Phosphorus constitutes a critical element essential for the survival of all living organisms, attributable to its indispensable function in energy transference, nucleic acid synthesis, and as a structural component of cellular architecture (Butusov and Jernelöv, 2013; Margalef et al., 2017). Given that organisms are capable of assimilating only dissolved phosphate, the enzymatic hydrolysis of organic phosphorus compounds is imperative to render this nutrient bioavailable (Cembella et al., 1982). Microbial entities and plant root systems secrete phosphatase enzymes, which facilitate the cleavage of phosphate moieties from organic molecular structures, thereby transforming phosphorus into bioavailable inorganic forms (Zhu et al., 2024). Among these microbial taxa, phosphate-solubilizing fungi, including both ectomycorrhizal and arbuscular mycorrhizal forms, play a pivotal role in phosphorus mineralization and solubilization processes, which collectively exert profound effects on plant growth, seed formation, crop phenological development, and resistance to phytopathogenic diseases (Etesami et al., 2021; Mehta et al., 2019).

Fungal phosphatases represent a heterogeneous assemblage of enzymes that facilitate the hydrolysis of phosphate esters, a process imperative for an array of molecular, ecological, and applied roles (de Assis et al., 2015). This group includes serine/threonine (Ser/Thr) protein phosphatases as well as acid/alkaline phosphomonoesterases, both of which are integral to signal transduction pathways, nutrient recycling, and metabolic regulation in fungi (Winkelstroter et al., 2015b). Whereas phosphatases, classified as a subgroup within esterases, specifically target phosphoric acid esters of alcohols and are systematically categorized into phosphomonoesterases, phosphodiesterases, and polyphosphatases based on the specificity of their substrates. Phosphomonoesterases, alternatively referred to as phosphatic monoester hydrolases, are further differentiated into acid phosphatases and alkaline phosphatases (ALPs), contingent upon their optimal pH activity (Bhalla et al., 2025). Predominantly, ALPs are of microbial derivation, whereas acid phosphatases (AcPases) are secreted by both plant and microbial sources (Rani et al., 2012). Fungal ectophosphatases, frequently identified as membrane-bound AcPases released by ectomycorrhizal fungi, play a critical role in the degradation of organic phosphorus compounds within the soil, facilitating their conversion into inorganic forms that are accessible for plant uptake (Freitas-Mesquita and Meyer-Fernandes, 2014; Ho and Zak, 1979). Within these symbiotic associations, ectophosphatase activity significantly augments plant nutrient absorption, particularly phosphorus, an effect often amplified by the presence of elevated nitrogen levels (Chiu and Paszkowski, 2019). Phosphatases are conventionally classified into five principal categories: alkaline phosphatases, high molecular weight acid phosphatases (HMW AcPases), low molecular weight acid phosphatases (LMW AcPases), purple acid phosphatases (PAPs), and protein phosphatases (PPs) (de Araujo et al., 1976; Li et al., 2021). Among these classifications, acid phosphatase activity is predominantly measured in environmental and biochemical research (Olczak et al., 2003). Although both AcPases and ALPs coexist within soil matrices, contemporary analytical methodologies frequently quantify only one enzyme class or substrate type at a time, predominantly emphasizing monophosphates. This focus persists despite the simultaneous hydrolysis of monoester and diester phosphates (Zheng et al., 2021).

From an applied perspective, fungal phosphatases exhibit significant potential for applications in biotechnology and industry, particularly concerning environmental remediation, biofertilizer development, and sustainable bioprocessing (Fu et al., 2024; Tian et al., 2024). Recent advancements in genetic engineering have facilitated the production of high-activity phosphatases within optimized fungal hosts, resulting in efficient biocatalysts for the treatment of wastewater, the recovery of phosphate from agricultural runoff, and the enhancement of soil fertility without the use of chemical additives (Fazeli-Nasab and Rahmani, 2021). Although many individual reviews have addressed phosphatase biochemistry, mycorrhizal phosphorus cycling, or biotechnological applications in isolation, none have provided the integrative framework necessary to translate molecular-level discoveries into tangible agronomic and environmental outcomes. This review addresses that critical deficiency by offering the first comprehensive synthesis that explicitly connects enzymatic mechanisms with ecological functions and biotechnological potential. In contrast to existing reviews that remain constrained within narrow disciplinary domains, we integrate five interrelated dimensions enzymatic heterogeneity, molecular regulation, methodological standardization, ecological roles, and translational applications that have thus far been treated in a fragmented manner in the literature. By systematically examining persistent knowledge gaps in non-model fungal phosphatases, their environmental responsiveness, the lack of standardized methodological approaches, and their underexplored biotechnological utility, this synthesis advances the conceptualization of fungal phosphatases as central leverage points for addressing global phosphorus sustainability and provides a foundational framework for future research that transcends traditional disciplinary boundaries.

A bibliometric analysis was conducted as summarized in Figure 1, utilizing specific keyword strings to explore the literature concerning fungal phosphatases. The search query composed of TITLE-ABS-KEY[(fung* OR mycorrhiz* OR “phosphate-solubilizing fungus” OR “phosphate solubilizing fungi”) AND (phosphatase* OR phytase* OR “acid phosphatase” OR “alkaline phosphatase” OR “purple acid phosphatase” OR phosphodiesterase* OR polyphosphatase* OR “protein phosphatase” OR dephosphorylat*] AND (phosphorus OR phosphate OR “P cycling” OR “phosphorus mobilization” OR “phosphate solubilization” OR “organic phosphorus” OR “phosphorus mineralization” OR “phosphate bioavailability” OR “phosphorus sustainability”) yielded 3,233 articles from the Scopus database and 2,273 articles from the Web of Science (WOS). After merging metadata from both databases, the consolidated dataset comprised 3,944 documents, reduced from an original size of 5,506 documents due to the elimination of 1,562 duplicates. The bibliometric analysis encompassed publications from 1944 to 2025 and included 1,083 distinct sources (journals, conference proceedings, and monographs), indicating a robust and sustained expansion of research activity in this area. An annual growth rate of 7.11% demonstrates a consistent increase in scholarly output and academic interest in this domain over the past 8 decades. The mean document age of 13.8 years suggests a mature yet continuously evolving field, wherein both seminal and recent contributions exert substantial influence. Scholarly impact is reflected by an average of 40.21 citations per document, evidencing considerable academic visibility and interdisciplinary connectivity across domains such as microbiology, soil science, and environmental biotechnology. The identification of 17,384 Keywords Plus and 8,585 Author Keywords indicates pronounced thematic heterogeneity and multidimensional research orientations, spanning molecular-scale investigations to applied studies, as illustrated in Figure 2. Authorship patterns further delineate the structure of the research community. A total of 11,595 authors have contributed to this body of literature, with only 137 authors responsible for single-authored documents. In absolute terms, single-authored publications number 156, representing less than 4% of the total output. These emphasize the predominance of collaborative research constellations and the centrality of co-authorship networks. The mean number of co-authors per article is 5.24, reinforcing the view that research on fungal phosphatases is intrinsically collaborative and integrative, frequently intersecting with microbiology, agronomy, ecology, and biochemistry. The international co-authorship rate of 16.24% indicates a moderate level of cross-national collaboration. This metric suggests an expanding, though not yet fully optimized, landscape of international research partnerships, likely driven by common global concerns related to phosphorus sustainability. At the same time, it highlights a substantial untapped potential for intensifying transnational networking, capacity building, and knowledge exchange. These patterns are broadly consistent with those documented in other emergent biotechnological domains that rely on interdisciplinary expertise and shared research infrastructure.

