Pho89 is 9.5 (Persson et al., 2003). These transporters are under strict phosphate control and are only derepressed at low phosphate concentration. In addition to its control by phosphate starvation, expression of pho84 and pho89 increases at alkaline pH, particularly the expression of pho89 is enhanced by alkaline pH even in mutants defective in the transcriptional activator Pho4 (Lamb et al., 2001; Serrano et al., 2002). The evolutive acquisition of high affinity and low affinity transport systems appears to confer metabolic advantage to yeasts and filamentous fungi (Levy et al., 2011) as it is also well documented in bacteria (Martín and Liras, 2021a).

Transport related nutrient effectors (named transfectors) have been discovered in S. cerevisiae and in several other eukaryotic cells. These proteins serve as nutrient sensors and are also involved in the nutrient transport mechanisms. In S. cerevisiae Pho84 is a transfector that is involved in phosphate sensing and transport, and as effector rapidly activates the protein kinase A pathway in concert with glucose (Giots et al., 2003). Arsenate, is a non-metabolizable structural analogue of phosphate that inhibits growth of fungi suggesting that arsenate binds to phosphate receptors. Mutants defective in the high affinity Pho84 transporter are unsensitive to arsenate suggesting that Pho4 is involved in both phosphate sensing and transport into the fungal cells (Giots et al., 2003; Wykoff et al., 2007; Popova et al., 2010). Phosphate containing molecules that interact with the Pho84 transporter can trigger the phosphate signaling without been transported, as is the case with glycerol-3-phosphate (Popova et al., 2010). These compounds behave as phosphate agonists of Pho84 and, therefore, affect the signaling activity of phosphate. In summary, this information indicates that agonists exert their function by interacting with the Pho84 phosphate binding site but do not require a complete transport into the cell.

Remarkably, the Pho89 symporter is under the control of a calcium TRP (Jiang and Pan, 2018) providing evidence of a close interaction between calcium transport and phosphate regulation. The Pho89 phosphate transporter in yeasts is a high affinity transporter functionally similar to the Pit transporter in bacteria (Martín and Liras, 2021) and to the mammalian Na⁺ P3, and is conserved from bacteria to vertebrates (Werner and Kinne, 2001). This protein has 10–12 transmembrane domains, depending on the species, and a large hydrophilic loop between regions TMS7 and TMS8 that has been shown to be located in the cytosolic side of the cell membrane (Chien et al., 1997; Persson et al., 1999). The Pho89 protein of S. cerevisiae has a Km for Pi of between 0.4 and 0.6 μM. This transporter is active at alkaline pH but lacks affinity for the substrate at pH below 4.5 in contrast to the proton dependent Pho84 that is very active at a wide pH range (Martínez and Persson, 1998).

In S. cerevisiae the Pho89 encoding gene is governed by a Crz zinc finger type transcriptional factor that is activated by the calcium-calcineurin system; accordingly, the pho89 gene is no expressed in crz1 mutants (Serrano et al., 2002). Notably, in S. cerevisiae expression of pho89 transporter gene is controlled by Crz1 transcriptional activator of calcium metabolism genes under conditions of alkaline pH providing evidence for another interaction between phosphate metabolism and calcium homeostasis (Serra-Cardona et al., 2014, Box 1). These findings agree with recent observations in C. albicans, in which the calcium permeable stress gated CaPhm7 affects the expression of pho89 (Jiang and Pan, 2018). These results suggests that Pho89 has a clearly distinct role than Pho84 and connects phosphate transport with calcium metabolism (Secco et al., 2012a).

The mechanisms of regulation of phosphate transport and metabolism by the master transcriptional regulator Pho4 are well documented. As indicated above the activation or inactivation of the pho genes takes place by phosphorylation or dephosphorylation of the Pho4 regulator. In phosphate sufficient conditions Pho4 is phosphorylated by protein kinases and then excluded from the nucleus to the cytosol, therefore, the PHO regulon genes remain unexpressed (Secco et al., 2012a). In phosphate depleted conditions Pho4 is unphosphorylated, binds to a nuclear import receptor and is introduced in the nucleus where attach to response elements (PRE) and activates different genes of the PHO regulon, including pho84 and pho89 (Mouillon and Persson, 2006; Tomar and Sinha, 2014). The phosphorylation of Pho4 is exerted by the cyclin-dependent kinase (CDK) complex Pho80-Pho85. This CDK complex phosphorylates serine/threonine residues in different sites of the Pho4 transcriptional factor and this causes distinct cellular localization (Komeili, 1999; Secco et al., 2012a; Secco et al., 2012b). The activity of the cyclin-dependent kinase complex CDK is controlled by Pho81, which is a CDK inhibitor. Under phosphate limiting condition the cyclin-dependent protein kinase complex is inhibited, resulting in a unphosphorylated active Pho4 and thus the genes of the PHO regulon are activated; vice versa, under phosphate sufficiency conditions the Pho81 activity is null and the cyclin-dependent protein kinase is active, resulting in a phosphorylated Pho4 that remains in the cytosol, and therefore the PHO regulon genes are not expressed (Schneider et al., 1994; O´Neill et al., 1996) (Figure 1).

