The TOR signaling pathway regulates vegetative development, aflatoxin biosynthesis, and pathogenicity in Aspergillus flavus

Guoqi Li; Xiaohong Cao; Elisabeth Tumukunde; Qianhua Zeng; Shihua Wang

doi:10.7554/eLife.89478.2

eLife assessment

This manuscript characterized signaling pathways for growth control and aflatoxin production in the important plant pathogen Aspergillus flavus. Associating tor and tapA with the control of aflatoxin production would be important. However, the copy number of the tor and tapA genes needs to be more clearly established, and without such work, the evidence remains incomplete.

https://doi.org/10.7554/eLife.89478.2.sa3

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Abstract

The target of rapamycin (TOR) signaling pathway is highly conserved and plays a crucial role in diverse biological processes in eukaryotes. However, the underlying mechanism of the TOR pathway in A. flavus remains elusive. In this study, we identified and characterized nine genes encoding various components of the TOR pathway in A. flavus, and investigated their biological function. The FK506-binding protein Fkbp3 and its lysine succinylation are important for aflatoxin production and rapamycin resistance. The Tor kinase plays a pivotal role in the regulation of growth, spore production, aflatoxin biosynthesis, and osmotic and rapamycin stress. As a significant downstream effector molecule of the Tor kinase, the Sch9 kinase regulates aflatoxin B₁ (AFB₁) synthesis, osmotic and calcium stress response in A. flavus, and this regulation is mediated through its S_TKc, S_TK_X domains, and the ATP binding site at K340. We also showed that the Sch9 kinase may have a regulatory impact on the high-osmolarity glycerol (HOG) signaling pathway. TapA and TipA, the other downstream components of the Tor kinase, play significant roles in regulating sclerotia formation and cell wall stress response in A. flavus. The members of the TapA-phosphatase complexes, SitA and Ppg1, are crucial for various biological processes in A. flavus, including vegetative growth, sclerotia formation, AFB₁ biosynthesis, and pathogenicity. Furthermore, we showed that SitA and Ppg1 are involved in regulating lipid droplets (LDs) biogenesis and cell wall integrity (CWI) signaling pathway. In addition, another phosphatase complex, Nem1/Spo7, plays critical roles in hyphal development, conidiation, aflatoxin, and lipid droplets biogenesis. This study provides an important insight into the regulatory network of the TOR signaling pathway and the molecular mechanism of aflatoxin biosynthesis in A. flavus.

Introduction

Aspergillus flavus is one of the most important phytopathogenic fungi that frequently contaminates various plants, causing economic losses in agricultural commodities and posing serious health concerns in developing countries ^[1]. A. flavus is also an opportunistic human pathogenic fungus that can cause aspergillosis in immune-compromised patients ^[2]. Aflatoxins (AFs), the toxic and carcinogenic secondary metabolites produced by A. flavus, are considered the most potent carcinogen present in nature, causing liver cancer in both humans and animals ^[3]. The aflatoxin biosynthesis is a complex process involving a series of reactions. Although the majority of the enzyme reactions and genes involved in aflatoxin biosynthesis have been elucidated ^[4], the related signaling pathway network and specific regulatory mechanisms governing aflatoxin biosynthesis remain elusive. Therefore, exploring the regulatory mechanism of the aflatoxin biosynthesis signaling pathway would provide new insights for preventing A. flavus and aflatoxins contamination.

In eukaryotes, the target of rapamycin (TOR) signaling pathway is highly conserved and plays essential roles in various significant biological processes, including ribosome biosynthesis, cell growth, and autophagy ^[5]. The serine/threonine-protein kinase Tor serves as a crucial protein component in the TOR signaling pathway, which interacts with other proteins to form multi-protein complexes ^[6]. In Saccharomyces cerevisiae, TOR exists in two distinct multiprotein functional complexes, namely TORC1 and TORC2 ^[7]. TORC1-mediated signaling governs cellular growth by modulating various growth-related mechanisms and exhibits sensitivity to rapamycin. Conversely, TORC2-mediated signaling primarily regulates cytoskeletal remodeling but remains unaffected by rapamycin ^[7]. In yeast, there are two tor genes, tor1 and tor2, whereas in higher eukaryotes such as plants, animals, and filamentous fungi, there is only one tor gene ^[8–10].

In the fungal kingdom, the TOR signaling pathway has been extensively investigated in three prominent species: the budding yeast S. cerevisiae ^[11–12], the fission yeast Schizosaccharomyces pombe ^[13–15], and the human pathogen Candida albicans ^[16–17]. In S. cerevisiae, macrolide antibiotic rapamycin interacts with the cytosolic protein Fkbp12 to form a complex, and the Fkbp12-rapamycin complex effectively inhibits the activity of the Tor kinase function by binding to the FRB domain ^[18]. The Tap42 phosphatase complexs serves as the primary target of the Tor kinase. Additionally, the Tap42 protein interacts with the Tip41 protein to collaboratively regulate phosphatases, including PP2A and Sit4 ^[19–20]. Phosphatase Sit4 can dephosphorylate the transcription factor Gln3, regulating the nitrogen source metabolism pathway ^[21]. In addition, another immediate effector of TORC1 is the AGC kinase Sch9, which participates in various cellular functions processes, such as ribosome biosynthesis, translation initiation, and entry into G₀ phase ^[22]. The TOR signaling pathway has been documented in various filamentous fungi, including Fusarium graminearum ^[23–24], Magnaporthe oryzae ^[25], Phanerochaete chrysosporium ^[26], Podospora anserine ^[27], A. nidulans ^[28], A. fumigatus ^[29–30], F. fujikuroi ^[31], Botrytis cinerea ^[32], F. oxysporum ^[33], and Mucor circinelloides ^[34]. However, the TOR signaling pathway has not yet been reported in A. flavus.

In eukaryotic cells, the TOR signaling pathway and the Mitogen Activated Protein Kinase (MAPK) signaling pathways play an important role in regulating adaptive responses to extra- and intracellular conditions ^[35]. Several studies have demonstrated that the TOR signaling pathway interacts with various other signaling pathways, such as MAPK and CWI. These pathways engage in crosstalk, forming a complex metabolic network that regulates cellular processes ^[36–40]. However, the underlying mechanisms of multiple crosstalks between the TOR and other signaling pathways in A. flavus remain unclear. Therefore, we intended to identify the genes associated with the TOR signaling pathway and elucidate their roles and contributions in regulating vegetative development and aflatoxin biosynthesis in A. flavus. Our results demonstrated that the TOR signaling pathway is involved in multiple cellular processes in A. flavus. Our findings revealed a complex interplay between the TOR pathway and other signaling pathways (such as HOG and CWI) in A. flavus, which play a crucial role in regulating cellular growth and survival under environmental stress conditions.

