The cAMP-dependent signaling pathway is a central regulatory system composed of several key proteins, including G protein-coupled receptors (GPCRs), heterotrimeric G proteins, adenylate cyclase (AC), cAMP-dependent protein kinase A (PKA), and multiple downstream effectors (Huang et al., 2019). This pathway governs essential cellular processes, including growth, differentiation, metabolism, and gene transcription (Daniel et al., 1998; McDonough and Rodriguez, 2011). Within this cascade, AC catalyzes the conversion of ATP into cAMP, which acts as a second messenger to activate PKA and initiate downstream signaling. Beyond its role as a signal generator, AC also functions as a metabolic sensor, integrating cellular energy status with regulatory pathways in eukaryotes (Hanoune et al., 1997). Evolutionarily conserved, AC is found across diverse organisms ranging from bacteria to humans (Casperson et al., 1983).

In fungi, the cAMP–PKA pathway orchestrates vegetative growth, conidiation, stress responses, and virulence (D’Souza and Heitman, 2001; Shimizu and Keller, 2001; Choi and Xu, 2010; Kim et al., 2011; Martin-Urdiroz et al., 2016). Disruption of AC in yeast impairs energy signaling, reduces PKA activity, and leads to severe growth and differentiation defects (Grahl et al., 2015). Similarly, AC inactivation diminishes virulence in multiple pathogenic fungi (Jurick and Rollins, 2007; Kohut et al., 2010; Hu et al., 2014; Wang et al., 2016; Yang et al., 2016; Guo et al., 2019). The pathway also regulates secondary metabolite production, further showing its broad influence on fungal physiology (Mukherjee et al., 2007; García-Rico et al., 2008; Garcia-Martinez et al., 2012; Kohut et al., 2010; Studt et al., 2013).

One of the most successful fungal pathogens, Alternaria alternata, infects a wide range of economically important crops, including citrus, apple, pear, rice, strawberry, tomato, broccoli, cauliflower, carrot, and potato, causing significant agricultural losses worldwide. Its pathogenic success largely stems from the production of diverse phytotoxins, particularly host-selective toxins (HSTs), which exhibit specific toxicity and unique mechanisms of action against target plants. Each A. alternata pathotype produces a distinct HST, enabling it to infect specific hosts (Hatta et al., 2002; Ito et al., 2004). For example, the tangerine pathotype causes Alternaria brown spot disease on citrus cultivars by producing ACT (Alternaria citri tangerine). This toxin induces lipid peroxidation and cell death (Lin et al., 2011). In addition to ACT, virulence depends on the fungal ability to detoxify reactive oxygen species (ROS) (Lin et al., 2009; Yang et al., 2009). Recent studies highlight further contributions of siderophore-mediated iron acquisition, peroxisomes, and autophagy to ROS tolerance and pathogenicity (Chen et al., 2013, 2014; Chung et al., 2020; Choo et al., 2023; Wu et al., 2022, 2023, 2024).

To survive diverse environmental challenges such as oxidative stress and nutrient limitation, fungal pathogens rely on autophagy. This conserved intracellular degradation pathway recycles cellular components and promotes survival under stress (Kraft et al., 2009; Das et al., 2012). Autophagy begins with the formation of a phagophore from ER-associated membranes, which expands into an autophagosome to sequester damaged organelles and proteins for degradation (Gómez-Sánchez et al., 2021). Beyond stress mitigation, autophagy supports fungal growth, conidiation, secondary metabolism, and virulence (Liu et al., 2007; Nguyen et al., 2011; Meng et al., 2025a, b). In A. alternata, autophagy is triggered by nitrogen deprivation and oxidative stress, helping eliminate ROS-damaged components and enhancing pathogenicity (Lu et al., 2022; Wu et al., 2022). Moreover, autophagy maintains iron homeostasis: disruption of autophagy-related (Atg) genes reduces siderophore biosynthesis, elevates ROS, and impairs growth under iron deficiency (Wu et al., 2024). Thus, autophagy alleviates oxidative damage and facilitates siderophore production and iron uptake, collectively strengthening virulence. Importantly, iron availability itself modulates autophagy, with both overload and deficiency disrupting autophagic processes and inducing stress (Krishan et al., 2015; Wang et al., 2022).