In addition to phosphorylation by the CDK complex the action of Pho4 is affected by trans-acting factor; they include the cooperation with the Pho2 homeodomain (Ogawa et al., 2000; Zhou and O´Shea, 2011). According to these authors Pho2 bind to a sequence distant 15 bp from the Pho4 binding site. When attached to this position Pho2 exert an activation of Pho4 binding to its target DNA. Furthermore, the interaction of Pho4 with its target sequence is controlled by the nucleosome in that DNA region. In S. cerevisiae nucleosome depleted regions there is a competition with the Cbf1 factor that recognize the same DNA sequence as Pho4, the relative abundance in the nucleus of these two proteins, Pho4 and ScBf1, determines the occupancy of the Pho4 binding site in the DNA and therefore the expression of the PHO regulon genes (Zhou and O´Shea, 2011). Detailed studies of the genes encoding enzymes of the PHO regulon have been performed only in a few model filamentous fungi, for example N. crassa (Tomar and Sinha, 2014). The core of the PHO regulon enzymes is conserved in the studied yeasts and most filamentous fungi but there are significant differences among them. The designation of functionally similar genes is sometimes different, e.g. the core PHO regulon genes in N. crassa are named nuc-1, preg, pgov and nuc-2 that correspond to the equivalent pho4, pho80, pho85 and pho81 genes of S. cerevisiae, respectively (Peleg et al., 1996; Gras et al., 2007; Tomar and Sinha, 2014). Although the core components of the S. cerevisiae PHO regulon are conserved in N. crassa it is unclear whether additional components occur in other filamentous fungi.

Calcium is taken up by the cells using different P-type membrane calcium translocases and calcium ATPase pumps (reviewed by Espeso, 2016). Excessive accumulation of calcium in the cells creates a calcium stress problem since high intracellular concentration of calcium interfers with nucleotide, polyphosphate and other macromolecules function. To avoid the calcium toxicity fungi and other eukaryotic cells have a system for calcium tolerance/sensitivity, the neuronal-calcium sensor (NCS). Transient increase in calcium causes activation of various Ca²⁺ binding proteins, including the neuronal calcium sensor-1 (NCS-1). Members of the NCS family contain an N-terminal myristoylation domain and four Ca²⁺ binding EF-hand domains (Burgoyne et al., 2004; Burgoyne, 2007). The NCSs are small proteins evolutively conserved across the species, and homologue proteins have been identified in yeast, filamentous fungi and mammals among other living beings (Sánchez-Gracia et al., 2010; Tamuli et al., 2011) In S. cerevisiae the NCS-1 protein is essential for growth and it is involved in the regulation of the activity and cellular location of the phosphatidyl-inositol-4-kinase (Huttner et al., 2003; Strahl et al., 2007). A knock-out mutant of N. crassa NCS-1 shows increased sensitivity to high calcium concentration and UV light (Tamuli et al., 2011) suggesting that NCS-1 is involved in calcium tolerance in this fungus. Similar increase sensitivity to calcium concentration has been observed in a NCS mutant of Magnaporthe grisea (Saitoh et al., 2003); however, the NCS null mutant of A. fumigatus does not show increased sensitivity to calcium concentration, indicating that the behaviour of NCS in A. fumigatus is different from that of other fungi (Mota et al., 2008). The NCS-1 homologous of A. fumigatus (NCS-A) is not essential for growth but plays an important role in sterols distribution in membrane domains and polar growth (Mota et al., 2008). The mechanism of NCS-mediated stress tolerance has been studied in N. crassa (Gohain and Tamuli, 2019). Increasing calcium levels enhances expression of the NCS-1 encoding gene. Importantly, these authors observed that the expression of the NCS-1 encoding gene is controlled by the calcineurin responsive zinc finger Crz1 transcriptional regulator (see below) indicating that there is a clear regulation of the NCS-1 protein by the calmodulin-calcineurin signaling pathway.