Results

Rapamycin exhibits inhibitory effects on the growth, sporulation, sclerotia formation, and aflatoxin production in A. flavus

Rapamycin, a secondary metabolite synthesized by Streptomyces hygroscopicus, exhibits efficacy in suppressing specific filamentous fungi. In F. graminearum, rapamycin has a significant inhibitory impact on growth and asexual reproduction ^[23]. To investigate the effects of rapamycin on vegetative development, sclerotia formation, and secondary metabolism in A. flavus, we conducted an assay to determine the sensitivity of the wild-type strain to rapamycin. We observed that the wild-type strain exhibited a high sensitivity to rapamycin. We found that 100 ng/mL of rapamycin significantly inhibited mycelial growth and conidia formation (Fig.1A, 1D, 1E). More importantly, the wild-type strain exhibited a significant reduction in sclerotia formation and aflatoxin synthesis (Fig.1B, 1C, 1F, 1G). Additionally, rapamycin resulted in a significant decrease in spore germination rate and sparser conidiophores (Fig. S1A, S1B). These results illustrated that the TOR signaling pathway may be involved in regulating the growth, development, and secondary metabolism in A. flavus. Calcofluor white is a chemifluorescent blue dye commonly employed for its nonspecific binding affinity to fungal chitin. Fluorescence microscopy analysis revealed a notable reduction in the blue fluorescence on the cell wall following treatment with rapamycin (Fig.S1C). The intracellular lipid droplets within the hyphae were labeled with BODIPY. We observed that the hyphae treated with rapamycin contained more lipid droplets compared to the control group (Fig. S1D). These findings suggest that the TOR pathway may play important roles in maintaining cell wall integrity and facilitating the biogenesis of lipid droplets.

Impacts of rapamycin on the growth, sporulation, sclerotia formation, and aflatoxin production of *Aspergillus flavus*
(A) Colony morphology of the WT strain cultured on PDA medium amended with different concentrations of rapamycin at 37°C for 5 days.
(B) Colony phenotype of the WT strain grown on CM medium amended with different concentrations of rapamycin at 37°C for 7 days.
(C) TLC assay of AFB₁ production by the WT strain cultured in YES liquid media amended with different concentrations of rapamycin at 29°C for 6 days.
(D) Statistical analysis of the colony diameter by the WT strain treated with different concentrations of rapamycin described in (A).
(E) Conidial quantification of the WT strain treated with different concentrations of rapamycin as mentioned in (A).
(F) Quantitative analysis of sclerotium formation by the WT strain treated with different concentrations of rapamycin described in (B).
(G) Relative quantification of AFB₁ production by the WT strain treated with different concentrations of rapamycin as mentioned in (C)
(H) Relative expression levels of conidia-related genes in WT strain treated with 100 ng/mL rapamycin after 3, 6, 9 h.
(I) Relative expression levels of sclerotia-related genes in WT strain treated with 100 ng/mL rapamycin after 3, 6, 9 h.
(J) Relative expression levels of aflatoxin biosynthesis regulatory and structural genes in WT strain treated with 100 ng/mL rapamycin after 3, 6, 9 h.

To investigate the potential regulatory role of the TOR pathway in modulating the sporulation, sclerotia formation, and aflatoxin biosynthesis in A. flavus, we examined the expression levels of conidia-related, sclerotia formation-related, and aflatoxin-related genes after 3, 6, and 9 hours of treatment with rapamycin. To determine the effects of rapamycin on conidiation, qRT-PCR was conducted to assess the transcript levels of two conidia-related genes, abaA and brlA. We also conducted qRT-PCR to evaluate the expression levels of three regulators (nsdC, nsdD, and sclR) involved in sclerotia development, and genes involved in AFB₁ biosynthesis, such as the regulatory gene aflS, as well as several structural genes (aflC and aflQ). The strains treated with rapamycin exhibited a significant reduction in the expression levels of genes related to sporulation, sclerotia formation, and aflatoxin synthesis (Fig. 1H, 1I, 1J). Overall, these results indicated that the TOR pathway plays a crucial role in regulating the growth, sporulation, sclerotia formation, and aflatoxin biosynthesis in A. flavus by modulating the expression of these genes.

The impact of Fkbp3 and its lysine succinylation on AFB₁ biosynthesis and rapamycin resistance in A. flavus

FK506-binding proteins (Fkbps) belong to a highly conserved immunophilin family, which are involved in various cellular functions, such as protein folding, immunosuppression, signaling transduction, and transcription ^[41]. Fkbps function as molecular switches by binding to target proteins and inducing conformational changes ^[41]. In S. cerevisiae, Fkbp12 and rapamycin interact to form a complex that exerts a negative regulatory effect on the activity of the Tor kinase ^[42]. The fkbp12 gene homologue fprA in A. nidulans shares high levels of homology with that from S. cerevisiae ^[28]. According to the protein sequence of FprA from A. nidulans, four genes encoding putative proteins with FK506 binding domain, named fkbp1 (AFLA_126880), fkbp2 (AFLA_087480), fkbp3 (AFLA_010910), and fkbp4 (AFLA_128200), have been identified from the A. flavus genome. We generated four fkbp knockout strains using homologous recombination, and all fkbp disruption strains were confirmed by PCR and sequencing analysis (Fig.S2). Phenotypic analyses showed that the mycelial growth and conidia formation of all the fkbp mutants were comparable to the wild-type strain on the PDA medium (Fig. 2A), suggesting that Fkbps have minimal influence on the process of vegetative development. We evaluated AFB₁ synthesis utilizing thin layer chromatography (TLC), and the findings demonstrated a noteworthy reduction in AFB₁ synthesis upon deletion of Fkbp3, in comparison to the wild-type strain (Fig.2B, 2D). Additionally, the quantity of sclerotia exhibited a significant decrease in all fkbp mutants compared to the wild-type strain (Fig.S3A, S3B). The aforementioned data indicated that Fkbp3 plays a crucial role in sclerotia formation and aflatoxin biosynthesis in A. flavus.