The cAMP–PKA pathway intersects with autophagy regulation, either promoting or inhibiting it depending on cell type and physiological context (Budovskaya et al., 2004; Grisan et al., 2021). While the role of the cAMP-PKA pathway in general cellular processes is well established, its specific involvement in siderophore biosynthesis remains less explored. In phytopathogenic fungi, siderophores are essential for iron uptake under deficiency, ensuring homeostasis and successful infection. In A. alternata, siderophore biosynthesis is regulated by several proteins, such as AaYap1, AaHog1, and AaNox (Chen et al., 2014), as well as AaHapX and AaSreA, which coordinate iron metabolism under fluctuating conditions (Chung et al., 2020; Wu et al., 2022). Recent findings in Mucor lusitanicus show that the cAMP–PKA pathway regulates the rfs gene, essential for rhizoferrin biosynthesis (Alejandre-Castaneda et al., 2022), suggesting a broader role for this pathway in iron regulation across fungi, including A. alternata.

In this study, we demonstrate that the cAMP–PKA signaling pathway, specifically the adenylate cyclase of A. alternata (AaAC), is indispensable for regulating both siderophore and ACT toxin biosynthesis. Interestingly, AaAC negatively affects autophagy under nutrient-rich conditions but does not influence oxidative stress resistance. These findings reveal a subtle interplay among signaling pathways, iron regulation, and pathogenicity, providing more profound insight into the molecular mechanisms underlying fungal virulence.

The cAMP-PKA signaling pathway plays an important, evolutionarily conserved role in regulating key cellular processes in numerous fungi (Martin-Urdiroz et al., 2016). Adenylate cyclase synthesizes cAMP and serves as a key component of the G protein–cAMP–PKA signaling pathway (McDonough and Rodriguez, 2011). In this study, we demonstrate that A. alternata AaAC functions as a master regulatory protein essential for its physiology, metabolism, and pathogenicity. It governs a wide array of cellular processes, including growth and development, maintenance of cell wall integrity, metabolic adaptation under stress conditions, suppression of autophagy, regulation of iron homeostasis, siderophore biosynthesis, and toxin production.

Although AC is conserved in fungi, its role in conidia formation could vary. AaAC is required for conidial formation. In contrast, an AC homolog in the citrus green mold pathogen Penicillium digitatum negatively affects conidial formation (Wang et al., 2016). AaAC has intricate interactions with iron metabolism, as AaAC-deficient mutants exhibit increased sensitivity to iron salts, suggesting a role in regulating iron levels under nutrient stress. Interestingly, BPS treatment alleviates this sensitivity, suggesting iron overload, rather than deficiency, exacerbates the growth defect in the absence of AaAC. Although AaAC appears non-essential for oxidative stress resistance, altered Congo red responses imply changes in cell wall dynamics. AaAC may function as a context-dependent modulator, possibly integrating nutrient and stress signals to maintain cellular homeostasis.

AaAC serves as a central regulator of siderophore biosynthesis and iron homeostasis, as evidenced by the absence of detectable siderophore activity in ΔAaAC. The lack of halos in ΔAaAC suggests a complete loss of siderophore production, implicating AaAC as essential for this process. The FeCl₃ reactivity assay offers a simple yet effective chemical validation of siderophore presence. The distinct dark red coloration observed in WT and CP1 filtrates, in stark contrast to the light brown hue of ΔAaAC, supports findings from the CAS plate assay. Further confirmation is provided by TLC and HPLC analyses, in which bands and peaks detected in WT and CP1 samples but absent in ΔAaAC appear to correspond to specific siderophore compounds whose synthesis depends on AaAC function. Furthermore, diminished siderophore production in strains deficient in Gα, the PKA catalytic subunit, or the PKA regulatory subunit indicates that siderophore biosynthesis is governed by a broader signaling network involving the G-protein- and cAMP-dependent pathway. The cAMP-PKA pathway is also involved in rhizoferrin siderophore secretion in Mucor lusitanicus (Alejandre-Castaneda et al., 2022), further supporting its role in siderophore biosynthesis.