Intracellular calcium binds to calmodulin, a protein encoded by the cmd1 gene (Cyert, 2001). Calmodulin is a small protein, 17 kDa, that is highly conserved in all eukaryotic organisms, and binds Ca²⁺ ions with high affinity (Kd 10⁻⁶ to 10⁻⁵ M). In S. cerevisiae it contains four amino acid motives, named EF-hands for calcium binding, although only three of the EF-hands in the calmodulin molecule are functional, the fourth lacks the Ca²⁺ binding loop (Kraus and Heitman, 2003). These motives consist in 12 amino acid loops that connect alpha helices and contain glutamic acid and aspartic acid residues that chelate the Ca²⁺ ion (Davis et al., 1986; Cyert, 2001).

Calmodulin, usually activated by Ca²⁺ acts by at least three different mechanisms: 1) interacting directly with proteins involved in the biosynthesis of bioactive metabolites, differentiation and pathogenicity, 2) Activating the calcium-calmodulin complex a class of protein kinases named calcium/calmodulin-dependent protein kinases, and 3) binding to the protein calcineurin what triggers the calmodulin-calcineurin cascade that regulates numerous genes in the cell (Figure 1).

Calmodulin interacts directly with many proteins in fungal cells. In the calcium-dependent processes calmodulin binds calcium through the EF hands what results in a change of calmodulin configuration that modulates its interaction with other proteins.

The direct interaction of calmodulin with secondary metabolites synthesizing enzymes has been studied in the insect pathogen Beauveria bassiana; this fungus produces several secondary metabolites, including compounds derived from the phenylpropanoid pathway and non-ribosomal peptides among others. The phenylpropanoid pathway starts from phenylalanine that is converted to cinnamic acid by the phenylalanine ammonia lyase (PAL) or a related tyrosine ammonia lyase (TAL) that forms coumaric acid (Figure 2A) (Martín and Liras, 2022a). Calmodulin interacts directly with the PAL enzyme of B. bassiana and inhibits its activity (Kim et al., 2015). Other interesting example is the formation of beauvericin, a product of B. bassiana with antimicrobial, antiviral and insecticidal activities. One structural component of beauvericin is 2-hydroxyisovaleric acid that is formed from 2-ketoisovaleric acid by the 2-ketoisovaleric acid reductase. Then a 325 kDa non-ribosomal peptide synthetase (NRPS) forms beauvericin (Figure 2B). Calmodulin binds the 2-ketoisovalerate reductase in vivo and in vitro and inhibits the biosynthesis of this precursor and of the final product, beauvericin (Kim et al., 2016). Salt stress and light inhibit the biosynthesis of 2-ketoisovalerate and beauvericin and this inhibition is reversed by calmodulin inhibitors indicating that the effect of these stressing factors is mediated by the interaction of calmodulin with the 2-ketoisovalerate reductase. In addition, calmodulin interact directly with the beauvericin synthetase. Calmodulin binding sequences have been found in the PAL, the 2-ketoisovalerate reductase and the beauvericin synthetase of B. bassiana (Figure 2) and similar sequences occur in many calmodulin binding proteins (Kim and Sung, 2018). The calmodulin binding domain consist in stretches of 16–35 amino acids that form a basic amphipathic α-helix (O´Day, 2003). The beauvericin synthetase contains a calmodulin-binding domain in the C-terminal region; binding of calmodulin to this region was confirmed by experiments using a synthetic binding domain and the formation of a complex was demonstrated by non-denaturing polyacrylamide gel electrophoresis (Kim and Sung, 2018). Formation of this complex takes place in presence of calcium ions but not in their absence. The direct interaction of calmodulin with enzymes involved in the biosynthesis of bioactive metabolites is of great interest although much more research needs to be done to advance in this field.

