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Review

Timing Is Everything: The Fungal Circadian Clock as a Master Regulator of Stress Response and Pathogenesis

by
Victor Coca-Ruiz
1,* and
Daniel Boy-Ruiz
2
1
Institute for Mediterranean and Subtropical Horticulture “La Mayora” (IHSM), CSIC-UMA, Campus de Teatinos, Avda. Louis Pasteur, 49, 29010 Málaga, Spain
2
Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, 11510 Puerto Real, Spain
*
Author to whom correspondence should be addressed.
Stresses 2025, 5(3), 47; https://doi.org/10.3390/stresses5030047 (registering DOI)
Submission received: 2 July 2025 / Revised: 22 July 2025 / Accepted: 29 July 2025 / Published: 1 August 2025
(This article belongs to the Collection Feature Papers in Plant and Photoautotrophic Stresses)

Abstract

Fungi, from saprophytes to pathogens, face predictable daily fluctuations in light, temperature, humidity, and nutrient availability. To cope, they have evolved an internal circadian clock that confers a major adaptive advantage. This review critically synthesizes current knowledge on the molecular architecture and physiological relevance of fungal circadian systems, moving beyond the canonical Neurospora crassa model to explore the broader phylogenetic diversity of timekeeping mechanisms. We examine the core transcription-translation feedback loop (TTFL) centered on the FREQUENCY/WHITE COLLAR (FRQ/WCC) system and contrast it with divergent and non-canonical oscillators, including the metabolic rhythms of yeasts and the universally conserved peroxiredoxin (PRX) oxidation cycles. A central theme is the clock’s role in gating cellular defenses against oxidative, osmotic, and nutritional stress, enabling fungi to anticipate and withstand environmental insults through proactive regulation. We provide a detailed analysis of chrono-pathogenesis, where the circadian control of virulence factors aligns fungal attacks with windows of host vulnerability, with a focus on experimental evidence from pathogens like Botrytis cinerea, Fusarium oxysporum, and Magnaporthe oryzae. The review explores the downstream pathways—including transcriptional cascades, post-translational modifications, and epigenetic regulation—that translate temporal signals into physiological outputs such as developmental rhythms in conidiation and hyphal branching. Finally, we highlight critical knowledge gaps, particularly in understudied phyla like Basidiomycota, and discuss future research directions. This includes the exploration of novel clock architectures and the emerging, though speculative, hypothesis of “chrono-therapeutics”—interventions designed to disrupt fungal clocks—as a forward-looking concept for managing fungal infections.

1. Introduction: The Fungal Clock as a Proactive Survival Mechanism

Endogenous circadian clocks, the internal timekeeping mechanisms found in nearly all domains of life, represent one of biology’s most elegant solutions to a fundamental challenge: life on a rotating planet [1]. These oscillators generate self-sustaining rhythms with a period of approximately 24 h, allowing organisms to synchronize their internal physiology with the predictable daily cycles of the external world [2]. However, the true adaptive value of the circadian clock lies not in passive timekeeping but in its capacity to anticipate change. By generating an internal representation of the 24 h day, the clock enables organisms to prepare for forthcoming environmental opportunities and threats, such as the rising sun, falling temperatures, or the activity patterns of hosts and competitors [3]. This proactive scheduling of biological processes, from metabolism to behavior, confers a significant fitness advantage over organisms that can only react to environmental stimuli after they occur [4].
The traditional view of stress response is often framed as a reactive process: a stimulus, such as high temperature or UV radiation, is detected and triggers a corresponding defensive pathway [5]. The circadian clock introduces a fundamentally different paradigm—one of proactive defense. Instead of waiting for cellular damage to occur and then initiating a costly repair process, a clock-equipped organism can pre-emptively upregulate protective mechanisms just before the stressor is expected to arrive. For example, it can synthesize antioxidant enzymes before dawn to counter the oxidative burst caused by morning light or accumulate compatible solutes to prepare for the desiccation that accompanies midday heat. This anticipatory strategy is far more efficient, conserving energy and minimizing the cellular damage that would otherwise accumulate.
This review will explore how fungi utilize this anticipatory capability to adapt to their environments. The fungal kingdom is characterized by extraordinary diversity in lifestyle and habitat, but a common thread is the constant exposure to a multitude of environmental stressors. For a filamentous fungus growing on a plant surface, the daily cycle presents a predictable series of stressors. Dawn brings intense light and ultraviolet (UV) radiation, which can cause direct DNA damage and generate harmful reactive oxygen species (ROS) [6]. The rising temperature throughout the day increases the risk of thermal damage and desiccation, imposing severe osmotic stress [7]. Concurrently, nutrient availability may fluctuate, and the fungus must compete with other microbes for limited resources [8]. For pathogenic fungi, the challenge is compounded by the host’s own rhythms. The host immune system is not static; its defensive capacity fluctuates over the 24 h cycle, creating windows of vulnerability that a pathogen can exploit [9]. From saprophytes decomposing organic matter to pathogens invading a living host, fungi must navigate these rhythmic abiotic and biotic pressures to survive, grow, and reproduce [10].
This review synthesizes the rapidly accumulating evidence positioning the fungal circadian clock as a central regulatory hub that integrates diverse environmental signals with the internal metabolic state to orchestrate a coherent, anticipatory survival strategy. We will move beyond the canonical Neurospora crassa model to critically synthesize knowledge across a broad phylogenetic spectrum, addressing the acknowledged taxonomic bias in current research and highlighting major knowledge gaps [11,12]. The following sections will examine the diverse molecular architectures of fungal clocks, the output pathways that translate time into action, and the clock’s role in ecological and pathogenic interactions and will conclude with a discussion of critical gaps and future directions that could leverage the fungal clock as a novel target for disease control. As summarized in Figure 1, the fungal circadian clock acts as a central hub that integrates key environmental and host signals to regulate fundamental biological outputs, including metabolism, development, stress response, and virulence. This model provides a conceptual framework for how the clock’s timing function enhances fungal resilience and adaptation.