The quantitative real-time PCR analysis reveals that deletion of AaAC disrupts the regulatory network governing iron homeostasis. Under iron-deficient conditions, there is a marked downregulation of genes encoding AaSit1, AaNps6, and AaHapX, key components of the iron acquisition and regulatory machinery. This indicates that deletion of AaAC impairs the fungal response to iron scarcity. ΔAaAC likely fails to properly sense or adapt to iron limitation, potentially compromising its survival and virulence in iron-depleted environments. In contrast, the observed upregulation of the gene encoding AaSreA, a known repressor of siderophore biosynthesis (Chung et al., 2020), further supports the hypothesis of a dysregulated iron response.

AaAC is not only involved in toxin biosynthesis but may also play a regulatory role in coordinating the expression of toxin-related genes. The TLC and HPLC analyses show that AaAC is necessary for the synthesis or accumulation of ACT toxins. The significant downregulation of toxin biosynthesis-related genes, ACTT2, ACTT5, ACTT6, and ACTTR, in ΔAaAC under iron-deficient conditions suggests that AaAC acts upstream in a regulatory cascade controlling their expression. Iron limitation is a known environmental cue that influences secondary metabolism (Canessa and Larrondo, 2013; Haas, 2015), and the observed transcriptional changes imply that AaAC may integrate ecological signals to modulate toxin production in response to such stress. Disruption of AaAC results in the loss of detectable toxin products and the downregulation of key biosynthetic genes, thereby reducing virulence. This signaling pathway likely influences the function of the PKA^r regulatory subunit rather than the PKA^c catalytic subunit, as deletion of PKAr, but not PKAc, significantly impairs A. alternata virulence (Tsai et al., 2013). Furthermore, the Gα subunit appears to play no role in toxin biosynthesis or virulence (Wang et al., 2010). This suggests that environmental signals regulating ACT toxin biosynthesis are not transduced through the Gα subunit. Nevertheless, AaAC remains a critical component in ACT toxin biosynthesis in A. alternata. A recent study by Yang et al. (2016) also shows that AC is involved in both aflatoxin biosynthesis and Aspergillus flavus virulence.

The inability of ΔAaAC to induce necrotic lesions strongly implicates AaAC as a critical determinant of pathogenicity in a detached calamondin leaf assay. Since ΔAaAC failed to produce conidia, pathogenicity was assessed by hyphal inoculation. This approach bypasses spore germination and directly tests the ability of vegetative mycelia to initiate infection. Pre-inoculation wounding did not restore lesion formation in ΔAaAC, indicating that the loss of pathogenicity is not due to impaired entry through intact plant surfaces. The failure of hyphae from ΔAaAC to cause disease suggests that AaAC may regulate factors beyond conidiation, including toxin production, host penetration, and iron acquisition. Thus, AaAC is involved in active virulence mechanisms rather than passive colonization.

The transcriptomic analysis confirms the central roles of AaAC in regulating siderophore biosynthesis and of the ACT toxin. The RNA-seq comparison between ΔAaAC and WT under iron-deficient conditions reveals broad, significant changes in gene expression, highlighting the profound influence of AaAC on cellular metabolism and secondary metabolite production. The enrichment of DEGs in many biosynthetic and metabolic pathways suggests that AaAC plays a pivotal role in coordinating metabolic responses to iron limitation. These pathways are critical for maintaining cellular homeostasis and supporting the biosynthetic demands of secondary metabolite production, particularly under nutrient-stress conditions. A key finding is the downregulation of multiple genes involved in siderophore biosynthesis and iron acquisition in ΔAaAC. The repression of these genes further indicates that AaAC is a positive regulator of siderophore-mediated iron uptake, which is a crucial function for fungal survival and virulence in iron-limited host environments. The upregulation of ornithine aminotransferase, which converts ornithine to proline, and arginase, which converts arginine to ornithine (Beckmann et al., 2013), suggests a compensatory metabolic shift. Ornithine is a key precursor in siderophore biosynthesis (Hissen et al., 2005). The deletion of AaAC may reroute nitrogen flux toward proline biosynthesis or to maintain ornithine pools in the absence of siderophore production. The redirection of metabolic flow could reduce intracellular ornithine availability for siderophore biosynthesis, thereby contributing to the observed reduction in siderophore biosynthesis. This metabolic reprogramming may indicate that ΔAaAC attempts to adapt to the loss of AaAC function.