Disruption of *fkbp3* significantly increases the resistance of *A. flavus* to rapamycin and FK506
(A) Phenotype of the WT and all mutant strains (Δ*fkbp1*, Δ*fkbp2*, Δ*fkbp3*, and Δ*fkbp4*) grown on PDA amended with rapamycin and FK506 at 37°C for 5 days.
(B) TLC analysis of AFB₁ production by the WT and all mutant strains (Δ*fkbp1*, Δ*fkbp2*, Δ*fkbp3*, and Δ*fkbp4*) cultured in YES liquid medium at 29°C for 6 days.
(C) The growth inhibition rate of the WT and all mutant strains (Δ*fkbp1*, Δ*fkbp2*, Δ*fkbp3*, and Δ*fkbp4*) under rapamycin and FK506 stress.
(D) AFB₁ quantitative analysis of the WT and all mutant strains (Δ*fkbp1*, Δ*fkbp2*, Δ*fkbp3*, and Δ*fkbp4*) as described in (B).
(E) Phenotype of the WT and all mutant strains (Δ*fkbp3*, K5A, K19A, K40A, K42A, K55A, and K65A) grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.
(F) The growth inhibition rate of the WT and all mutant strains (Δ*fkbp3*, K5A, K19A, K40A, K42A, K55A, and K65A) under rapamycin stress.
(G) TLC assay of AFB₁ production by the WT and all mutant strains (Δ*fkbp3*, K5A, K19A, K40A, K42A, K55A, and K65A) cultured in YES liquid medium at 29°C for 6 days.
(H) Relative quantification of AFB₁ production in the WT and all mutant strains (Δ*fkbp3*, K5A, K19A, K40A, K42A, K55A, and K65A) as mentioned in (G).

To determine the specific Fkbp responsible for rapamycin sensitivity, all mutant strains were cultured on PDA solid plates amended with 100 ng/mL rapamycin. The strain with disrupted fkbp3 showed significant resistance to rapamycin, while the deletion mutants of fkbp1, fkbp2, and fkbp4 did not exhibit any alteration in resistance to rapamycin (Fig.2A, 2C). These results demonstrated that Fkbp3 serves as the principal target for rapamycin in A. flavus. In the FK506-sensitivity analysis, we observed a significant suppression of mycelial growth and conidia formation in A. flavus with the addition of 10 ng/mL FK506 to the PDA medium. Additionally, the deletion of fkbp3 led to the heightened resistance to FK506 compared to the wild-type strain (Fig.2A, 2C). These findings suggested that Fkbp3 is mainly involved in the modulation of FK506 resistance in A. flavus.

Previous studies have shown that lysine succinylation plays a significant role in aflatoxin production and pathogenicity in A. flavus ^[43]. Based on the analysis of our succinylome data, we have identified six reliable succinylation sites (K5, K19, K40, K42, K55, and K65) on the Fkbp3 protein. To identify the function of Fkbp3 succinylation sites, site-directed mutations were constructed according to the homologous recombination strategy. Compared to the wild-type strain, all point mutants exhibited a comparable phenotype in vegetative growth and conidiation. Interestingly, we observed that the K19A mutation resulted in increased resistance to rapamycin (Fig. 2E, 2F). Additionally, the K19A strain exhibited a significant reduction in the production of AFB₁ compared to the wild-type strain (Fig. 2G, 2H). These findings suggested that the succinylation of Fkbp3 at K19 plays a crucial role in rapamycin resistance and aflatoxin biosynthesis. Furthermore, we found that various point mutants, including K40A, K42A, and K55A, exhibited reduced levels of AFB₁ production in comparison to the wild-type strain (Fig. 2G, 2H), suggesting that the succinylation of Fkbp3 at other sites also contributes to AFB₁ biosynthesis in A. flavus. The above results indicated that the K19 site likely plays a more prominent role in Fkbp3 succinylation compared to other sites.

The Tor kinase plays a critical role in A. flavus

In S. cerevisiae, the Tor kinase can interact with a diverse range of proteins, resulting in the formation of complex structures known as TORC1 and TORC2 ^[44]. TORC1 can detect various signals, including nutrients, growth factors, and environmental stress. It plays a crucial role in regulating cellular processes such as gene transcription, protein translation, ribosome synthesis, and autophagy ^[45]. To identify the ortholog of the Tor protein in A. flavus, the S. cerevisiae Tor protein was utilized as search queries in the A. flavus genome database using the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The genome of A. flavus contains a solitary ortholog of the tor gene (AFLA_044350), which encodes a protein exhibiting 48.91% similarity to S. cerevisiae Tor2. We used the absolute quantitative PCR technique to identify the copy number of the tor gene at the genome level in A. flavus (Table S1). To elucidate the function of the Tor kinase in A. flavus, we employed gene deletion techniques to target the tor gene. While our attempt to obtain a null mutant was unsuccessful, we constructed a single copy deletion strain, tor+/−, through homologous recombination, which contains one copy of the tor gene (Table S1). Compared to the wild-type strain, the tor+/− strain exhibited a significantly slower growth rate on PDA (Fig. 3C). Additionally, the tor+/− strain produced fewer conidia and sclerotia compared to the wild-type strain (Fig. 3B, S4). The results from thin-layer chromatography (TLC) revealed a noteworthy decrease in AFB₁ synthesis in the tor+/− strain compared to the wild-type strain (Fig. 3D, 3F). Interestingly, the tor+/− mutant exhibited a notable increase in sensitivity to rapamycin and decreased sensitivity to osmotic stress (Fig. 3C, 3E, S5). Furthermore, the qRT-PCR results demonstrated a significant downregulation in the expression levels of the genes associated with sexual sporulation and aflatoxin synthesis in the tor+/− mutant compared to the wild-type strain (Fig. S6). The transcriptional level of the tor gene in the tor+/− mutant was downregulated compared to the level in the wild-type strain (Fig. S6). These findings suggested that the Tor kinase plays a crucial role in various biological processes in A. flavus, including vegetative growth, asexual development, sclerotia formation, response to environmental stress, and biosynthesis of aflatoxins.

The Tor kinase plays a vital role in *A. flavus*
(A) The structure diagram of the Tor kinase.
(B) Morphology of conidiophores in the WT, *tor+*/-, and *tor*^ΔFRBstrains observed by microscope.
(C) Phenotype of the WT, *tor+*/-, and *tor*^ΔFRBstrains grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.
(D) TLC assay of AFB₁ production by the WT, *tor+/−*, and *tor*^ΔFRBstrains cultured in YES liquid medium at 29°C for 6 days.
(E) The growth inhibition rate of the WT, *tor+*/-, and *tor*^ΔFRBstrains under rapamycin stress.
(F) AFB₁ quantitative analysis of the WT, *tor+*/-, and *tor*^ΔFRBstrains described in (D).