2. The Molecular Architecture of Fungal Timekeeping: Conservation and Divergence

The molecular machinery that generates circadian rhythmicity in fungi is a testament to both deep evolutionary conservation and remarkable niche-specific adaptation. The transcription-translation feedback loop (TTFL) first elucidated in the filamentous fungus Neurospora crassa has served as a powerful paradigm, yet studies in other fungi, and even in convergent fungus-like eukaryotes such as oomycetes, reveal a fascinating diversity of timekeeping strategies [13].

2.1. The Neurospora crassa Paradigm: The FRQ-WCC Oscillator

For decades, the bread mold Neurospora crassa has been the preeminent model for dissecting the eukaryotic circadian clock, revealing a mechanism centered on a negative feedback loop [14,15]. This canonical system, often referred to as the FREQUENCY/WHITE COLLAR (FRQ/WCC) oscillator, is composed of distinct positive and negative elements.
Positive Arm: The primary positive-acting component is the White Collar Complex (WCC), a heterodimer of two GATA-type zinc-finger transcription factors, WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) [16]. WC-1 contains a Light-Oxygen-Voltage (LOV) domain, which functions as a blue-light photoreceptor, directly linking the clock to its most dominant environmental cue [17]. The WCC binds to specific promoter elements (Clock boxes or C-boxes) in its target genes to activate their transcription.
Negative Arm: The core of the oscillator’s negative feedback is that the WCC drives the transcription of its own inhibitor, the frequency (frq) gene. The FRQ protein, upon translation in the cytoplasm, forms a complex with the FRQ-interacting RNA helicase (FRH) and casein kinases [18]. This complex then enters the nucleus and physically interacts with the WCC, repressing its transcriptional activity and thereby shutting down its own expression.
Pacing the Clock: The ~24 h periodicity of the clock is not determined by a simple on/off switch but by a crucial, regulated time delay. FRQ undergoes progressive, time-dependent phosphorylation at over 100 sites by several kinases, including Casein Kinase 1 (CK1) and Casein Kinase 2 (CK2) [19,20]. This series of phosphorylation events governs its stability, its nuclear localization, and its ability to inhibit the WCC. Once FRQ becomes hyperphosphorylated, it is targeted for ubiquitination and subsequent proteasomal degradation, which releases the WCC from inhibition and allows a new cycle of frq transcription to begin [21,22]. This elegant, phosphorylation-based time delay is the key to generating a robust, near-24 h rhythm. Recent work has added another layer of complexity, showing that FRQ, an intrinsically disordered protein, can undergo liquid-liquid phase separation (LLPS), forming condensates that may modulate kinase activity and contribute to temperature compensation [23].