Very small increases in cytosolic calcium content triggers the rapid formation of several protein kinases, including the calcium/calmodulin-dependent protein kinases (named CamKs). These enzymes are serine/threonine protein kinases that are involved in distinct mechanisms of control of calcium homeostasis in eukaryotic cells. Most of these calcium/calmodulin dependent protein kinases have been characterized in mammals but there is little information of this group of proteins in filamentous fungi (Tamuli et al., 2011). Studies on the structure of these proteins indicate that they contain an amino terminal protein kinase domain linked to an autoinhibitory domain that overlaps with the calmodulin-binding domain (Hook and Means, 2001). These enzymes show an elaborated mode of action: the autoinhibitory domain binds to the catalytic centre and maintains the protein kinase in an inactive state until calcium and calmodulin bind to the calmodulin binding site and, therefore, activate the protein kinase activity. These calcium/calmodulin protein kinases differ in their range of substrate specificity (Tamuli et al., 2011). Protein kinases of this class were found in S. cerevisiae, Schizosaccharomyces pombe (Melcher and Thorner, 1996; Rasmussen, 2000) and also in several filamentous fungi (Table 1). In N. crassa two different CamKs protein kinases have been identified that regulate the circadian clock (Yang et al., 2001) and sexual development of ascospores (Tamuli et al., 2011). Recently, three calmodulin-dependent protein kinases have been studied in Alternaria alternata, a fungus that causes pears infection. Gene disruption studies indicated that mutation of one of the kinases gene does not affect the overall growth but reduces hyphal differentiation, sporulation and melanin formation (Jiang et al., 2023). Expression of the three kinases is intense during the infection process; in addition inhibition of CamKs activity supress the infection process indicating that these kinases play key roles in pathogenicity. Some of these enzymes are also important in the development of invasion structures in pathogenic fungi. In C. gloeosporioides an inhibition of the CamKs delays spore germination and formation of the appressorium. Treatment of C. gloeosporioides with inhibitors of these protein kinases reduces also the formation of melanin associated with the penetration of the appressorium during infection of the hosts (Kim et al., 1998). The available information suggests that there are different roles played by these calcium/calmodulin protein kinases in fungi. In summary, phosphorylation of proteins by these calcium calmodulin-dependent protein kinases provides a novel example of interaction between calcium homeostasis and phosphate regulation (Box 1).

Fungi sense different nutrients in the medium and transduce the signal by cascades that produce adequate responses in the cell, some of them mediated by the calmodulin-calcineurin pathway (Martín et al., 2019). In contrast with the little information available on regulation of fungal metabolites by direct interaction of calmodulin with the antibiotic synthetases, the molecular mechanism of calcineurin-mediated regulation has received much attention in several fungi. Calcineurin is a heterodimeric protein phosphatase that consist in a catalytic subunit CnA and a regulatory subunit CnB. The catalytic subunit CnA contains the phosphatase active center and an associate regulatory region which are separated by a small amino acid stretch. This regulatory region serves to link together the CnA and CnB subunits and, in addition, contains domains for interaction with calmodulin and for self-inhibition (Klee et al., 1998). Calcineurin is the target of the immunosuppressants cyclosporin and FK506 (tachrolimus) and these compounds have been extensively used in research to block the activity of calcineurin in vivo. The calcineurin in S. cerevisiae regulates expression of numerous genes through the transcriptional factor Crz1 (calcineurin responsive zinc finger). For example, it regulates cell wall biosynthesis, ion homeostasis, vesicles traffic and protein degradation (Yoshimoto et al., 2002).

The calmodulin-calcineurin signaling pathway in filamentous fungi and yeasts is triggered by external stressing factors that are recognized at the membrane level by G protein couple receptors (GPCR) (Krishnan et al., 2012; Martín et al., 2019). Some phospholipases, particularly phospholipase C and the secretory phospholipase La2 (SPLa2) exert important roles in calcium homeostasis (Barman and Tamuli, 2015). In mammals, the GPCRs activate the membrane phospholipase C that forms inositol-1,4,5 triphosphate (IP3, so-called calcium releasing factor); this factor interacts with specific receptors resulting in cytosolic accumulation of Ca²⁺. Interaction of IP3 with the IP3 receptor (IP3R) changes the configuration of this receptor opening calcium channels (Prole and Taylor, 2011, 2019; Taylor and Machaca, 2019); as a result, calcium is released from the endoplasmic reticulum and is redistributed to the cytosol and different organelles. The IP3 receptors of mammals are phosphorylated by the cAMP-dependent protein kinase A (Taylor, 2017). This phosphorylation of IP3R is another example of the interaction of phosphate modification of proteins in calcium homeostasis. Phosphorylation of the IP3 receptors in humans enhance the IP3-mediated calcium release (Betzenhauser et al., 2010; Taylor, 2017) (Box 1). The IP3 receptor contain an ATP-binding site and it has been shown that ATP regulates IP3-mediated calcium release. In addition to ATP other adenine nucleotides and also GTP appears to exert a regulatory function on IP3R affinity, although the selectivity of the nucleotide binding is still unclear.