The Tor protein is characterized by the presence of various conserved domains, including the DUF3385, FAT, FKBP12-rapamycin binding (FRB), kinase catalytic, and FATC domains, which are located at the N-terminus (Fig. 3A). To investigate the functional significance of the FRB domain within the Tor kinase, the FRB domain was eliminated using homologous recombination. Compared to the tor+/− strain, the tor^ΔFRB mutant displayed comparable deficiencies in vegetative growth, conidiation, sclerotia formation, osmotic stress, and rapamycin sensitivity (Fig. 3B, 3C, S4, S5). Based on these observations, we speculated that the FRB domain plays a pivotal role in the modulation of the Tor kinase activity, thereby influencing fungal development and rapamycin resistance in A. flavus.

The Sch9 kinase participates in aflatoxin biosynthesis and the HOG pathway
(A) The structure diagram of the Sch9 kinase.
(B) Phenotype of the WT, Δ*sch9*, *sch9*^ΔC2, *sch9*^ΔS_TKc, *sch9*^ΔS_TK_X, and *sch9*^K340A strains grown on PDA medium amended with 100 ng/mL rapamycin at 37°C for 5 days.
(C) TLC assay of AFB₁ production from the WT, Δ*sch9*, *sch9*^ΔC2, *sch9*^ΔS_TKc, *sch9*^ΔS_TK_X, and *sch9*^K340A strains cultured in YES liquid medium at 29°C for 6 days.
(D) The phenotypes of the WT, Δ*sch9*, *sch9*^ΔC2, *sch9*^ΔS_TKc, *sch9*^ΔS_TK_X, and *sch9*^K340Astrains on YGT media amended with 1 M NaCl and 1 M KCl for 3 days.
(E) The phosphorylation levels of Hog1 in the WT and Δ*sch9* strains were determined with or without osmotic stress.
(F) The growth inhibition rate of the WT, Δ*sch9*, *sch9*^ΔC2, *sch9*^ΔS_TKc, *sch9*^ΔS_TK_X, and *sch9*^K340A strains under rapamycin stress.
(G) Quantitative analysis of AFB₁ as shown in (C).
(H) The growth inhibition rate of the WT, Δ*sch9*, *sch9*^ΔC2, *sch9*^ΔS_TKc, *sch9*^ΔS_TK_X, and *sch9*^K340A strains under osmotic stress.

TapA and TipA regulate sclerotia formation in *A. flavus*
(A) Phenotype of the WT, *tapA* +/−, and Δ*tipA* strains grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.
(B) Phenotypic characterization of the WT, *tapA* +/−, and Δ*tipA* strains grown on CM medium at 37°C for 7 days.
(C) Morphology of conidiophores in the WT, *tapA* +/−, and Δ*tipA* strains observed by microscope.
(D) TLC assay of AFB₁ production by the WT, *tapA* +/−, and Δ*tipA* strains cultured in YES liquid medium at 29°C for 6 days.
(E) The growth inhibition rate of the WT, *tapA* +/−, and Δ*tipA* strains under rapamycin stress.
(F) Amount of sclerotia produced by the WT, *tapA* +/−, and Δ*tipA* strains.
(G) AFB₁ quantitative analysis of the WT, *tapA* +/−, and Δ*tipA* strains described in (D).

The Sch9 kinase is involved in aflatoxin biosynthesis and the HOG pathway

Sch9, a Ser/Thr kinase belonging to the AGC family, is directly phosphorylated by TORC1. The phosphorylation of Sch9 is inhibited by rapamycin, as well as by carbon or nitrogen starvation ^[22]. The putative sch9 gene (AFLA_127440) in A. flavus encodes a protein consisting of 706 amino acids, exhibiting 80.31% identity to the Sch9 protein in A. nidulans. To investigate the role of Sch9 in A. flavus, we attempted to create a sch9 deletion mutant using a homologous recombination strategy. The Δsch9 strain exhibited typical vegetative growth and conidiation similar to the wild-type strain (Fig. 4B). TLC assay and quantitative analysis showed a significantly decreased aflatoxin production in the Δsch9 strain compared to the wild-type strain (Fig. 4C, 4G). These results indicated that Sch9 regulates aflatoxin biosynthesis in A. flavus.

In yeast, Sch9 plays an important role in the transcriptional activation of osmostress inducible genes ^[46]. To characterize the roles of Sch9 in stress response, we examined the sensitivity of the sch9 deletion strain to various stresses including osmotic stress and calcium stress. Compared with the wild-type strain, the Δsch9 mutant significantly increased sensitivity to calcium stress (Fig. S7), and decreased sensitivity to osmotic stress induced by NaCl and KCl (Fig.4D, 4H). In A. flavus, the Sch9 kinase consists of several domains, including the protein kinase C conserved region 2 (C2), protein kinase (S_TKc), AGC-kinase C-terminal (S_TK_X) domain, and an ATP binding site (Fig. 4A). To explore the role of these domains and the ATP binding site in Sch9 kinase, we constructed deletion strains for both domains and a sch9^K340A mutant. The results revealed that the sch9^ΔS_TKc strain, sch9^ΔS_TK_X strain, and sch9^K340A mutant exhibited similar phenotypes in osmotic stress and AFB₁ production as the Δsch9 mutant (Fig.4B, 4C, 4D). Therefore, we speculate that the protein kinase domain, kinase C-terminal domain, and the ATP binding site at K340 play a crucial role in modulating the impact of Sch9 on aflatoxin biosynthesis and stress response in A. flavus.

To investigate the potential function of Sch9 in the HOG and TOR pathways in A. flavus, we conducted a sensitivity analysis of the Δsch9 strain to rapamycin. Compared to the wild-type strain, the sch9 deletion strain exhibited a slightly higher sensitivity to rapamycin (Fig. 4B, 4F). The MAKP Hog1 is an element of the high osmolarity glycerol (HOG) pathway. To verify the connection between Sch9 and the HOG-MAPK pathway, the phosphorylation levels of Hog1 kinase were measured under osmotic stress (1M NaCl). Western blot analysis showed that the phosphorylation level of Hog1 increased significantly in the Δsch9 strain (Fig. 4E). This finding suggested that Sch9 plays a pivotal role in modulating the response of Hog1 to osmotic stress. Taken together, these results reflected that Sch9 regulates osmotic stress response via the HOG pathway in A. flavus.