Fungal phosphatases demonstrate significant structural and functional heterogeneity, indicative of their participation in a multitude of physiological, biochemical, and ecological functions (Chen et al., 2024). These enzymes are systematically categorized according to their catalytic attributes, substrate specificity, and molecular features (Millan, 2006). Classification methodologies, as depicted in Figure 3, such as the Enzyme Commission (EC) framework and biochemical classifications, offer systems for distinguishing predominant phosphatase categories, including phosphomonoesterases, phosphodiesterases, and protein phosphatases. This heterogeneity encompasses variations in optimal pH, metal ion dependencies, molecular mass, and catalytic mechanisms, which collectively underpin enzyme function and ecological roles.

The EC classification system systematically categorizes hydrolases that act on ester bonds into distinct subgroups based on the type of bond cleaved and the chemical properties of the substrates involved (McDonald et al., 2015). Within this classification scheme, fungal phosphatases are classified among esterases that specifically hydrolyze phosphoric acid esters of alcohols, and they are allocated to three primary EC classes: phosphomonoesterases (EC 3.1.3), phosphodiesterases (EC 3.1.4), and other phosphatases/related hydrolases (Leake and Miles, 1996). Leake and Miles (Leake and Miles, 1996) provided direct evidence of extracellular phosphodiesterase production by an ericoid mycorrhizal fungus when utilizing DNA as a phosphorus source, thereby demonstrating the functional expression of EC 3.1.4 activities by fungal taxa in natural environments. Various studies on organophosphate esterases, including monoesterases (alkaline phosphatases) and triesterases in microorganisms, underscore the ecological significance and diversity of phosphatase reactions categorized under the EC 3.1. designation (Tehara and Keasling, 2003). Whereas, phosphomonoesterases (EC 3.1.3) facilitate the hydrolysis of phosphomonoesters, resulting in the production of inorganic phosphate and an alcohol (Kuznetsova et al., 2005). In fungal systems, these enzymatic activities manifest as both extracellular and intracellular enzymes and are conventionally classified as acid phosphatases and alkaline phosphatases (ALPs), distinguished by their pH optima and molecular characteristics (Leake and Miles, 1996). Empirical investigations have documented the activities of fungal acid and alkaline phosphomonoesterases: whereas they detailed that extracellular phosphomonoesterase and phosphodiesterase activities associated with DNA/phosphate utilization by mycorrhizal fungi, illustrating the contribution of fungal phosphomonoesterases to the mobilization of organic phosphorus (Leake and Miles, 1996). Extensive microbiological research on organophosphate esterases emphasizes alkaline phosphatase (a monoesterase) as a predominant enzyme studied for its role in releasing inorganic phosphate under conditions of phosphate scarcity, in alignment with EC 3.1.3 categorization (Ragot, 2016; Tehara and Keasling, 2003). The bifurcation into acid versus alkaline phosphatases is indicative of observable differences in pH optima and ecological distribution: fungi inhabiting acidic environments typically exhibit acid phosphatase activity, whereas those in more alkaline habitats may express ALPs, a trend corroborated by analyses of fungal phosphatase activity in environmental and culture-based studies (Della Monica et al., 2018). While, phosphodiesterases (EC 3.1.4) are enzymes that catalyze the hydrolysis of phosphodiester bonds within nucleic acids, phospholipids, or cyclic nucleotides (Morton, 1965). The activities of fungal phosphodiesterases have been directly observed; Leake and Miles (Leake and Miles, 1996) identified both extracellular and cell-wall-associated phosphodiesterase produced by Hymenoscyphus ericae, which facilitated growth on DNA as the exclusive phosphorus source. Further biochemical analyses of fungal phosphodiesterase fractions, such as those from Aspergillus niger, reveal heterogeneous diesterase activities with varying substrate specificities, indicating that fungal EC 3.1.4 enzymes exhibit significant biochemical diversity (Jarosch, 2016). Additionally, multiple studies have demonstrated overlapping or dual activities among some microbial phosphodiesterases, challenging strict EC classification but highlighting the true diversity of these enzymes (Garner et al., 2024). Such dual functionality has been documented both in non-fungal microorganisms and within fungal systems, suggesting that fungal phosphodiesterases may similarly exhibit broad substrate specificity and play crucial roles in the recycling of nucleic acid-derived phosphorus (Tehara and Keasling, 2003). In addition, fungi exhibit a range of phosphatase activities and associated hydrolases that are integral to phosphate metabolism and their ecological adaptability. Furthermore, multifunctional hydrolases that integrate diesterase, phosphatase, and nucleotidase functions are also present. Recent advancements in the identification of microbial enzymes in bacteria highlight the biochemical diversity of these enzymes and suggest the presence of similar capabilities or functional analogs in fungal genomes (Freeman et al., 2025). Studies on fungal pathogens have shown that disruption of polyphosphate utilization affects phosphate homeostasis within cells and influences virulence, thereby underscoring the biological significance of polyphosphatase activity in fungi (Zyla et al., 1995). Additionally, enzymes capable of hydrolyzing organophosphates, such as triesterases and phosphotriesterases, are being explored within microbial systems for applications in environmental detoxification and nutrient recycling (Tehara and Keasling, 2003).