2.2. Beyond the Paradigm: Clock Diversity Across the Fungal Kingdom

The FRQ-WCC model, while foundational, is not universal. The presence, absence, or modification of this system across different fungal lineages provides profound clues about their distinct ecological strategies and evolutionary histories. The architecture of a fungus’s clock appears to be a direct reflection of the most critical rhythmic variable in its environment.
Filamentous Ascomycetes: The FRQ-WCC system is well-conserved among many filamentous ascomycetes, particularly plant pathogens. In Botrytis cinerea, a functional clock with homologs of frq, wc-1, and wc-2 is essential for regulating virulence [24]. Similarly, Fusarium oxysporum possesses multiple frq homologs and a WCC that are indispensable for its pathogenicity [25]. In the nematode-trapping fungus Arthrobotrys oligospora, the blue-light receptor CryA, a cryptochrome often associated with circadian systems, regulates developmental processes like conidiation and trap formation in response to light, though a full clock mechanism has not been elucidated [26]. In contrast, Aspergillus flavus exhibits a clear circadian rhythm in development but lacks an frq ortholog, suggesting a non-canonical, FRQ-less oscillator [27]. Penicillium claviforme displays a non-circadian rhythm inducible by light and, remarkably, by static magnetic fields [28].
Basidiomycota and Early-Diverging Fungi: These phyla remain largely uncharacterized in fungal chronobiology. While genomic surveys identify putative clock gene homologs, functional data is sparse [29,30]. Nonetheless, physiological evidence exists, such as the nocturnal discharge of basidiospores in Pellicularia filamentosa [31] and the clock-controlled bioluminescence in the mushroom Neonothopanus gardneri [32]. In early-diverging Mucoromycota, the dung fungus Pilobolus displays a classic circadian rhythm in sporangium discharge, suggesting ancient origins for fungal timekeeping [33].
Non-Canonical Clock Mechanisms in Yeast: The budding yeast Saccharomyces cerevisiae lacks obvious homologs of frq and wcc [34]. Instead, it exhibits robust, temperature-compensated metabolic oscillations, known as Yeast Respiratory Oscillations (YROs), which are thought to be rooted in metabolic feedback loops rather than a dedicated TTFL [35]. The fission yeast Schizosaccharomyces pombe presents another alternative, with an ultradian clock operating on a 30-minute period, driven by the interplay of major cellular signaling pathways [36,37].
Emerging Clocks in Symbionts: The presence of the complete FRQ-WCC gene set in arbuscular mycorrhizal fungi (AMF), such as Rhizoglomus irregulare, is particularly telling [38,39]. As an obligate symbiont in the dark soil environment, its clock is likely entrained not by light but by rhythmic metabolic signals from its host plant, coordinating their mutualistic exchange [40].
Recent reviews emphasize that clocks can be “pervasive, and often times evasive,” meaning they may only reveal rhythmicity under specific, ecologically relevant conditions not always replicated in the laboratory. This suggests that the absence of an observable rhythm is not evidence of its absence [41].

2.3. Entrainment: Synchronizing with the External World

For an internal clock to be useful, it must be synchronized, or entrained, to the external 24 h day. Fungi use several environmental cues, known as zeitgebers, to achieve this.
Light: As the most reliable environmental signal, light is the dominant entrainment cue for most surface-dwelling fungi. In the Neurospora model, the WCC’s function as a direct blue-light photoreceptor provides an elegant mechanism for entrainment. A pulse of light rapidly induces frq transcription, and the effect on the clock’s phase depends on when the pulse is received, effectively aligning the internal rhythm with the external light cycle [3].
Temperature: Temperature is another critical zeitgeber. Fungal clocks exhibit temperature compensation, a hallmark property meaning that the period of the rhythm remains relatively constant across a range of physiological temperatures [42]. This prevents the clock from running at different speeds on warm versus cool days. However, the clock is still sensitive to changes in temperature, which can entrain the rhythm, a feature likely crucial for fungi where light cues are weak or absent [43].
Table 1 provides a comparative overview of the core clock machinery, highlighting the conserved paradigm and key evolutionary divergences that reflect niche-specific adaptations.

3. Beyond Transcription: Non-Transcriptional and Metabolic Oscillators

While the TTFL has been the focus of chronobiology for decades, a growing body of evidence points to the existence and fundamental importance of non-transcriptional oscillators (NTOs), which can function independently of gene feedback loops. This is particularly relevant for fungi like yeasts but also for understanding the ancestral origins of timekeeping across all eukaryotes, including oomycetes and slime molds [13].
The Yeast Respiratory Oscillator (YRO): A TTFL-Independent Metabolic Clock: As noted, Saccharomyces cerevisiae lacks FRQ/WCC homologs but exhibits robust metabolic oscillations (YROs) in continuous culture [44]. The entire metabolome and a large portion of the transcriptome oscillate, with clear temporal separation between reductive/biosynthetic phases and oxidative/respiratory phases [45]. This system is thought to be driven by feedback loops within core metabolic pathways, making it a prime example of a non-transcriptional, metabolic clock well-suited to its nutrient-driven lifestyle [44].
Peroxiredoxin Rhythms: A Universal and Ancestral Timekeeping Marker? The discovery of transcription-independent circadian rhythms in the oxidation state of peroxiredoxin (PRX) proteins has provided a potentially unifying concept [46]. These highly conserved antioxidant proteins show ~24 h rhythms in their hyperoxidation state (PRX-SO2/3) across all domains of life, including eukaryotes like fungi (Neurospora), plants, and animals, as well as bacteria and archaea [47]. Critically, these PRX rhythms persist even in mutants lacking core TTFL genes (e.g., Neurospora without frq), demonstrating they are generated by an NTO [46]. This has led to a “Divergence” model for clock evolution, which posits that an ancestral, post-translational oscillator (PTO) based on metabolic/redox cycles existed first, and that TTFLs evolved later in different lineages as specialized modules that coupled to this ancient core [48]. This model helps explain how Aspergillus can have a clock without frq and how Neurospora can maintain redox rhythms without its TTFL. The clock is thus likely a layered system, with an ancient metabolic core and lineage-specific transcriptional modules coupled to it.
Comparative Insights from Other Eukaryotes: The principles of NTOs are not confined to fungi. Oomycetes, such as the plant pathogen Phytophthora infestans, are fungus-like but belong to a different eukaryotic supergroup (Stramenopila). While their life cycles are regulated by environmental cues like light and temperature, and they possess genes with PAS domains (often found in photoreceptors and clock proteins), a canonical circadian clock has not been definitively characterized [13]. Similarly, slime molds (Dictyostelium discoideum), which are amoebozoans, exhibit ultradian (shorter than 24 h) oscillations in cAMP signaling that drive their collective behavior and development, but these are not considered true circadian rhythms [49,50]. The existence of these diverse timing mechanisms in related eukaryotic microbes highlights the evolutionary plasticity of biological oscillators and suggests that comparative studies across these groups could reveal conserved principles of non-transcriptional timekeeping.