TapA and TipA participate in regulation of sclerotia development and cell wall stress response

Tap42, a protein that associates with phosphatase 2A, serves as the direct target of the Tor kinase in yeast. Tap42 plays a crucial role in various Tor functions, particularly in the regulation of transcriptional processes ^[47]. The genome of A. flavus contains a homolog of tap42 named tapA (AFLA_092770), which is predicted to encode a 355 amino-acid protein (sharing 31.9% identity with S. cerevisiae Tap42). To further determine the function of the TapA regulator in the TOR signaling pathway of A. flavus, we conducted targeted gene deletion experiments on tapA. We failed to obtain a null mutant, therefore we generated the tapA+/− strain, which contains one copy of the tapA gene (Table S2). Compared to the wild-type strain, the tapA+/− strain exhibited diminished hyphal growth and conidiation (Fig. 5A, 5C), indicating that TapA is important for vegetative growth and conidiation. The results of the rapamycin sensitivity test indicated that the tapA+/− strain displayed reduced sensitivity to rapamycin (Fig.5A, 5E). In addition, the TapA+/− strain was unable to generate any sclerotia (Fig. 5B, 5F), and almost lost its capability in AFB₁ production compared to the wild-type strain (Fig. 5D, 5G), these findings indicated that TapA is essential for sclerotia formation and biosynthesis of AFB₁. The tapA+/− mutant was more insensitive to cell wall stress, including CFW and CR (Fig. S8). Furthermore, the levels of nsdC, nsdD, and sclR expression were significantly decreased in tapA+/− strain compared to those in the wild-type strain (Fig. S9). The transcriptional level of the tapA gene in the tapA+/− strain was drastically downregulated compared to the wild-type strain (Fig. S9). These findings have demonstrated the significantly positive impact of TapA on the growth, sporulation, sclerotia formation, cell wall stress, and AF biosynthesis in A. flavus.

In S. cerevisiae, Tip41 was identified as a Tap42-interacting protein and has been measured to function as a negative regulator of Tap42, which downregulates TORC1 signaling via activation of PP2A phosphatase ^[48]. The putative orthologue of tip41 in A. flavus named tipA (AFLA_047310). The amino acid sequence of TipA exhibits a similarity of 40.49% to Tap41 in S. cerevisiae. To elucidate the function of the TOR signaling pathway protein TipA in A. flavus, we generated the tipA deletion mutants. The ΔtipA strain exhibited a slightly reduced number of conidia produced on PDA compared to the wild-type strain (Fig. 5A). Meanwhile, the ΔtipA strain exhibited decreased sensitivity to rapamycin (Fig. 5A). The ΔtipA strain was unable to produce any sclerotia (Fig. 5B). In addition, the ΔtipA strain was more sensitive to cell wall and cell membrane stress, including CR and SDS (Fig. S8). Additionally, the expression levels of nsdC and nsdD exhibited a decrease in ΔtipA strain compared to the wild-type strain (Fig. S9). These results indicated that TipA is crucial for the sclerotial formation and cell wall stress in A. flavus.

Phosphatase SitA and Ppg1 are involved in growth, conidiation, and sclerotial formation

In S. cerevisiae, Tap42 has been identified to interact with the catalytic subunit of type 2A protein phosphatases, including Pph3, Pph21, and Pph22, as well as the type 2A-like phosphatases Sit4 and Ppg1^[19]. Phosphatase sitA (AFLA_108450) and ppg1 (AFLA_132610) were identified in A. flavus, encoding putative orthologs of sit4 and ppg1 in S. cerevisiae. To determine the biological functions of phosphatase SitA and Ppg1 in A. flavus, we disrupted the sitA and ppg1 by homologous recombination strategy. To further investigate the relationship between phosphatase SitA and Ppg1, we additionally generated complemented and double deletion mutants. The vegetative development phenotype analysis showed that the ΔsitA, Δppg1, and ΔsitA/Δppg1 strains grew significantly more slowly than the wild-type strain on PDA (Fig. 6A, 6D). Additionally, the ΔsitA, Δppg1, and ΔsitA/Δppg1 strains were more sensitive to rapamycin (Fig. 6A, 6E). In addition, the ΔsitA, Δppg1, and ΔsitA/Δppg1 strains formed lower number of conidiophores (Fig. 6A). To gain further insight into the role of SitA and Ppg1 in the process of conidiation, qRT-PCR was employed to determine the transcript levels of conidial synthesis-related genes (abaA and brlA). The expression levels of the abaA and brlA genes were significantly downregulated in the ΔsitA, Δppg1, and ΔsitA/Δppg1 mutants when compared to the wild-type and complemented strains (Fig. 6G). The analysis of sclerotial formation revealed that the ΔsitA, Δppg1, and ΔsitA/Δppg1 mutants exhibited a complete inability to produce any sclerotia (Fig. 6B). To further prove these findings, we conducted qRT-PCR to determine the expression levels of three regulators (nsdC, nsdD, and nclR) of sclerotia development. Our findings indicated that the expression levels of nsdC, nsdD, and sclR were all significantly reduced in the ΔsitA, Δppg1, and ΔsitA/Δppg1 mutants (Fig. 6H), which is consistent with the observed decrease in sclerotia production in the ΔsitA, Δppg1, and ΔsitA/Δppg1 mutants. These results showed that SitA and Ppg1 are crucial for hyphal development, conidiation, and sclerotia formation in A. flavus.

The impact of the phosphatases SitA and Ppg1 on the growth, conidiation, and aflatoxin biosynthesis in *A. flavus*
(A) Colony morphology of the WT, single knockout, and double knockout strains cultured on PDA medium amended with 100 ng/mL rapamycin at 37℃ for 5 days.
(B) Colony morphology of the WT, single knockout, and double knockout strains cultured on WKM medium at 37℃ for 7 days.
(C) TLC analysis of AFB₁ production from the WT, single knockout, and double knockout strains cultured in YES liquid medium at 29℃ for 6 days.
(D) Growth diameter of the WT, single knockout, and double knockout strains on PDA media.
(E) The growth inhibition rate of the WT, single knockout, and double knockout strains under rapamycin stress.
(F) AFB₁ quantitative analysis of the WT, single knockout, and double knockout strains.
(G) Relative expression levels of conidia synthesis genes in the WT, single knockout, and double knockout strains.
(H) Relative expression levels of sclerotia synthesis genes in the WT, single knockout, and double knockout strains.
(I) Relative expression levels of aflatoxin biosynthesis genes in the WT, single knockout, and double knockout strains.

SitA and Ppg1 play crucial roles in aflatoxin biosynthesis and pathogenicity

We assayed AFB₁ biosynthesis in the ΔsitA, Δppg1, and ΔsitA/Δppg1 mutants. TLC analyses revealed that the ΔsitA and ΔsitA/Δppg1 strains exhibited a decrease in AFB₁ production, whereas the Δppg1 strain showed a significant increase in AFB₁ production compared to the wild-type and complemented strains (Fig. 6C, 6F). The expression levels of genes related to aflatoxin synthesis, including structural genes (aflO and aflP) and regulatory genes (aflR and aflS), were determined by qRT-PCR in the ΔsitA, Δppg1, and ΔsitA/Δppg1 mutants. The results revealed a significant downregulation of these genes in the ΔsitA and ΔsitA/Δppg1 mutants, while upregulated in the Δppg1 mutants, compared to the wild-type and complemented strains (Fig. 6I). These results indicated that SitA and Ppg1 play different roles in regulating aflatoxin biosynthesis in A. flavus.