The research delineates the biochemical classification of fungal phosphatases, focusing on ALPs, AcPases, and PAPs, with distinctions made according to molecular weight. ALPs are classified as phosphomonoesterases that exhibit optimal catalytic function at alkaline pH levels, facilitating the release of inorganic phosphate from an extensive array of monoester substrates (Saavedra and Baltar, 2025). The biochemical identification and characterization of fungal ALP activities have been documented through both environmental and laboratory investigations. For instance, the study by Della Monica et al. (2018), highlights the presence of alkaline phosphatase activity across various fungal strains and observes that these alkaline activities can coexist with acid phosphatase activities, albeit frequently exhibiting lower activity compared to acid phosphatases under numerous tested conditions, this due to differences in optimal pH ranges, substrate specificity, and regulatory control mechanisms as ALPs are most active under alkaline conditions, which are less common in many natural soils and rhizosphere environments, whereas AcPases function efficiently under the acidic conditions typical of most soils. Furthermore, the biochemical profiling of ectophosphatase activities in Candida albicans reveals an alkaline, Cu²⁺ dependent ectophosphatase, which is stimulated by Mg²⁺, setting it apart from an acidic, Cu²⁺ independent activity, thereby providing functional evidence for distinct ALP-like activities at the fungal cell surface (Freitas-Mesquita et al., 2025). The co-regulation by divalent metal ions and responsiveness to classical phosphatase inhibitors are consistent biochemical characteristics of these fungal alkaline activities, as noted in targeted ectophosphatase characterizations (Freitas-Mesquita et al., 2025). Additionally, alkaline phytases, which are a functionally related group, hydrolyze phytate most effectively at higher pH levels and have been biochemically differentiated from histidine-acid phytases in comparative analyses (Oh et al., 2004). While acid phosphatases (AcPases) are a class of phosphomonoesterases that exhibit optimal activity under acidic pH conditions and are ubiquitously present among fungi, playing a crucial role in the mineralization of organic phosphorus within acidic environments (Oliveira et al., 2025). Empirical evidence indicates that the activity of fungal acid phosphatases frequently surpasses that of alkaline phosphatases across a wide array of fungal species and environmental conditions. This pattern is corroborated by the findings of Della Monica et al. (2018), who reported consistently higher acid phosphatase activity compared to alkaline activity across various pH treatments and time points in culture assays. Cell-wall-associated acid phosphatases, such as those found in Aspergillus fumigatus (PhoAp), have been subject to molecular and biochemical characterization (Freitas-Mesquita and Meyer-Fernandes, 2014). PhoAp is identified as a glycosylphosphatidylinositol-anchored glycoprotein with a molecular weight of approximately 80 kDa, demonstrating enzymatic activity on both phosphate monoesters and diesters and subject to repression by phosphate availability, thereby illustrating typical regulatory and biochemical characteristics of fungal AcPases (Bernard et al., 2002). Historical and ecological investigations of ericoid mycorrhizal fungi similarly highlight the role of extracellular acid phosphatases in mobilizing organic phosphorus sources to support fungal growth (Lemoine et al., 1992). Studies focusing on ectophosphatases have concluded that acid phosphatase activities generally exhibit insensitivity to certain divalent metal ions, such as Mg²⁺, which typically enhance alkaline phosphatase activities, thereby further distinguishing these biochemical classes (Freitas-Mesquita et al., 2025). Moreover, purple acid phosphatases (PAPs) are a category of metallophosphatases, distinguished by a distinctive purple hue that results from their binuclear metal center, typically comprising iron-zinc or iron-manganese complexes (Schenk et al., 2008). These enzymes have been extensively characterized in both plant and bacterial systems. Current research is increasingly identifying fungal sequences related to acid phosphatase families, inclusive of PAPs, through comprehensive sequence curation and phylogenetic analyses. A recent curated phylogenetic study focusing on fungal acid phosphatase and phytase sequences has delineated several distinct clades, which encompass groups corresponding to classical acid phosphatases, phytases, and sequences annotated as PAPs (Gontia-Mishra and Tiwari, 2013). This suggests that fungal genomes encode enzymes homologous to PAPs and that these enzymes represent a discernible biochemical and phylogenetic subset within the broader category of fungal acid phosphatases (Gomez-Gallego et al., 2025). Investigations centered on plant PAPs have established specific sequence motifs and catalytic characteristics that facilitate the identification of PAP homologues in non-plant genomes, thereby substantiating the detection of fungal PAPs through bioinformatic profiling (Schenk et al., 2000).