The transcriptomic analysis also demonstrates that AaAC is essential for the expression of genes involved in ACT toxin biosynthesis. The downregulation of genes encoding key enzymes such as SAM synthase, HMGS, HMGH, NRPS, and cytochrome P450 monooxygenases in ΔAaAC suggests that AaAC is a master regulator of this virulence-associated secondary metabolite pathway. Given the role of the ACT toxin in host tissue necrosis and disease progression, downregulation of the biosynthetic genes in the mutant strain may significantly attenuate pathogenicity. The antiSMASH-based prediction of biosynthetic gene clusters further supports the broad regulatory scope of AaAC. The observed downregulation of clusters 4 (NRPS), 32 (dimethylcoprogen), and 30 (ACT toxin) in ΔAaAC indicates that AaAC functions as a global transcriptional regulator of secondary metabolism. This suggests that AaAC may function upstream of multiple biosynthetic pathways, potentially through direct transcriptional activation or by modulating other regulatory elements.

The reduction in siderophore and toxin production observed in ΔAaAC may result from metabolic disruptions triggered by impaired cAMP signaling. In the absence of adenylate cyclase, intracellular cAMP levels may decline, thereby reducing protein kinase A activity and potentially compromising cellular energy sensing and metabolic homeostasis (Dai et al., 2025). This imbalance may impair mitochondrial function and diminish tricarboxylic acid cycle flux, thereby limiting the availability of key metabolic intermediates such as acetyl-CoA. As acetyl-CoA is a critical precursor in nonribosomal peptide synthesis (Gründlinger et al., 2013), which is required for both siderophore and toxin biosynthesis, its reduced availability could directly impact metabolite output. Thus, the defect in siderophore production in ΔAaAC likely arises from a combination of transcriptional repression and a shortage of essential biosynthetic substrates, both of which contribute to metabolic dysfunction.

Beyond its roles in regulating siderophores and the ACT toxin, AaAC acts as an autophagy suppressor under nutrient-rich conditions. Fluorescence microscopy reveals only low levels of autophagy in the WT strain cultured in nutrient-rich PDB medium. The formation of autophagosomes and activation of autophagy are induced by nitrogen starvation or oxidative stress, consistent with known fungal survival strategies under adverse environmental conditions (Palmer et al., 2008; Bartoszewska and Kiel, 2010). Strikingly, ΔAaAC exerts autophagy across all tested conditions, including nutrient-rich PDB. This constitutive autophagy phenotype indicates that AaAC is required to repress autophagosome formation under nutrient-sufficient conditions, and its absence leads to deregulated autophagy regardless of environmental cues. The Western blot analysis of GFP-Atg8 proteolysis further substantiates the microscopy findings. The suppression of autophagy by AaAC under favorable growth conditions may serve to conserve cellular resources and maintain metabolic efficiency. Autophagy is energetically costly, and its unnecessary activation could disrupt cellular homeostasis. Therefore, AaAC likely contributes to the fine-tuning of autophagic responses, ensuring that this catabolic process is only engaged when necessary. Although autophagy is typically regarded as a mechanism that promotes cellular survival under stress, excessive or dysregulated autophagy could lead to cell death (Kraft et al., 2009; Bartoszewska and Kiel, 2011; Das et al., 2012). Deregulated autophagy in ΔAaAC may influence its development, stress tolerance, and virulence.

Our findings establish AaAC as a central regulator within the cAMP–PKA signaling pathway, orchestrating diverse physiological and pathogenic processes in A. alternata. Beyond its conserved role in fungal growth and development, AaAC integrates nutrient and stress signals to maintain cellular homeostasis, particularly by regulating iron metabolism, siderophore biosynthesis, and toxin production. Its deletion disrupts metabolic balance, represses key biosynthetic genes, abolishes siderophore and toxin output, and severely attenuates pathogenicity. AaAC is also a key suppressor of autophagy under nutrient-sufficient conditions, thereby preventing unnecessary activation of this energetically demanding process. These results highlight AaAC as a key hub that coordinates environmental stress responses with secondary metabolism, underscoring its essential role in fungal virulence and survival.