4. Circadian Gating of Cellular Defense: Anticipatory Regulation of Stress Responses

One of the most critical functions of the fungal circadian clock is the temporal regulation of stress response pathways. Rather than maintaining a constant state of high alert, which is metabolically expensive, the clock “gates” the expression and activity of defensive systems, deploying them only when they are most likely to be needed. This represents a profound optimization of cellular economy. This anticipatory defense is a key contributor to fungal fitness. The molecular underpinnings of circadian gating in stress responses are illustrated in Figure 2.

4.1. Anticipating Oxidative Threats

Exposure to sunlight and metabolic activity inevitably generates reactive oxygen species (ROS), which can damage vital cellular components [51]. The circadian clock plays a central role in managing this threat. In Neurospora, cellular ROS levels themselves have been shown to oscillate in a circadian manner [52]. This rhythm is actively managed by the clock through the timed expression of key antioxidant enzymes. The gene encoding catalase-1 (cat-1), an enzyme that neutralizes H2O2, is a well-characterized clock-controlled gene (ccg) whose transcript levels peak in anticipation of dawn, preparing the cell to detoxify the ROS that will be generated by light exposure [14].

4.2. Managing Osmotic and Desiccation Stress

For terrestrial fungi, the daily cycle of temperature and humidity imposes a predictable rhythm of osmotic stress. The primary defense against this is the High-Osmolarity Glycerol (HOG) pathway, a highly conserved mitogen-activated protein kinase (MAPK) cascade [53]. In Neurospora, the clock has co-opted this pathway for anticipatory defense. The terminal MAPK of the cascade, OS-2, undergoes robust rhythmic phosphorylation that peaks around subjective dawn, even in the complete absence of any osmotic stress [54]. This clock-driven “pre-activation” of the HOG pathway primes the fungus for the desiccation stress that will accompany the rising sun and temperatures of the day. This rhythmic phosphorylation of OS-2 in turn drives the rhythmic expression of downstream target genes, such as ccg-1, ensuring that the full defensive program is in place before it is critically needed [55].

4.3. Adapting to Nutritional Fluctuations

The circadian clock and cellular metabolism are deeply and bidirectionally intertwined. In Neurospora, global transcriptomic analyses have revealed that as much as 40% of the genome can be expressed under circadian control, with a profound enrichment for genes involved in metabolic pathways [56]. The clock appears to partition metabolism across the day, favoring catabolism during the active “day” phase and biosynthesis during the “night” phase. A critical molecular link that allows the clock to adjust to the cell’s nutritional status has recently been uncovered. The evolutionarily conserved GCN2 signaling pathway, which is activated by amino acid starvation, is essential for maintaining a robust circadian rhythm under nutrient-poor conditions. The mechanism involves the GCN2-regulated transcription factor CPC-1, which, under starvation, helps recruit the SAGA histone acetyltransferase complex to the frq promoter to modulate histone H3 acetylation, ensuring the promoter remains accessible for rhythmic binding by the WCC [57].

4.4. Responding to Chemical Stress

The relationship between the circadian clock and the response to xenobiotic chemicals, such as fungicides, is an emerging area of research. Studies in Cordyceps militaris have shown that exposure to various fungicides can alter the expression of the core clock protein CmFRQ [58]. This suggests that chemical stress can directly perturb the timekeeping machinery. Conversely, it is plausible that the clock regulates detoxification pathways, such as those involving cytochrome P450 enzymes or efflux pumps, to anticipate exposure to naturally occurring toxins. This temporal regulation could have a significant impact on the efficacy of agricultural and clinical antifungal treatments.

5. The Chrono-Pathogenesis of Fungal Infections

The interaction between a pathogenic fungus and its host is a dynamic and intricate process. Success for the pathogen depends not only on its suite of virulence factors but also on the timing of infection. A growing body of evidence reveals that fungal pathogens possess their own circadian clocks that rhythmically control their virulence, a concept termed “chrono-pathogenesis” [59]. This internal timing allows the pathogen to coordinate its infection programs with the daily rhythms of its host, particularly the host’s immune system, which itself exhibits strong circadian fluctuations. This temporal coordination suggests a co-evolutionary dynamic where timing is a critical selective pressure, opening up novel avenues for disease control by disrupting this pathogenic timing.