We conducted pathogenicity tests on peanuts and maize to examine the pathogenicity of the phosphatases SitA and Ppg1 on crops. The results showed that the wild-type and complemented strains demonstrated complete virulence on all peanut and maize seeds. In contrast, the ΔsitA, Δppg1, and ΔsitA/Δppg1 mutants displayed compromised colonization on peanut and maize seeds (Fig. 7A). In addition, the number of conidial productions in these mutants on the infected peanut seeds also dramatically decreased compared to those of the wild-type and complemented strains (Fig. 7C). The thin-layer chromatography analyses revealed that the Δppg1 mutant exhibited increased production of AFB₁ in peanuts and maize. Conversely, the levels of aflatoxin produced by the ΔsitA and ΔsitA/Δppg1 mutants on peanut and maize were significantly reduced compared to the the wild-type and complemented strains (Fig. 7B, 7D). This observation aligns with the above results of aflatoxin biosynthesis from all mutant strains in the YES medium. All these data showed that SitA and Ppg1 are crucial for crop seeds’ pathogenicity.

Pathogenicity analysis of the phosphatase SitA and Ppg1 in *A. flavus*
(A) Phenotypes of peanut and maize infected by the WT, single knockout, and double knockout strains.
(B) TLC was used to detect the AFB₁ from peanut and maize infected by the WT, single knockout, and double knockout strains.
(C) Quantitative analysis of conidia production from peanut and maize infected by the WT, single knockout, and double knockout strains.
(D) Quantitative analysis of AFB₁ from peanut and maize infected by the WT, single knockout, and double knockout strains.

SitA and Ppg1 are involved in regulating cell wall integrity

To explore the role of phosphatases SitA and Ppg1 in response to cell wall stress, all strains were cultivated on PDA amended with CFW, CR, and SDS. We found that the relative growth inhibition of ΔsitA, Δppg1 and ΔsitA/Δppg1 mutants induced by cell wall stress were significantly increased compared to the wild-type and complemented strains (Fig. 8A, 8B). Further investigations using qRT-PCR revealed a notable reduction in the expression levels of genes related to the synthesis of cell walls, specifically chitin synthase genes chsA, chsD, chsE, and β-1,6, in the ΔsitA, Δppg1, and ΔsitA/Δppg1 strains (Fig. 8C). Western blot analysis also revealed that the phosphorylation level of Slt2 was significantly elevated in the ΔsitA, Δppg1, and ΔsitA/Δppg1 strains under cell wall stress (200 μg/ml CFW) (Fig. 8D). In conclusion, the phosphatases SitA and Ppg1 play a crucial role in regulating cell wall integrity.

Sensitivity of phosphatase SitA and Ppg1 to cell wall damaging agents in *A. flavus*
(A) Morphology of the WT, single knockout, and double knockout strains grown on YES media supplemented with 200 µg/mL CR, 200 µg/mL CFW, or 100 µg/mL SDS at 37°C for 5 days.
(B) The growth inhibition rate of the WT, single knockout, and double knockout strains under cell wall and cell membrane stress.
(C) Relative expression levels of cell wall synthesis genes (*chsA*, *chsD*, *chsE* and *β-1,6*) in the WT, single knockout, and double knockout strains.
(D) The phosphorylation level of Slt2 in the WT, single knockout, and double knockout strains was detected with or without cell wall stress.

The phosphatase complex Nem1/Spo7 are important for growth, aflatoxin biosynthesis, and LD biogenesis

Lipid droplets (LDs) are highly dynamic spherical organelles, which appear in the cytosol of most eukaryotic cell types^[49]. Rapamycin treatment led to the accumulation of lipid droplets in various filamentous fungi ^[24]. The FgTOR pathway regulates the phosphorylation status of FgNem1 mainly via the FgPpg1/Sit4-FgCak1/other kinase cascade in F. graminearum ^[24]. In this study, we observed that the treatment of hyphae with rapamycin resulted in the accumulation of LD biogenesis (Fig. 9A). This finding suggested that the TOR pathway may regulate the LD biogenesis. However, the underlying regulatory mechanism of LD biosynthesis remain largely unknown in A. flavus. To investigate the regulatory mechanism of the TOR pathway in A. flavus that controls LD biogenesis, we examined LD accumulation in Δsch9, ΔsitA, and Δppg1 mutants. BODIPY staining assays revealed that LD biogenesis was not observed in the ΔsitA and Δppg1 strains upon treatment with rapamycin, in contrast to the wild-type and Δsch9 strains (Fig. 9A, 9B, 9C, 9D). These results indicated that LD biogenesis induced by rapamycin is predominantly reliant on the phosphatases SitA and Ppg1 in A. flavus. In F. graminearum, the phosphatase FgNem1/FgSpo7 complex was involved in the induction of LD biogenesis by rapamycin. Phosphatase nem1 (AFLA_002649) and spo7 (AFLA_028590) were identified in A. flavus, which encode putative orthologs of nem1 and spo7 in F. graminearum. To examine the role of the nem1 and spo7 genes in A. flavus, we generated Δnem1 and Δspo7 mutants using homologous recombination. The Δnem1 and Δspo7 mutants displayed a notable reduction in both vegetative growth and conidiation compared to the wild-type strain (Fig. 10A, 10C, 10D). TLC assay revealed a significantly decreased aflatoxin production in the Δnem1 and Δspo7 compared to the wild-type strain (Fig. 10B). In addition, BODIPY staining showed no visible lipid droplets in the hyphae of Δnem1 and Δspo7 after rapamycin treatment (Fig. 10E). Taken together, these results indicated that Nem1/Spo7 are involved in vegetative growth, conidiation, aflatoxin production, and LD biogenesis.