Serine/threonine protein phosphatases represent several conserved families, including the PPP family members such as PP2A, PP2B (calcineurin), and PP1, as well as PPM/PP2C family members, which are also detected in fungi, as depicted in Figure 4 (Chen et al., 2016; Yang and Arrizabalaga, 2017). These phosphatases play pivotal roles in processes such as signal transduction, stress response, morphogenesis, and virulence (Ortiz-Urquiza and Keyhani, 2015). Calcineurin, also known as protein phosphatase 2B (PP2B), serves as a prototypical example of a fungal Ser/Thr PP, it functions as a heterodimer, consisting of a catalytic A subunit and a regulatory B subunit, and is activated by calcium and calmodulin, with its sequence and quaternary structure being conserved across eukaryotic organisms (Fox and Heitman, 2005; Rusnak and Mertz, 2000). Genetic and biochemical disruptions of calcineurin lead to impairments in thermotolerance, hyphal growth, and virulence in a range of fungal pathogens, including Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus, this underscores the crucial role of calcineurin in fungal physiology and pathogenicity (Bader et al., 2003; Fox et al., 2001; Odom et al., 1997). For example, mutants lacking the calcineurin A and B subunits fail to grow at host temperatures and exhibit a virulence in C. neoformans, highlighting its indispensability for infection-related attributes (Odom et al., 1997; Rasmussen et al., 1994). Furthermore, loss or pharmacological inhibition of calcineurin in Candida species increases cellular sensitivity to antifungal agents and diminishes virulence in animal models (Brown et al., 2013; Yu et al., 2014). Beyond calcineurin, other Ser/Thr phosphatases regulate nutrient-sensing and developmental programs. TOR pathway–regulated PP2A and PP2C phosphatases modulate responses to nutrient availability and carbon source, and their activities change under rapamycin or nutrient deprivation, linking Ser/Thr dephosphorylation to metabolic and developmental control (Brown et al., 2013). In Aspergillus and other filamentous fungi, multiple Ser/Thr phosphatases contribute to germination, hydrolytic enzyme production, and primary metabolism; mutations in these phosphatases alter growth on cellulose or affect secretion of hydrolytic enzymes, underscoring their biochemical integration with fungal ecology and biotechnology-relevant processes (de Assis et al., 2015). Protein tyrosine phosphatases, encompassing both classical and low-molecular-weight PTPs, are integral components of fungal genomes, where they have been associated with specialized regulatory functions, notably in secondary metabolism and developmental processes (Yu and Zhang, 2018). Filamentous fungi are known to encode a diverse array of PTP family members, including both dual-specificity and classical PTPs (Arino et al., 2011). Comparative genomic analyses, such as those conducted in Aspergillus fumigatus, have identified a substantial repertoire of PTP genes, underscoring the significance of tyrosine dephosphorylation as a crucial facet of the fungal phosphatase repertoire (Winkelstroter et al., 2015a). Empirical data indicate that the inhibition of tyrosine phosphatase activity can modulate secondary metabolite synthesis, exemplified by the impact of PTP inhibitors on aflatoxin production in Aspergillus flavus, thereby implicating PTPs in the regulation of biosynthetic pathways with virulence-associated or ecological relevance (Inoguchi et al., 2019). Dual-specificity phosphatases (DUSPs), often referred to as MAP kinase phosphatases (MKPs) in the context of their action on mitogen-activated protein kinases (MAPKs), are enzymes that catalyze the removal of phosphate groups from both phosphoserine/threonine and phosphotyrosine residues (Jeffrey et al., 2007). These enzymes serve as critical regulators of MAPK-mediated signaling pathways in fungi. DUSPs are characterized by a conserved domain architecture featuring family-specific interaction motifs that facilitate substrate recognition and enable regulatory coupling to stress response mechanisms (Huang and Tan, 2012). For instance, yeast proteins such as Sdp1 demonstrate the role of MKPs in linking oxidative or other types of stress to selective inactivation of MAPKs (González-Rubio et al., 2019). In fungal systems, MKPs are instrumental in modulating MAPK pathways responsible for maintaining cell wall integrity, osmotic balance, and developmental processes (Gonzalez-Rubio et al., 2019). Genomic and functional analyses have identified fungal DUSPs that exert influence over these pathways, thus establishing a connection between dual-specificity dephosphorylation processes and adaptive responses as well as pathogenic characteristics (Gonzalez-Rubio et al., 2019).

Fungal protein phosphatases exhibit a remarkable structural diversity that is fundamental to their catalytic mechanisms and regulatory capabilities. Calcineurin (PP2B), a notable example, contains a dinuclear metal center at its active site and necessitates calcium ions and calmodulin for its activation. The conserved architecture of calcineurin accounts for its susceptibility to immunosuppressive complexes, such as FK506–FKBP12 or cyclosporin A (CsA) cyclophilin, which inhibit phosphatase activity by stabilizing inhibitory ternary complexes (Fox and Heitman, 2005; Rusnak and Mertz, 2000). This structural foundation has been leveraged to design fungal-selective calcineurin inhibitors, specifically modified FK506/FK520 analogs, which minimize immunosuppression while maintaining antifungal effectiveness. This demonstrates the potential of detailed structural insights into phosphatase-inhibitor interactions to facilitate the development of targeted therapeutic strategies (Hoy et al., 2022; Rivera et al., 2023). Ser/Thr phosphatase families such as PP2A, PP1, and PP2C exhibit variations in their catalytic metal requirements, regulatory subunits, and structural configurations. Specifically, PP2C phosphatases, classified as type 2C, belong to the PPM enzyme family and are characterized by their single-polypeptide structure and reliance on metal cofactors. These enzymes achieve substrate specificity not through stable regulatory subunits but by expressing multiple isoforms (Arino et al., 2011). Furthermore, low molecular weight protein tyrosine phosphatases and dual-specificity phosphatases are identified by distinct active-site motifs and substrate binding domains, which determine their affinity for phosphotyrosine, phosphoserine, and/or phosphothreonine residues. Structural and biochemical analyses across diverse eukaryotic systems have provided foundational models for elucidating the enzymatic mechanisms of fungal phosphatases (Gonzalez-Rubio et al., 2019; Lee et al., 2020). The interplay between conserved catalytic cores and adaptable regulatory modules accounts for the diverse cellular localizations, such as cytosolic, cell-wall/ectophosphatases, and membrane-associated and specialized functional roles observed in fungal species (Freitas-Mesquita et al., 2025).

The availability of phosphate serves as a predominant transcriptional signal that regulates phosphatase expression within eukaryotic organisms (Azevedo and Saiardi, 2017). Comparative genomic analyses have revealed the conservation of PHO/PHR-like components in both fungi and their symbiotic counterparts. Genomic assessments of arbuscular mycorrhizal fungi have reported the preservation of PHO signaling components, in conjunction with the TOR, cAMP-PKA, and SNF1 pathways (Zhou et al., 2021). This suggests the existence of a phosphate-responsive regulatory module capable of modulating phosphatase gene expression in AM fungi, and by extension, other fungal taxa (Zhou et al., 2021). Studies on phosphorus starvation in plants and fungi indicate that the genes encoding purple acid phosphatase and acid phosphatase are transcriptionally activated under conditions of Pi limitation, thereby demonstrating a conserved Pi-responsive regulatory mechanism of phosphatase expression across biological kingdoms (Huong To et al., 2022; Hurley et al., 2010). Furthermore, sequence-based analyses of fungal acid phosphatases have identified Pi-repressible enzymes, such as PhoAp in Aspergillus fumigatus, that are inhibited by the presence of phosphate and activated during Pi deprivation, offering direct fungal instances of PHO-like regulation of phosphatase genes (Bernard et al., 2002). While the mediation of PHO/PHR responses by specific transcription factors has been well-documented, in plants, regulators belonging to the PHR family, such as PHR2, have been shown to directly regulate PAP gene expression under conditions of Pi deprivation (Huong To et al., 2022). This mechanism is paralleled by orthologous systems, wherein transcription factors upregulate Pi-scavenging enzymes as part of the eukaryotic phosphate starvation responses (Wang et al., 2003). While the transcriptional machinery involved in PHO responses, particularly Pho4 in Saccharomyces, is thoroughly characterized, comparative studies in arbuscular mycorrhizal fungi and filamentous fungi reveal conserved sets of nutrient-responsive regulators. These regulators are postulated to perform analogous functions in the regulation of phosphatase gene expression (Zhou et al., 2021). As an empirical example, the phosphate-repressible transcriptional regulation of PhoAp in Aspergillus fumigatus illustrates the transcription factor-mediated regulation of fungal acid phosphatase genes under Pi-deprivation conditions (Bernard et al., 2002).