5.1. Transcriptional Cascades: The WCC as a Master Regulator

The outcome of a host–pathogen interaction is not a fixed property but varies dramatically depending on the time of day the infection occurs. This is because both the pathogen’s capacity to cause disease and the host’s ability to defend itself are under circadian control. A successful pathogen has evolved to launch its attack not at random but during a period when its own virulence is maximal and the host’s defenses are at their minimum level.
The interaction between Botrytis cinerea and Arabidopsis thaliana provides a compelling demonstration. The size of the necrotic lesions produced by the fungus varies depending on the time of inoculation, with the most severe disease occurring when the fungus is at its subjective dawn. Elegant experiments using clock-null mutants of both the fungus and the plant have unequivocally demonstrated that it is the fungal clock that is the primary determinant of this rhythm. Similar principles govern the pathogenesis of Fusarium oxysporum on tomato, where disruption of core clock genes renders the fungus almost completely nonpathogenic. The Fusarium clock orchestrates this timed attack by rhythmically controlling the expression of transcription factors that overcome host defenses (e.g., FoZafA for zinc starvation) and regulate toxin production (e.g., FoCzf1 for fusaric acid). Evidence for clock-controlled virulence is also emerging in other important pathogens, such as Magnaporthe oryzae, the rice blast fungus, where light cycles and clock components influence conidiation, development, and pathogenicity, with virulence peaking at different times of day depending on the host (rice vs. barley) (reviewed in Wilson & Talbot, 2009; see also Goodwin, 2022, for recent data) [60,61].

5.2. The Temporal Interplay Between Host and Pathogen and the Chrono-Therapeutic Hypothesis

The success of chrono-pathogenesis relies on the fungus exploiting the host’s own rhythms. The mammalian immune system, for example, is profoundly regulated by the circadian clock, with key processes such as the metabolism of tryptophan into the immunomodulatory kynurenine pathway, and the trafficking and activity of innate immune cells like neutrophils, exhibiting robust daily oscillations [62]. This creates predictable times of day when the immune response is stronger or weaker [63,64].
This has led to the emerging, though still speculative, hypothesis of “chrono-therapeutics” [65]. This concept proposes a therapeutic strategy that does not aim to kill the pathogen directly but rather to perturb its internal clock. By disrupting the pathogen’s timing, its pathogenic program would become misaligned with the windows of host vulnerability, potentially rendering it less effective. This approach of temporal disruption, rather than direct inhibition, might be less prone to the rapid evolution of resistance that plagues many conventional fungicides [66]. Potential molecular targets could include conserved clock proteins or kinases, but specificity is a major challenge, as hosts possess homologous proteins. A more viable avenue may be to target clock-regulated output pathways that are essential for virulence but more divergent between fungus and host, such as autophagy, for which inhibitors like ebselen have recently been identified [67,68]. However, it must be emphasized that this concept remains hypothetical and requires substantial experimental validation in vivo before it can be considered a viable strategy.

6. Molecular Output Pathways: From Oscillation to Action

The core circadian oscillator is ultimately a timekeeping device. For this timekeeping to have a physiological impact, its temporal information must be translated into rhythmic changes in cellular function. Fungi have evolved a sophisticated, multi-layered system of output pathways to achieve this, creating a clear hierarchy of control.

6.1. Transcriptional and Epigenetic Control

The most direct output from the core FRQ-WCC oscillator is transcriptional. The WCC, as the primary clock-controlled transcription factor, binds to the promoters of a large set of clock-controlled genes (ccgs) [69]. A key feature of this network is that many of the direct targets of the WCC are themselves transcription factors. This architecture creates downstream transcriptional cascades, allowing a single central oscillator to generate complex and diverse phase relationships in the expression of thousands of downstream genes [70]. A second layer of control involves the dynamic, rhythmic regulation of the chromatin environment. As discussed previously, the link between the GCN2 nutrient-sensing pathway and the circadian clock in Neurospora is mediated by rhythmic histone H3 acetylation at the frq promoter. It is highly likely that this principle extends to the clock’s output, with the clock rhythmically directing chromatin modifications at the promoters of ccgs to fine-tune their expression.

6.2. Post-Translational Regulation

The clock’s influence is not limited to transcription. It can also impose rhythmicity on post-translational events, allowing for rapid modulation of protein activity. The rhythmic activation of the OS-2 MAPK in Neurospora is a prime example. The clock directs the rhythmic phosphorylation of OS-2, meaning that the activity of this key stress-signaling kinase oscillates throughout the day. This rhythmic signal can then propagate further. It has been shown that the OS-2 pathway leads to the phosphorylation of eukaryotic elongation factor-2 (eEF-2), a critical regulator of protein synthesis. As a result, the overall translational activity of the cell exhibits a circadian rhythm, demonstrating a powerful layer of clock control that gates the final step of gene expression [71].