Phosphatase SitA and Ppg1 are involved in regulating lipid droplet biogenesis
(A) The intracellular lipid droplets were stained by boron dipyrromethene difluoride (BODIPY) in hyphae of the WT strain and observed under fluorescence microscopy. The phenotype of lipid droplets accumulation in the mycelia of the WT strain treated with or without 100 ng/mL rapamycin for 6 h (Bar, 10 µm).
(B) Phenotype of lipid droplets accumulation in the mycelia of the Δ*sch9* strain treated with or without 100 ng/mL rapamycin for 6 h (Bar, 10 µm).
(C) Phenotype of lipid droplets accumulation in the mycelia of the Δ*sitA* strain treated with or without 100 ng/mL rapamycin for 6 h (Bar, 10 µm).
(D) Phenotype of lipid droplets accumulation in the mycelia of the Δ*ppg1* strain treated with or without 100 ng/mL rapamycin for 6 h (Bar, 10 µm).

Phosphatase complex Nem1/Spo7 play significant role in growth, conidiation, aflatoxin, and lipid droplet biogenesis
(A) Phenotype of the WT, Δ*nem1*, and Δ*spo7* strains grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.
(B) TLC assay of AFB₁ production from the WT, Δ*nem1*, and Δ*spo7* strains cultured in YES liquid medium at 29°C for 6 days.
(C) Growth diameter of the WT, Δ*nem1*, and Δ*spo7* strains on PDA media.
(D) Statistical analysis of the sporulation by the WT, Δ*nem1*, and Δ*spo7* strains.
(E) Phenotype of lipid droplets accumulation in the mycelia of the WT, Δ*nem1*, and Δ*spo7* strains treated with or without 100 ng/mL rapamycin for 6 h (Bar, 10 µm).

Discussion

Aflatoxins (AFs) are a class of toxic secondary metabolites synthesized by A. flavus, including aflatoxin B₁, B₂, G₁, and G₂, among which AFB₁ has been regarded as the most pathogenic and toxic aflatoxin^[50]. Therefore, understanding the regulatory mechanisms involved in fungal secondary metabolites biosynthesis would be instrumental for effectively addressing aflatoxin control in A. flavus. In this study, we demonstrated that the target of rapamycin (TOR) signaling pathway plays a pivotal role in regulating growth, sporulation, sclerotia formation, aflatoxin production, and various stress response in A. flavus. Through conducting functional studies on genes within the TOR signaling pathway, we explored the conservation and complexity exhibited by the TOR signaling pathway in A. flavus. In addition, we constructed a crosstalk network between the TOR and other signaling pathways (HOG, CWI) and analyzed the specific regulatory process. We proposed that the TOR signaling pathway interacts with multiple signaling pathways, including HOG and CWI pathway, to modulate the cellular responses to diverse environmental stresses (Fig. 11).

The proposed model of the target of rapamycin (TOR) pathway in *A. flavus*
Based on the above results, we propose a hypothesis regarding the TOR signaling pathway model in *A. flavus.* Rapamycin forms a complex with Fkbp3, and this particular complex can bind to the Tor kinase, thereby impeding its regular functionality. Sch9, functioning as a downstream element of the Tor kinase, regulates aflatoxin biosynthesis and HOG signaling pathway. As another important target of the Tor kinase, TapA-phosphatase complex is involved in the regulation of growth, sporulation, sclerotia, aflatoxin production, CWI signaling pathway, lipid droplet synthesis, and other processes. In conclusion, the TOR signaling pathway plays a crucial role in various aspects of *A. flavus*, including vegetative development, stress response, aflatoxin biosynthesis, and pathogenicity.

We have observed that rapamycin has a significant impact on the growth and sporulation of A. flavus (Fig. 1A). Additionally, it exhibits a strong inhibitory effect on sclerotia production and aflatoxin biosynthesis (Fig. 1B, 1C). The inhibitory effect has been documented in various fungal species, including Candida albicans ^[51], Cryptococcus neoformans ^[51], Podospora anserine ^[27], A. nidulans ^[28], Magnaporthe oryzae ^[39], and F. graminearum ^[23]. This suggested that the TOR signaling pathway may have a conserved function in filamentous fungi. The number of FK506-binding proteins varies among different species: there are four Fkbps protein in S. cerevisiae and three Fkbps protein in S. pombe ^[52]. In S. cerevisiae, rapamycin can bind to Fkbp protein, resulting in the irreversible inhibition of the G₁ phase of the cell cycle and the regulation of cell growth ^[53]. In A. nidulans, the fkbp12 gene homologue fprA displays considerable sequence similarity with homologues identified in the S. cerevisiae ^[28]. In this study, based on the homologous alignment of the protein sequence of FprA, it was observed that four fkbp genes present in A. flavus. Among these, deletion of fkbp3 exhibited enhanced tolerance to rapamycin (Fig. 2A). So we speculated that rapamycin forms a complex with Fkbp3 in A. flavus, subsequently targeting downstream target genes in order to exert its biological function. We found that Fkbp3 is involved in sclerotia and aflatoxin biosynthesis in A. flavus (Fig. 2A, S3), and subsequent analysis revealed that the underlying mechanism may be associated with succinylation modification. The succinylation sites of Fkbp3 were identified through the utilization of succinylation proteomics data. By constructing point mutant strains and conducting phenotype experiments, we found that the succinylation of Fkbp3 at residue K19 is involved in rapamycin resistance and aflatoxin biosynthesis (Fig. 2E, 2G). Previous studies have demonstrated that Fkbp12 has been identified as the receptor for FK506 and rapamycin, which inhibits calcineurin and target of rapamycin complex 1 (TORC1), respectively ^[53]. The deletion of Fkbp12 confers the resistance to rapamycin in C. albicans ^[51] and C. neoformans ^[51]. The entomopathogenic fungus Beauveria bassiana contains three putative fkbp genes, and disruption of Bbfkbp12 significantly increases the resistance to rapamycin and FK506 ^[54]. The plant pathogenic fungus F. graminearum harbors three Fkbps proteins, among which deletion of Fgfkbp12 leads to resistance to rapamycin ^[23]. Thus, we propose that these Fkbps may exhibit analogous functionalities in conferring resistance to rapamycin. In Botrytis cinerea, deletion of Bcfkbp12 cause a reduction of the virulence of strain T4, while knockout of the fkbp12 gene did not affect the pathogenic development of the strain B05.10 ^[32]. These findings demonstrated that the fkbp12 homologous genes exhibited conservation in terms of rapamycin resistance. However, there is significant variation in other biological functions among fungal species, which may be attributed to species or strain evolutionary divergence.