Protein phosphatases are intricately regulated through phosphorylation networks and interactions with kinase signaling pathways. Nutrient signaling via the Target of Rapamycin (TOR) pathway exerts control over the PP2A/PP2C family of phosphatases. Specifically, the inhibition of TOR, achieved through agents such as rapamycin or nutrient deprivation, modulates the interaction of regulatory factors, such as Tap42, with PP2A-type phosphatases. This modulation results in the swift activation or altered targeting of PP2A and PP2A-like enzymes, thereby linking nutrient availability to phosphatase activity and subsequent transcriptional responses (Cutler et al., 2001; Wang et al., 2003). Dual-specificity phosphatases (MKPs) are upregulated in response to stress, functioning to dephosphorylate Mitogen-Activated Protein Kinases (MAPKs). In the yeast model, the stress-inducible MKP Sdp1 mediates the downregulation of Slt2 MAPK phosphorylation following heat or oxidative stress, illustrating the role of phosphatase induction and activity in terminating kinase signaling during adaptive responses (Hahn and Thiele, 2002).

Extracellular and cell wall-associated fungal phosphatases frequently necessitate processing through the secretory pathway and subsequent glycosylation to achieve appropriate localization and enzymatic activity. Specifically, the Aspergillus fumigatus cell wall acid phosphatase, PhoAp, is characterized as a glycoprotein possessing a glycosylphosphatidylinositol (GPI) anchor. The glycosylation and processing of this anchor are pivotal in determining its association with the cell wall or its release into the culture filtrate, a process essential to its function as an ectophosphatase involved in mobilizing organic phosphorus (Bernard et al., 2002). More generally, phosphatidic acid phosphatases and secreted acid phosphatases in eukaryotic organisms undergo post-translational modifications that significantly impact their stability, secretion, and extracellular functionality. This is consistent with the observed reliance of extracellular phosphatase activity on processes of maturation and trafficking (Gomez-Gallego et al., 2025; Hurley et al., 2010). The assembly of multimeric complexes involving regulatory subunits and specific protein-protein interfaces constitutes the primary mechanism by which highly conserved catalytic phosphatase cores achieve substrate specificity, precise localization, and regulated activity. Type-1 phosphatases (PP1) rely on an array of regulatory subunits that dock through conserved motifs such as RVXF to direct the catalytic subunit PP1c to specific substrates and cellular compartments (Gibbons et al., 2005). Similarly, fungal PP1 systems, including the Glc7 and PPZ families, exhibit specialized regulatory partners across different species, with structural variations, such as the N-terminal extensions observed in PPZ, contributing to distinct regulatory mechanisms (Casamayor et al., 2022). Phosphatases of the PP2A family are organized into heterotrimeric holoenzymes, wherein B-type regulatory subunits, including the B56/PR61 classes, dictate substrate selection and biological outcomes. In fungi and fungal pathogens, the PP2A regulatory subunit B56 (MoB56) and the atypical catalytic subunit Ppg1 form complexes that are crucial for growth and pathogenicity in Magnaporthe oryzae, highlighting the biological significance of specific regulatory subunit-catalytic subunit pairings (Shomin-Levi and Yarden, 2017; Wang et al., 2003). Proteins of the Tap42 (yeast)/α4 family associate with PP2A/PP4/PP6-related catalytic subunits, thereby linking nutrient/TOR signaling to phosphatase regulation. These Tap42–phosphatase interactions are essential for numerous TOR-regulated developmental responses and Sit4/PP2A-type functions in fungi (Cutler et al., 2001; Wang et al., 2003). Collectively, these findings underscore that assembly with regulatory subunits, along with modulators such as Hal3/Cab3 involved in PPZ regulation, and the presence of multifunctional regulatory proteins, are fundamental to the specificity and regulation of fungal phosphatases (Casamayor et al., 2022).

Phosphatases are integral components and effectors within conserved nutrient and stress signaling pathways. The TOR signaling pathway directly modulates the activity of protein PP2A and PP2C, thereby influencing cellular decisions regarding growth, autophagy, and differentiation in response to nutrient availability. The activation of PP2A after TOR inhibition is instrumental in mediating extensive metabolic reprogramming (Cutler et al., 2001; Wang et al., 2003). Calcineurin (PP2B), a calcium and calmodulin-activated serine/threonine phosphatase, is essential for thermotolerance, ion homeostasis, hyphal growth, and pathogenicity in various fungal species, effectively converting Ca²⁺ signals into appropriate stress-adaptive responses (Bader et al., 2003; Odom et al., 1997; Rusnak and Mertz, 2000). Dual-specificity phosphatases modulate MAPK pathways that respond to osmotic, cell wall, and oxidative stressors by terminating MAPK activation to restore homeostasis (Hahn and Thiele, 2002). These instances underscore the pivotal role of phosphatases in sensing nutrient and stress conditions and orchestrating suitable cellular responses. The regulation of phosphatases is intricately connected with the metabolic pathways of carbon, nitrogen, and lipids. In the context of filamentous fungi, Serine/Threonine phosphatases are crucial in modulating germination and primary metabolic processes in response to carbon sources, such as the involvement of PP2A/PP4 in carbon catabolite repression/carbon catabolite derepression (CCR/CCDR). Recent research highlights the role of PP4 complexes in governing carbon catabolite repression mechanisms in the fungus Magnaporthe oryzae, thereby illustrating the direct association between phosphatase activity and carbon utilization pathways (Huang et al., 2025). Furthermore, PP2A regulatory modules, such as B56, interact with kinases like NDR kinases, including COT1, to regulate polar growth, branching, and development, effectively linking phosphatase function with both morphogenetic and metabolic states (Shomin-Levi and Yarden, 2017). Within symbiotic and rhizosphere environments, fungal phosphatases collaborate with host transporters and microbial communities to facilitate the mobilization of organic phosphorus, thus integrating phosphatase activity into ecosystem-scale nutrient cycling (Cao et al., 2022; Ren et al., 2018). Additionally, the mobilization of polyphosphate and the action of polyphosphatases impact phosphate homeostasis and the expression of virulence phenotypes in pathogenic organisms, further exemplifying the complex metabolic interactions involved (Ahmed et al., 2022).