6.3. Developmental Rhythms: Conidiation and Hyphal Branching

Among the most visible outputs of the fungal clock are developmental programs, including the formation of spores (conidiation) and the regulation of mycelial architecture. In Neurospora, the clock times the daily burst of asexual conidiation to occur just before dawn, when high humidity and cool temperatures are ideal for spore dispersal and viability [72]. This is mechanistically driven by the rhythmic expression of a cascade of developmental regulatory genes [73]. Rhythmic growth also directly influences colony morphology. In a clock mutant of Podospora anserina, concentric bands of growth are formed due to rhythmic changes in hyphal extension rate and branching [74,75]. In Neurospora, a homeostatic mechanism that maintains a constant density of hyphal branches has been shown to require the activity of circadian clock-associated proteins, further linking the clock to the control of colony architecture [76].
This multi-layered system—transcriptional, post-translational, and epigenetic—explains both the clock’s robustness and its flexibility. Table 2 systematically connects specific stresses and pathogenic functions to the known molecular players regulated by the circadian clock.

7. Discussion

The evidence synthesized in this review indicates that the fungal circadian clock is a master regulatory system, fundamental to survival and success [77,78]. It is far more than a simple mechanism for timing developmental events. The clock is a central coordinator that integrates external environmental cues with the internal physiological state of the cell to generate a proactive, anticipatory set of physiological responses [79]. By rhythmically gating cellular defenses, the clock prepares the fungus for predictable daily periods of oxidative, thermal, and osmotic stress. By partitioning metabolic activities across the 24 h day, it ensures maximal efficiency in nutrient utilization and growth. And in pathogenic species, the clock functions as a key regulator of virulence programs, a process termed chrono-pathogenesis, that times infection to exploit temporal weaknesses in host defenses [24].
In any complex ecosystem, organisms compete for resources in space. Figure 3 depicts the ecological impact of circadian coherence: fungi with synchronized clocks exhibit improved tolerance to heat, drought, and salinity, while clock disruption predicts reduced resilience. The circadian clock allows them to also compete in, and partition, the dimension of time. By scheduling key activities for specific times of day, fungi can occupy a unique temporal niche. This temporal partitioning reduces direct competitive pressure and minimizes exposure to predictable threats. In the context of a holobiont, such as the mycorrhizal symbiosis, the clock’s role in temporal coordination becomes even more critical, ensuring that the metabolic activities of the fungus and its host plant are synchronized for their mutual benefit [80].
Our understanding of the fungal circadian clock has advanced significantly, moving from the initial characterization of the core oscillator in Neurospora crassa to a broader appreciation of its central role in fungal biology. It is now established that the clock is a proactive, anticipatory system that provides a major fitness advantage. Recent reviews have emphasized the vast extent of circadian regulation in fungi and called for more comparative studies to understand the evolution and diversity of these networks [81,82].
Despite this progress, many fundamental questions remain unanswered. The molecular basis of the clock is understood in only a handful of filamentous ascomycetes. The mechanisms of timekeeping in the vast majority of the fungal kingdom, including the entire phylum Basidiomycota, remain uncharacterized. While genomic surveys confirm the presence of core clock gene homologs in many basidiomycetes, their functionality is largely unproven [83]. Intriguing evidence of rhythmic behaviors, such as the clock-controlled bioluminescence in the mushroom Neonothopanus gardneri, strongly suggests that functional clocks are present [32]. Elucidating the molecular machinery in these economically and ecologically important fungi is a major priority.
While transcriptomic studies have identified hundreds of clock-controlled genes, this is only the first step. The complete network of clock outputs, including the full complement of rhythmically modified proteins (the “chrono-proteome”) and metabolites (the “chrono-metabolome”), is far from mapped. The discovery of clock genes in non-photosynthetic, subterranean AMF is particularly significant. It strongly implies that the fungal clock is entrained by rhythmic signals from its host plant, likely related to the daily flux of photosynthates. However, the molecular nature of this temporal communication is completely unknown [84].
Addressing these knowledge gaps will require a multi-pronged approach leveraging cutting-edge technologies:
Comparative Genomics and Transcriptomics: Applying long-read sequencing and time-series RNA-seq to a much broader phylogenetic diversity of fungi will be crucial for identifying novel clock components and new clock architectures [85].
Chrono-Proteomics and -Metabolomics: The use of high-resolution mass spectrometry to perform time-series analyses of the entire proteome, phosphoproteome, and metabolome of fungi grown in constant conditions will be essential for mapping the full extent of the clock’s output pathways.
Structural Biology: Determining the high-resolution 3D structures of key clock protein complexes, such as the WCC and the FRQ-FRH complex, using techniques like cryo-electron microscopy. This will provide deep mechanistic insights into their function.
In Vivo Infection Models: Developing and employing advanced imaging techniques, such as live-cell microscopy with dual-reporter systems, will allow researchers to dissect the temporal dynamics of the host-pathogen interaction in real-time.
By pursuing these avenues, the scientific community can move toward a comprehensive understanding of this fundamental aspect of fungal biology, paving the way for innovative solutions to long-standing challenges in agriculture and human health.