In fungi calcineurin works through regulation of different transcriptional factors, of which the best known is Crz1 in S. cerevisiae, named CrzA in some filamentous fungi (Stathopoulos and Cyert, 1997). Crz1 contains a C2H2 zinc finger motif that binds to calcineurin-dependent regulatory elements (CDRE) (Fedotova et al., 2017; Gohain and Tamuli, 2019). The nuclear concentration of CrzA changes in a pulsatile mode rather than maintaining a constant level. This means that the CrzA-mediated calcineurin signaling is modulated by the frequency, duration and amplitude of the pulses (Dalal et al., 2014).

Following the identification of Crz1 in S. cerevisiae, the crzA gene was cloned from A. fumigatus and several other filamentous fungi (Soriani et al., 2008). Disruption and complementation of crzA in A. fumigatus proved that this gene is involved in cell tolerance to high calcium and manganese concentrations. In addition, the crzA mutant showed an altered expression of several genes encoding calcium transport at high calcium concentration, and decreased virulence. A detailed characterization of the role of CrzA has been made in A. nidulans using crzA defective mutants (Hagiwara et al., 2008; Spielvogel et al., 2008) demonstrating that CrzA was the regulator of calcium homeostasis. The crzA-defective mutant was highly sensitive to external calcium or manganese concentrations and to alkaline pH. This mutant was also altered in a vacuole calcium exchanger (Hagiwara et al., 2008). Spielvogel et al. (2008) observed that the crzA mutant had an aberrant morphology related to decreased expression of the chitin synthetase gene. Interestingly, GFP fluorescent protein labelled CrzA was located in the cytosol when the cells were cultured in low calcium concentration but upon calcium addition was rapidly internalized into the nucleus and therefore exerts its regulation by binding to CDRE sequences (Figure 1). The CrzA gene of Aspergillus parasiticus is required for the production of aflatoxin under calcium-stress conditions (Chang, 2008) and in Fusarium oxysporum CrzA is needed for the production of deoxynivalenol (DON) and for virulence (Chen L. et al., 2019) (see Section 3.7).

Several authors have studied the effect of alkaline pH or high calcium concentration on the control of the calmodulin-calcineurin cascade. It is well known that in S. cerevisiae alkaline pH triggers a rapid uptake of calcium and the calcium increment in the cytosol triggers the calcineurin mediated cascade (Serrano et al., 2002; Yoshimoto et al., 2002; Viladevall et al., 2004).

The role of Aspergillus oryzae calcineurin in response to stress has been studied in detail by Juvvadi et al. (2003). In A. oryzae sequences for binding stress response regulatory elements (STRE) and for heat stress factor binding have been found in the upstream region of the cnA gene of A. oryzae, encoding the calcineurin catalytic subunit CnA. The calcineurin activity in the wild type A. oryzae increased in response to alkaline pH, salt concentration, and high temperatures. Blocking the cnA mRNA by expression of the antisense RNA decreases the activity of the calcineurin under stressing conditions and caused reduced growth. In contrast overexpression of cnA resulted in an increased calcineurin activity under stressing conditions and produces tolerance to the calcineurin inhibitor FK506. These results support the conclusion that adaptation to different stressing factors in this fungus is mediated by the calcineurin signaling pathway.

There are many examples of the effect of stressing factors and alkaline pH on the production of antimicrobial fungal products. One of them is the formation of the cysteine rich antifungal protein AFP that is produced by several species of Aspergillus and Penicillium. The AFP protein is of interest because it shows antifungal activity against pathogenic yeasts and filamentous fungi (Huber et al., 2018).