The complexity of fungus phosphatase regulating mechanisms relates to the combination of the external phosphate availability, global nutrient conditions, cellular metabolic conditions, and various stress inputs, each of which is signaled by partially different but overlapping cells of molecular circuits (Martin, 2023). Two main regulatory centers in these include the phosphate-responsive phosphatase regulator (involving the PHO regulator) and the nutrient-sensing TOR in the regulation of phosphatase gene transcription and enzyme action under phosphate scarcity (Rouached et al., 2010; Zhou et al., 2021). Comparative genomic studies of arbuscular mycorrhizal fungi show that the common phosphate starvation signaling components are conserved with TOR, cAMP-PKA, and SNF1 pathways, along with the view that a generalized signaling architecture exists that interacts phosphate starvation signals with the cellular energy and nutritional status of the cell (Zhou et al., 2021). In this allosteric mechanism, the dissociation of the Tap42 adaptor protein of PP2A and PP2C phosphatases is stimulated by TOR inactivation, which occurs, among other stimuli, in response to amino-acid or nitrogen deprivation (Santhanam et al., 2004). Whereas the resulting actin release and activation of these phosphatases promotes PHO regulon induction, acting by dephosphorylating and hence activating transcription factors, including Pho4, or functional equivalents PHR orthologs (Lamarche et al., 2008). Furthermore, the TOR Tap42 -PP2A axis is described as a molecular coincidence detector and consolidates signaling of convergent nutrient starvation and, therefore, phosphatase gene expression not only robustly reacts to a combination of nutrient stress and phosphate limitation (Beck and Hall, 1999; Bonneau et al., 2013). Such dual-input control logic will probably be especially useful in oligotrophic settings, in which co-limitation by phosphate and nitrogen occurs. In addition to being involved in nutrient sensing, environmental stresses such as osmotic perturbation, oxidative stress, and pathogen challenge activate mitogen-activated protein kinase (MAPK) cascades that cross with PHO and TOR signaling through common phosphatase effectors (Mohammadi et al., 2021; Zhang et al., 2021). Appropriate examples of these nodes are dual-specificity phosphatases (DUSPs) and MAPK phosphatases (MKPs), such as the enzymes Sdp1 and Ptc1 in yeast, and these are the points that integrate stress-derived with nutrient-derived inputs (Gonzalez-Rubio et al., 2019). While HOG1 MAPK is hyperphosphorylated and inactivated by a combination of dephosphorylation by Ptc1, one of the type 2C phosphatases, PTP2C, and the partially overlapping protein-tyrosine phosphatases, PTP2 and PTP3 (Tatebayashi and Saito, 2023). It is significant to note that the same PP2C phosphatases, which are triggered on TOR inhibition when nutrient-deprived cells are exposed to nutrient deprivation, also catalyze the end of MAPK signaling when cells are exposed to acute stress; therefore, the nutrient-limited conditions can precondition cells in a functional linkage where cells can respond to stress forcefully or abnormally in response to nutrient deprivation. In filamentous and hyaline fungi like Aspergillus and Magnaporthe, the same principle of integrating rise and fall signals is demonstrated by PP2A -PP4 phosphatase complexes to give rise to both carbon catabolite repression and osmotic stress responses (Anjago et al., 2018; Franck et al., 2015). Such observations confirm a paradigm that phosphatase assemblies can be considered multivalent information-processing centers and not simplistic and single input effectors. This regulatory network is further extended by the cAMP-protein kinase A (PKA) and SNF1/AMPK interactive pathways linked to glucose and general availability of carbon and cellular energy depletion respectively, respectively, and instruct the activity of phosphatases to participating metabolic choices (Nicastro, 2015). In the context of phosphate homeostasis particularly, cAMP- PKA signaling governs secretion of hydrolytic enzymes such as extracellular phosphatases during the condition of carbon starvation resulting in the linkage of carbon status to phosphate uptake (Perez-Diaz et al., 2023; Steyfkens et al., 2018). Simultaneously, SNF1-regulated phosphorylation cascades adjust the activity status of catabolic pathways, such as that of the mineralization of organic phosphorus (Nicastro, 2015). A joint response of cAMP-PKA, SNF1, and TOR in a common group of regulatory phosphatases-primarily PP2A, PP2C, and their proximate effectors- indicate that fungus cells utilize a hierarchical state of decision-making processes where diverse nutrient (phosphate, nitrogen, carbon, energy) and stress signals are collectively integrated to produce more coordinated physiological responses.

The initial focus is on the role of Protein Tyrosine Phosphatase (PTC) Genes in the Mitogen-Activated Protein Kinase (MAPK) signaling pathways within Saccharomyces cerevisiae. An Overview of the Four MAPK Pathways in Yeast reveals that Saccharomyces cerevisiae encompasses four unique MAPK signaling pathways, each mediating cellular responses to specific environmental cues: the Pheromone Response Pathway (Fus3/Kss1 MAPKs), which orchestrates the mating response to peptide pheromones; the Filamentous Growth Pathway (Kss1 MAPK), which is activated under conditions of nutrient scarcity and promotes invasive growth; the High Osmolarity Glycerol (HOG) Pathway (Hog1 MAPK), which is triggered by osmotic stress to maintain cellular homeostasis; and the Cell Wall Integrity (CWI) Pathway (Slt2/Mpk1 MAPK), which is essential for responding to cell wall perturbations and stress. Each of these pathways adheres to a conserved three-tier kinase module: MAPKKK → MAPKK → MAPK. However, the precise modulation of these pathways by phosphatases is paramount to avert hyperactivation and subsequent cellular dysfunction (González-Rubio et al., 2023; Tatjer et al., 2016). In the context of identifying and classifying PTC Genes within the KEGG MAPK Pathway, analysis has identified many genes, as depicted in (Table 1), which function as phosphatases involved in the regulation of MAPK pathways:

The comprehensive functional characterization of PTC/phosphatase genes in Saccharomyces cerevisiae highlights their essential regulatory roles within the mitogen-activated protein kinase signaling cascades, as illustrated in (Figure 5). Among these, YDL006W (PTC1) is classified as a Type 2C protein phosphatase, belonging to the magnesium-dependent serine/threonine phosphatase family (PPM family, PP2C) (Warmka et al., 2001). This enzyme plays a pivotal role in the MAPK signaling network, primarily targeting Hog1 MAPK within the High Osmolarity Glycerol (HOG) pathway, which mediates osmotic stress responses. Moreover, Ptc1 also acts on Slt2 MAPK in the Cell Wall Integrity (CWI) pathway (Tatjer et al., 2016), and has broader roles in SNF1/AMPK-mediated glucose regulation (Ruiz et al., 2013). Mechanistically, Ptc1 catalyzes the dephosphorylation of both activating tyrosine and threonine residues on Hog1, thereby terminating the osmotic stress response. In the absence of Ptc1, Hog1 remains hyperphosphorylated, leading to excessive MAPK signaling, disruption of the G2/M cell cycle checkpoint, impaired mitochondrial inheritance, and defective daughter cell separation under heat stress (Gonzalez-Rubio et al., 2023; Warmka et al., 2001). Furthermore, YER075C (PTP3) encodes a tyrosine-protein phosphatase that functions as a phosphotyrosine-specific classical PTP. Its primary target is also Hog1 MAPK within the HOG pathway, where it acts redundantly with PTP2 (YOR208W) to mediate Hog1 inactivation. PTP3 localizes to the cytoplasm and functions as part of a dual-component negative regulatory system with PTP2, dephosphorylating the activating tyrosine residues of Hog1 to maintain the kinase in a hypophosphorylated, inactive state under normal conditions. Additionally, both PTP2 and PTP3 are transcriptionally induced under osmotic stress, ensuring the timely deactivation of the HOG pathway (Jacoby et al., 1997). YOR208W (PTP2), a phosphotyrosine-specific classical phosphatase, also targets Hog1 MAPK as its primary substrate and plays a secondary role in calcium signaling through co-regulation with Msg5. Unlike PTP3, PTP2 exhibits both nuclear and cytoplasmic localization, allowing it to fine-tune Hog1 activity in multiple cellular compartments. It dephosphorylates the activating tyrosine residues of Hog1, thereby restricting maximal kinase activation during osmotic stress (Jacoby et al., 1997). In contrast, YNL053W (MSG5) is a dual-specificity phosphatase capable of dephosphorylating both tyrosine and serine/threonine residues. MSG5 primarily targets Fus3 MAPK in the pheromone response pathway and Slt2 MAPK in the CWI pathway (Gonzalez-Rubio et al., 2019). Moreover, it contributes to the maintenance of low basal signaling through the CWI pathway and exists in two isoforms with distinct regulatory dynamics. Mechanistically, MSG5 acts as a rapid feedback inhibitor—deactivating Fus3 following pheromone stimulation and preventing excessive Slt2 activation through dephosphorylation of both activating residues. While YMR036C (MIH1), a CDC25-like phosphatase, is a phosphotyrosine-specific enzyme primarily targeting Cdc28p, the cyclin-dependent kinase that governs mitotic and meiotic progression. While not directly part of the MAPK cascade, MIH1 coordinates cell cycle transitions with MAPK-regulated stress responses. It dephosphorylates the inhibitory Tyr-15 residue on Cdc28p, thereby promoting the G2/M transition and ensuring synchronization between cell cycle progression and stress signaling pathways. Finally, YIL113W (SDP1) encodes a stress-inducible dual-specificity phosphatase with broad substrate specificity across multiple MAPK cascades. SDP1 expression is transcriptionally upregulated under various stress conditions, providing a rapid, inducible negative feedback mechanism to prevent prolonged MAPK activation. Additionally, it complements the constitutively expressed phosphatases such as Ptc1, Msg5, and PTP2/3, ensuring dynamic and stress-responsive regulation of MAPK activity.

Additionally, in Saccharomyces cerevisiae, phosphatase genes play a pivotal role in mRNA surveillance pathways, notably through their involvement in the nonsense-mediated decay (NMD) process. This pathway functions as an essential quality control mechanism, systematically eliminating aberrant mRNAs that harbor premature termination codons (PTCs) to prevent the synthesis of truncated proteins that could be deleterious. Furthermore, NMD exerts regulatory influence over approximately 10% of normal endogenous transcripts, thereby facilitating the fine-tuning of gene expression (Chang et al., 2007; Lejeune, 2022). Through KEGG pathway analysis, several phosphatase genes have been identified as integral to mRNA surveillance. These include TPD3, PPH21, PPH22, GLC7, CDC55, SSU72, RTS1, and PPQ1 as presented in (Table 2), each of which contributes distinct regulatory functions within the pathway.