8. Conclusions

The fungal circadian clock has emerged as a central regulator of stress responses across diverse environmental conditions. Far from functioning solely as a developmental timer, it operates as a proactive system that anticipates and mitigates predictable stressors. By rhythmically gating defense pathways, modulating developmental programs, and temporally organizing gene expression, the clock significantly enhances fungal resilience and ecological fitness. In pathogenic fungi, the clock has further evolved to coordinate the timing of virulence factor expression to exploit host vulnerabilities. This concept of chrono-pathogenesis informs our understanding of host–pathogen dynamics under stress and points to new therapeutic avenues. The molecular architecture of these clocks is surprisingly diverse, reflecting adaptation to specific ecological niches. Acknowledging this diversity and the significant gaps in our knowledge, particularly outside of a few model ascomycetes, is essential for future progress. Overall, the study of the fungal circadian clock represents a powerful molecular framework that links environmental perception with stress adaptation and disease, offering novel strategies to enhance stress tolerance and disease control in both agricultural and clinical settings.

Author Contributions

Conceptualization, V.C.-R.; methodology, V.C.-R.; formal analysis, V.C.-R.; investigation, V.C.-R.; resources, V.C.-R. and D.B.-R.; data curation, V.C.-R.; writing—original draft preparation, V.C.-R.; writing—review and editing, V.C.-R.; visualization, V.C.-R. and D.B.-R.; supervision, V.C.-R.; project administration, V.C.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

All the research data can be found in the text.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
FRQFREQUENCY
WCCWHITE COLLAR COMPLEX
TTFLTranscription-Translation Feedback Loop
ROSReactive Oxygen Species
YROsYeast Respiratory Oscillations
AMFArbuscular Mycorrhizal Fungi
MAPKMitogen-Activated Protein Kinase
PP2AProtein Phosphatase 2A
eEF-2Eukaryotic Elongation Factor 2
ccgClock-Controlled Genes
NTOsNon-Transcriptional Oscillators
GCN2General Control Nonderepressible 2
CPC-1Cross-Pathway Control 1
SAGASpt-Ada-Gcn5 Acetyltransferase (complex)