The AFP protein is a small protein (6.2 kDa in P. chrysogenum) with potent antifungal activity toward Fusarium and Aspergillus species (Theis et al., 2005), some of which are plant pathogens. Therefore, it has been suggested to use AFP in the protection of vegetables such as tomato, grape and rice plant infections by Fusarium oxysporum and Botrytis cinerea . Expression of the AFP encoding gene is upregulated at alkaline pH (Meyer and Stahl, 2002a; Mayre et al., 2002b) and is abolished by the calcineurin inhibitor FK506 suggesting that transcription of the encoding gene is regulated by the calmodulin/calcineurin cascade (Meyer et al., 2005). This hypothesis is supported by the finding in the upstream region of the AFP encoding gene of a CDRE sequence that is the binding site for the CrzA transcriptional regulator. The mode of action of the AFP protein in relation to calcium metabolism has been elucidated in N. crassa. The AFP protein was shown to increase significantly the intracellular calcium concentration of N. crassa resting cells. The response of calcium metabolism to the addition of AFP change by chelating the extracellular calcium with EDTA, or other calcium specific chelators; calcium chelation counteract the AFP perturbation of calcium homeostasis indicating that calcium ions are involved and required for the AFP toxicity effect. The calcium transport blocker diltiazem exerts the same effect on calcium homeostasis as the addition of AFP (Binder et al., 2010). The addition of both AFP and diltiazen exert an cumulative effect on calcium homeostasis. In summary, in N. crassa the effect of AFP is due to the transport of calcium into the cells or its release from internal stores into the cytosol, where it triggers the regulation of calcium homeostasis, although the molecular mechanism by which AFP affects the calcium sensing and/or transport is not yet clear.

The infection and virulence of several animal and plant pathogenic fungi is regulated by calcium. Early studies on calcineurin mediated regulation in the pathogenic filamentous fungi M. grisea and B. cinerea indicated that calcineurin controls the formation of invasion structures. The development of these structures and the infection in plants is inhibited by cyclosporin (Viaud et al., 2002, 2003; Schumacher et al., 2008). In these infections, calcium regulates growth rate, morphology, differentiation of the filamentous fungus and attachment and penetration in the plant (Gupta et al., 2022). This is a very complex phenomenon that requires many enzymes, and bioactive metabolites that play important roles in intercellular communications (Table 2), however, in only a few fungal infections there is clear evidence of the involvement of calcineurin regulation in secondary metabolites production. Here we describe a few of the more representative examples. A. parasiticus produces several toxins, including aflatoxin. Mutants of two strains deficient in crzA, grown in media supplemented with calcium produce very low amounts of aflatoxin and O-methylsterigmatocystin. Transcriptional studies of the aflatoxin biosynthetic genes revealed that three of them, nor1, ver1 and omtA were very poorly transcribed in a crzA defective mutant indicating that expression of the aflatoxin genes was under the control of the calcium/calcineurin signaling pathway in calcium stressing conditions (Chang, 2008).

An important plant pathogen is Fusarium graminearum that produces the Fusarium head blight disease in wheat and other cereals (Chen et al., 2019). This fungus synthesizes several trichothecenes which are highly toxic and carcinogenic to humans and other mammals. The trichothecenes include the toxins deoxynivalenol (DON), nivalenol, and zearalenone (Goswami and Kistler, 2004; Pestka and Smolinski, 2005). DON and its acetylated derivatives three acetyl deoxynivalenol and 15 acetyl deoxynivalenol are highly toxic compounds.

Recently, Chen et al. (2019a, b) studied the role of Crz1 of F. graminearum and observed that in addition to the effect on growth and differentiation, the Crz1 mediated regulation reduces drastically the trichothecenes formation. Importantly, expression studies of the F. graminearum crz1 mutant compared to the parental strain revealed that twelve trichothecene try genes involved in the biosynthesis of DON have significant reduced expression (Chen et al., 2019). These results connect previous studies on regulation of DON biosynthesis (Hu et al., 2014; Yu et al., 2014; Zheng et al., 2016) with the calcium-calcineurin mediated control of this toxin (Imazaki et al., 2010).

Melanin is a dark pigment that has important roles in protecting the fungal cells against UV irradiation and in the resistance of different microorganisms to antimicrobial agents (Nosanchuk and Casadevall., 2006). In Verticillium dahliae, that causes the Verticillium wilt in trees, the crzA gene is involved in the biosynthesis of microconidia melanin which is important for the pathogenesis of this fungus (Xiong et al., 2015). Similar observations were also made in the mutants defective in crzA of Penicillium digitatum that also forms melanized microconidia (Zhang et al., 2009). Another important fungal pathogen is the citrus pathogen Alternaria alternata that synthesizes the polyketide AK toxin. This fungus contains a PLC-phospholipase similar to the well characterized enzyme of N. crassa that forms IP3. IP3 diffuses and binds to IP3 receptors and induces release of calcium ions from the vacuoles. The phospholipase of A. alternata is involved in calcium homeostasis, pathogenicity and virulence (Tsai and Chung, 2014).