Whereas, as illustrated in (Figure 6), the gene YAL016W (TPD3) encodes the regulatory subunit A of the heterotrimeric protein phosphatase 2A (PP2A) complex, which functions as the structural scaffold that facilitates the interactions among the catalytic subunits, specifically Pph21 and Pph22, and the regulatory subunits, Cdc55 and Rts1. TPD3 is integral to the regulation of translation and protein biosynthesis, exerting an indirect influence on nonsense-mediated mRNA decay (NMD) by preserving translational fidelity (Schuller et al., 2004). Also, the genes YDL134C (PPH21) and its paralog YDL188C (PPH22) encode the catalytic subunits of protein phosphatase 2A (PP2A). PPH21 plays a crucial role in the modulation of mRNA stability, the regulation of cell cycle checkpoints, and the translation process under stress conditions. In contrast, PPH22, despite being functionally redundant with PPH21, is specifically activated under DNA replication stress conditions, thereby establishing a connection between genomic stability and mRNA quality control mechanisms. Moreover, both catalytic subunits are essential for the orchestration of the dephosphorylation of checkpoint proteins, which are critical for the synchronization of cell cycle progression with mRNA metabolic activities (Haluska et al., 2021; Shen et al., 2025). Furthermore, the gene YER133W, also known as GLC7, encodes a Type 1 serine/threonine phosphatase that plays a critical role in the regulation of translation termination factors. This regulatory function is essential for the accurate recognition of nonsense codons and the subsequent activation of nonsense-mediated mRNA decay. Additionally, Glc7 exhibits interactions with cleavage and polyadenylation factors, thereby establishing a linkage between transcription termination and mRNA surveillance mechanisms (Guan et al., 2006). In a parallel manner, the gene YGL190C, referred to as CDC55, acts as a regulatory subunit B of PP2A. CDC55 specifically directs the activity of PP2A towards substrates involved in translation and RNA metabolism, thereby orchestrating the synchronization of cell cycle progression with mRNA quality control checkpoints and modulating the components of the Mitotic Exit Network (Baro et al., 2018; Philip et al., 2022). The gene YNL222W, also known as SSU72, encodes a C-terminal domain (CTD) phosphatase that is instrumental in the processing of mRNA 3′-end and transcription termination. This enzyme achieves its function by dephosphorylating the CTD of RNA Polymerase II, specifically targeting the Tyr-1 residues and possibly the Ser-5 residues. This activity serves as a nexus between transcriptional regulation and mRNA surveillance (Ganem et al., 2003). In parallel, YOR014W, denoted as RTS1, acts as an alternative regulatory B′ of protein phosphatase 2A (PP2A). RTS1 confers distinct substrate specificity compared to CDC55, facilitating the organization of septins during cytokinesis and contributing to DNA damage response mechanisms. This action further links genome integrity maintenance with mRNA quality control (Aouida et al., 2019). Additionally, the gene YPL179W, identified as PPQ1, encodes a phosphatase related to protein phosphatase 1 (PP1). PPQ1 is postulated to function within protein quality control pathways that are complementary to the activity of Glc7, thereby aiding in the integrated regulation of both mRNA surveillance and proteostasis.

Fungal phosphatases constitute key biochemical mediators at the interface of intracellular signal transduction, nutrient metabolism, and the global biogeochemical cycling of phosphorus. The fungal phosphatase repertoire is highly diverse and encompasses phosphomonoesterases (including acid and alkaline phosphatases, purple acid phosphatases, and phytases), phosphodiesterases, polyphosphatases, as well as protein dephosphorylating enzymes such as serine/threonine- and tyrosine-specific phosphatases. Comparative genomic and phylogenetic investigations reveal pronounced sequence divergence and lineage-specific gene family expansions among fungal clades, indicative of substantial structural and regulatory heterogeneity that underlies fungal ecological plasticity and phosphorus acquisition strategies. Fungi occupy a central position in the biotransformation of both organic and inorganic phosphorus forms. By secreting extracellular phosphomonoesterases and phosphodiesterases, fungal communities mineralize a broad spectrum of organic phosphorus substrates, including phytate, nucleic acids, and phospholipids, thereby liberating orthophosphate for subsequent assimilation by fungi and plants. Enzymes with high substrate specificity, particularly phytases, play a pivotal role in phytate depolymerization, with direct consequences for soil phosphorus availability, the development of biofertilizers, and the efficiency of phosphorus utilization in animal nutrition. In parallel, numerous fungi facilitate the mobilization of mineral-associated phosphate pools (e.g., calcium-, iron-, and aluminium-bound phosphates) through the secretion of organic acids and the modulation of rhizosphere pH, processes that collectively enhance soil productivity and nutrient bioavailability. The expression and catalytic activity of fungal phosphatases are stringently controlled by interconnected signaling and regulatory networks, including the target of rapamycin, calcineurin, and mitogen-activated protein kinase pathways, together with carbon nutrient-sensing systems. These regulatory circuits integrate phosphatase function with cellular energy status, nutrient supply, and abiotic and biotic stress responses, thereby enabling fungi to dynamically optimize phosphorus acquisition under temporally and spatially variable environmental conditions.

Recent advances in analytical methodologies such as refined colorimetric and fluorometric assays, zymography, mass spectrometry-based proteomics, and high-resolution microscopy have substantially improved both the quantitative and spatial characterization of phosphatase activity. These technologies facilitate the discrimination of individual isozymes, elucidate their localization and function in situ, and enhance the reproducibility and comparability of experimental data across studies. From ecological and agronomic perspectives, mycorrhizal and saprotrophic fungi represent major drivers of terrestrial phosphorus cycling. A substantial body of evidence indicates that phosphate-solubilizing and phytase-secreting fungi can increase phosphorus use efficiency, promote seedling establishment, and augment crop yields. Nevertheless, the performance of fungal inoculants in field conditions is highly context-dependent, being strongly influenced by soil physicochemical properties, plant genotype, resident microbiomes, and climatic factors. This variability underscores the necessity of site-specific validation and optimization across distinct edaphic and agroclimatic settings. When appropriately integrated into management practices, enhanced fungal-mediated phosphorus mobilization may permit reductions in inorganic phosphorus fertilizer inputs by approximately 20%–40% in certain production systems, while concurrently mitigating phosphorus losses via runoff and reducing the risk of eutrophication. Furthermore, extensive fungal hyphal networks contribute to the long-term stability and functioning of ecosystems by promoting soil aggregate formation, stabilizing soil organic matter, and facilitating carbon sequestration.

Despite substantial progress, several critical knowledge gaps persist that constrain the reliable, scalable, and field-relevant application of fungal phosphatases. Future research should prioritize: (i) multi-location and multi-crop field trials across a broad spectrum of soil types (pH 4.5–8.5) to translate laboratory-scale efficacy into quantifiable agronomic outcomes and to realistically evaluate the potential for synthetic fertilizer substitution; (ii) integrated multi-omics investigations (transcriptomics, proteomics, and metabolomics), coupled with quantitative measurements of phosphatase activity and plant nutrient uptake, to elucidate mechanistic links between molecular regulatory networks and phosphorus mobilization under environmental conditions; (iii) comparative genomics combined with functional validation, including CRISPR-based engineering of phosphatase-producing fungal strains, to concomitantly enhance multiple phosphatase systems and introduce broad-spectrum stress-tolerance traits; and (iv) international harmonization of phosphatase assay methodologies through standardized protocols, inter-laboratory proficiency testing, and the development of field-portable diagnostic platforms, thereby enabling regulatory implementation and robust cross-study comparability. Addressing these research priorities will be crucial for transitioning fungal phosphatase research from predominantly mechanistic insights toward dependable, scalable, and sustainable phosphorus management strategies in agricultural systems.