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Figure 1. The fungal circadian clock as a central regulatory hub. The circadian clock is a master regulator that receives and integrates diverse input signals, such as light, temperature, nutrient availability, and host signals. In response, it rhythmically orchestrates fundamental biological processes (outputs), including metabolism, development, stress response, and virulence, to synchronize the fungus with its environment.
Figure 1. The fungal circadian clock as a central regulatory hub. The circadian clock is a master regulator that receives and integrates diverse input signals, such as light, temperature, nutrient availability, and host signals. In response, it rhythmically orchestrates fundamental biological processes (outputs), including metabolism, development, stress response, and virulence, to synchronize the fungus with its environment.
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Figure 2. Stress-specific molecular cascades. This figure details the molecular mechanisms connecting the circadian clock to specific stress responses in three subpanels. (A) Oxidative stress: it shows how reactive oxygen species (ROS) influence the central oscillator to activate antioxidant genes. (B) Osmotic stress: it illustrates the regulation of the HOG-MAPK pathway by the clock to manage cell wall integrity. (C) Nutritional/pathogen stress: it represents how the clock temporally controls nutrient acquisition pathways and virulence factors, synchronizing these functions with external or host conditions.
Figure 2. Stress-specific molecular cascades. This figure details the molecular mechanisms connecting the circadian clock to specific stress responses in three subpanels. (A) Oxidative stress: it shows how reactive oxygen species (ROS) influence the central oscillator to activate antioxidant genes. (B) Osmotic stress: it illustrates the regulation of the HOG-MAPK pathway by the clock to manage cell wall integrity. (C) Nutritional/pathogen stress: it represents how the clock temporally controls nutrient acquisition pathways and virulence factors, synchronizing these functions with external or host conditions.
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Figure 3. Environmental impact and resilience to climate change. This scenario diagram illustrates the ecological implications of the circadian clock’s function. Two paths are compared: on the left (Resilience), a synchronized clock allows fungi to anticipate environmental changes (e.g., temperature), adapting their phenology to survive and thrive. On the right (Vulnerability), the disruption of the clock by climate change causes desynchronization, which increases the fungus’s vulnerability to stress and threatens its function in the ecosystem, with potential repercussions for biodiversity and agriculture.
Figure 3. Environmental impact and resilience to climate change. This scenario diagram illustrates the ecological implications of the circadian clock’s function. Two paths are compared: on the left (Resilience), a synchronized clock allows fungi to anticipate environmental changes (e.g., temperature), adapting their phenology to survive and thrive. On the right (Vulnerability), the disruption of the clock by climate change causes desynchronization, which increases the fungus’s vulnerability to stress and threatens its function in the ecosystem, with potential repercussions for biodiversity and agriculture.
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Table 1. Conservation and function of core circadian clock components across fungal species.
Table 1. Conservation and function of core circadian clock components across fungal species.
PhylumSpeciesCore Oscillator TypeWC-1/WC-2 Homologs (Presence/Function)FRQ Homolog(s) (Presence/Function)Primary Entrainment Cue(s)Key Rhythmic Output(s)Validation StatusReference(s)
AscomycotaNeurospora crassaFRQ/WCC TTFLPresent/positive element, photoreceptorPresent/negative elementLight, temperatureAsexual sporulation (conidiation), stress response, hyphal branchingExperimental[14]
Botrytis cinereaFRQ/WCC TTFLPresent/positive element homologsPresent/negative element, virulence regulatorLightVirulence, pathogenesisExperimental[10,24]
Fusarium oxysporumFRQ/WCC TTFLPresent/positive element homologs essential for virulencePresent/primary negative element essential for virulenceLight, host signals (inferred)Virulence, toxin production, zinc homeostasisExperimental[25]
Aspergillus flavusNon-FRQ (Unknown)Present/putativeAbsentLight, temperatureSclerotia developmentExperimental[27]
Penicillium claviformeUnknownUnknownUnknownLight, magnetic fieldCoremia formationExperimental[28]
Saccharomyces cerevisiaeMetabolic NTO (YROs)AbsentAbsentTemperature, metabolic cyclesRespiratory oscillations, gene expressionExperimental[35]
Schizosaccharomyces pombeUltradian OscillatorUnknownUnknownTemperature, signaling pathwaysCell signaling oscillationsExperimental[36]
Arthrobotrys oligosporaUnknown (CryA involved)Present (putative)UnknownLightConidiation, Trap formationExperimental[26]
Rhizoglomus irregulareFRQ/WCC TTFL (putative)Present/expressed in symbiotic stagesPresent/expressed in symbiotic stagesHost metabolic signals (hypothesized)Coordination with host (hypothesized)Genomic/inferred[39]
BasidiomycotaPellicularia filamentosaUnknownUnknownUnknownLight (inferred)Nocturnal basidiospore dischargeExperimental[31]
Neonothopanus gardneriUnknownUnknownUnknownLight, temperatureBioluminescence (luciferase, luciferin cycling)Experimental[32]
MucoromycotaPilobolus spp.UnknownUnknownUnknownLightSporangium discharge (phototropism)Experimental[33]
Table 2. Clock-regulated stress response pathways and virulence factors in fungi.
Table 2. Clock-regulated stress response pathways and virulence factors in fungi.
Stress/FunctionKey Clock-Controlled Gene/ProteinMechanism of RegulationFungal SpeciesValidation StatusReference(s)
Oxidative StressCatalase-1 (cat-1)Transcriptional regulation by WCCNeurospora crassaDirectly validated[52]
Osmotic StressOS-2 (MAPK)Rhythmic phosphorylation (activation)Neurospora crassaDirectly validated[53]
Osmotic Stressccg-1 (osmotic-responsive gene)Transcriptional regulation downstream of rhythmic OS-2 activationNeurospora crassaDirectly validated[55]
Nutritional Stressfrequency (frq)Epigenetic (histone acetylation) via GCN2/CPC-1/SAGA pathwayNeurospora crassaDirectly validated[57]
Virulencebcfrq1 (clock core component)Required for rhythmic virulenceBotrytis cinereaDirectly validated[24]
Zinc StarvationFoZafA (transcription factor)Rhythmic transcription regulated by the clockFusarium oxysporumDirectly validated[25]
Toxin ProductionFoCzf1 (transcription factor)Rhythmic transcription regulated by the clockFusarium oxysporumDirectly validated[25]
TranslationeEF-2 (elongation factor)Rhythmic phosphorylation downstream of rhythmic OS-2 activationNeurospora crassaDirectly validated[71]
DevelopmentConidiation and hyphal branchingRhythmic growth and gene expressionNeurospora crassa, Podospora anserinaDirectly validated[75]
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Coca-Ruiz, V.; Boy-Ruiz, D. Timing Is Everything: The Fungal Circadian Clock as a Master Regulator of Stress Response and Pathogenesis. Stresses 2025, 5, 47. https://doi.org/10.3390/stresses5030047

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Coca-Ruiz V, Boy-Ruiz D. Timing Is Everything: The Fungal Circadian Clock as a Master Regulator of Stress Response and Pathogenesis. Stresses. 2025; 5(3):47. https://doi.org/10.3390/stresses5030047

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Coca-Ruiz, Victor, and Daniel Boy-Ruiz. 2025. "Timing Is Everything: The Fungal Circadian Clock as a Master Regulator of Stress Response and Pathogenesis" Stresses 5, no. 3: 47. https://doi.org/10.3390/stresses5030047

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Coca-Ruiz, V., & Boy-Ruiz, D. (2025). Timing Is Everything: The Fungal Circadian Clock as a Master Regulator of Stress Response and Pathogenesis. Stresses, 5(3), 47. https://doi.org/10.3390/stresses5030047

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