Another example of calcium-calcineurin Crz-mediated regulation of pathogenicity is the production of virulence factors by C. neoformans. This fungus is a basidiomycete that causes meningoencefalitis in humans, a severe disease with high mortality (Fu et al., 2018). During the infection C. neoformans release pigments, enzymes and several virulence factors that are secreted in vesicles, one of the major virulence factors is glucuronosyl-manan that is released forming the extracellular capsule, a pathogenicity determinant that has potent immunosuppressive activity (Rodrígues et al., 2008).

In N. crassa there are detailed studies on the effect of calcineurin on the sexual and asexual spore development (Tamuli et al., 2016) and on the formation of some secondary metabolites, e.g. carotenoids ( Barman and Tamuli, 2015 ). The N. crassa carotenoids include the neurosporene astaxanthin and some carotenoid precursors (Avalos et al., 2013) that are responsible of the orange colour of N crassa. These carotenoids prevent damage produced by UV light irradiation (Barman and Tamuli, 2015).

Calcineurin affects many reactions in the cell and therefore its activity is likely to be regulated by different cell factors. A family of these factors, named calcipressins, bind the calcineurin CnA subunit inhibiting its phosphatase activity (Juvvadi et al., 2014, 2015). In yeasts one of the Crz regulated genes encodes a calcineurin modulator, Rcn1 (RcnA in some fungi), named for regulator of calcineurin, that belongs to the calcipressins family. These are cytosolic proteins that do not enter in the nucleus and, therefore, appear to modulate the calcineurin activity by direct or indirect interaction with this phosphatase. The CrzA protein of A. fumigatus affects also expression of some genes involved in different aspects of calcium metabolism (Soriani et al., 2010). A. fumigatus rcnA null mutants are affected in the expression of several genes, including the gene for the calcineurin subunit A (Pinchai et al., 2009) and, therefore, these authors proposed that RcnA might exert a feedback regulation of the calcineurin-mediated regulatory cascade.

Members of the Rcn family are conserved in all eukaryotes from yeast to humans (Kingsbury and Cunningham, 2000). Several studies provide evidence on the role of calcipressing in fungal biology including S. cerevisiae (Mehta et al., 2009) and in the fungi A. fumigatus, A. nidulans, C. neoformans and Magnaporte oryzae (Pinchai et al., 2009; Liu et al., 2022). The exact mechanism of regulation of calcineurin by Rcn1 is complex. This protein contains a proline-serine repeated motif which seems to be phosphorylated in two different serine residues by the mitogen-activated protein kinase (MAPK). As a consequence of the phosphorylation of Rcn1 the interaction with the calcineurin subunit A is modified and calcineurin phosphatase activity is modulated; when Rcn1 is unphosphorylated the regulator exert a negative effect on calcineurin while when Rcn1 is phosphorylated the regulator does not inhibit the calcineurin phosphatase activity (Hilioti et al., 2004; Kishi et al., 2007; Li et al., 2011).

Recently the mechanism of regulators of calcineurin on the biosynthesis of secondary metabolites has been elucidated in M. oryzae , a fungus that causes the rice blight disease (Zhang et al., 2016b, 2019). During infection M. oryzae attaches to the rice plant cells and form a melanin rich appressorium that penetrates the plant tissue (Zhang et al., 2019). Deletion of the MoRCN1 gene causes a decrease in virulence and reduces the formation of several secondary metabolites. Transcriptomic analysis of this M. oryzae mutant has revealed that the Rcn1 regulator enhances expression of 491 genes and causes downregulation of 377 genes. The Rcn1 regulated genes encode enzymes involved in the biosynthesis of lysine, serine, threonine and the aromatic amino acids, and several genes involved in lipids biosynthesis and fatty acid degradation that may provide precursors for secondary metabolites, including genes for the biosynthesis of staurosporin, indolditerpenoids, meroterpenoids and aflatoxins. The affected genes encode also an ABC transporter; these transporters are frequent in many secondary metabolite gene clusters (García-Estrada et al., 2011; Abu Ammar et al., 2013; Martín, 2020).