The Tor kinase functions as a fundamental component of the TOR signaling pathway, which exhibits evolutionary conservation across fungi, plants, and mammals ^[55]. TOR assembles into two distinct multi-protein complexes known as TORC1 and TORC2. A characteristic that distinguishes TORC1 is its interaction with KOG1 (Raptor in mammals) and its sensitivity to the rapamycin-FKBP12 complex ^[56]. Almost all other eukaryotes, including plants, worms, flies, and mammals, have a single tor gene, whereas S. cerevisiae has two tor genes ^[45]. The Tor kinase is widely conserved both in terms of its structural characteristics and its role as the target of Fkbp-rapamycin. Typically, the Tor kinase exhibits comparable structural characteristics, including the HEAT-like, FAT, FRB, kinase, and FATC domains. The FRB domain is the binding region of the Fkbp-rapamycin complex, and rapamycin resistance is usually caused by the disruption of the FRB domain ^[45]. The comparison of homology and the analysis of phylogenetics revealed that there is only one predicted tor gene in A. flavus. To ascertain its functionality, we generated the tor single-copy deletion mutant by homologous recombination. We discovered that the Tor kinase is involved in the vegetative growth and asexual development of A. flavus (Fig. 3B, 3C). In addition, the tor+/− mutant shows a high sensitivity to rapamycin (Fig. 3C). Our findings have demonstrated the significant involvement of Tor kinase and its FRB domain in the processes of morphogenesis, aflatoxin biosynthesis, and multiple stress responses in A. flavus. We speculate the common expectation that the deletion of the tor and FRB domain should result in insensitivity and resistance to rapamycin, as it disrupts the binding site for Fkbp-rapamycin. However, we observed that the tor+/− and FRB domain-deleted mutant were more sensitive to rapamycin. This intriguing result suggests that there are additional factors or complexities involved in TOR signaling pathway regulation in A. flavus. We hypothesize that this result is related to the double copy of the tor gene. Previous findings demonstrated that the Tor kinase could function as a molecular switch connecting an iron cue to defend against pathogen infection in C. elegans ^[57]. In A. fumigatus, the Tor kinase represents a central regulatory node controlling genes and proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis, and degradation, as well as adaptation to low iron conditions^[58]. In summary, the Tor kinase acts as a central regulator in the TOR signaling pathway, playing a crucial role in the regulation of the cell cycle, protein synthesis, and cellular energy metabolism.

In S. cerevisiae, the protein kinase Sch9 is a major downstream effector of TORC1, and this kinase is known to have crucial functions in stress resistance, longevity, and nutrient sensing ^[46]. In addition, the TORC1-Sch9 pathway acts as a crucial mediator of chronological lifespan ^[59]. In filamentous fungi, an intricate network of interactions exists between the TOR and HOG pathways. In F. graminearum ^[38], FgSch9 and FgHog1 mutants displayed heightened susceptibility to osmotic and oxidative stresses. The phosphorylation level of FgHog1 increased significantly in FgSch9 mutant. Additionally, the affinity capture-MS assay showed that the Tor kinase was among the proteins co-purifying with FgSch9. These results suggest that FgSch9 serves as an intermediary for the TOR and HOG signaling pathways ^[38]. Furthermore, FgSch9 plays a crucial role in the regulation of various biological processes such as hyphal differentiation, asexual development, virulence, and DON biosynthesis^[38]. In A. fumigatus, the SchA mutant was sensitive to rapamycin, high concentrations of calcium and hyperosmotic stress, and SchA was involved in iron metabolism. The SchA mutant exhibited enhanced phosphorylation of SakA in response to osmotic stress ^[51]. In this study, we discovered that Sch9 is involved in regulating aflatoxin biosynthesis (Fig.4C), and responses to osmotic stress and rapamycin stress (Fig.4B, 4D). The S_TKc domain deletion strain (sch9^ΔS_TKc), S_TK_X domain deletion strain (sch9^ΔS_TK_X), and sch9^K340A mutant displayed a similar phenotype to the Δsch9 strain (Fig. 4B, 4C, 4D). These findings revealed that Sch9 plays a significant role in the biological synthesis of aflatoxin and in responding to various stresses. This role is primarily mediated through its kinase domain, kinase-C domain, and ATP binding site k340. We also found that the phosphorylation level of Hog1 increased significantly in the Δsch9 strain under osmotic stress (Fig. 4E). Based on the above results, we hypothesized that Sch9 was involved in the osmotic stress response by modulating the HOG pathway.

In S. pombe, the protein Tip41, which interacts with Tap42, plays a significant role in the cellular responses to nitrogen sources by regulating type 2A phosphatases ^[60]. In S. cerevisiae, Tap42-phosphatase complexes associate with TORC1, whereas Tip41 functions to attenuate TORC1 signaling by stimulating the PP2A phosphatase ^[48]. Unlike its yeast counterpart Tip41, TIPRL, a mammalian Tip41-like protein, exerts a positive influence on mTORC1 signaling by interacting with PP2Ac ^[61]. While the interaction between Tip41 and Tap42 was not observed in the filamentous fungi. The yeast two-hybrid (Y2H) assay showed that FgTip41 interacts with the phosphatase FgPpg1 rather than FgTap42, while the FgTap42 bind with phosphatases FgPp2A, implying that FgTap42, FgPpg1, and FgTip41 form a heterotrimer in F. graminearum ^[23]. In the rice blast fungus M. oryzae, MoTip41 does not interact with the MoTap42. However, MoTip41 has the ability to bind with the phosphatase MoPpe1, thereby facilitating crosstalk between the TOR pathway and the cell wall integrity pathway ^[40]. In this study, we found that the TapA and TipA played a crucial role in sclerotia production and cell wall stress in A. flavus (Fig. 5B, S8). The tapA single-copy deletion mutant and the tipA deletion mutant exhibited decreased sensitivity to rapamycin (Fig. 5A). However, the interaction between TapA and TipA is not yet clear in A. flavus. We speculated that the interrelations and functions of TapA and TipA in A. flavus are comparable to their ortholog in other filamentous fungi.

In S. cerevisiae, Tap42 can bind and regulate various PP2A phosphatases, including Pph3, Pph21, Pph22, Sit4, and Ppg1. This interaction plays a crucial role in the TOR signaling pathway, particularly in response to rapamycin treatment or nutrient deprivation ^[48]. The phosphatase Sit4 plays a crucial role in regulating cell growth, morphogenesis, and virulence in C. albicans ^[62]. FgSit4 and FgPpg1 are associated with various cellular processes, including mycelial growth, conidiation, DON biosynthesis, and virulence in F. graminearum ^[23]. In addition, FgSit4 and FgPpg1 are involved in the regulation of various signaling pathways, such as the CWI pathway and lipid droplets biogenesis regulation pathway ^[23]. In this study, we found that the phosphatase SitA and Ppg1 play critical role in vegetative growth, conidiation, sclerotia formation, and aflatoxin biosynthesis in A. flavus (Fig.6A, 6B, 6C). Additionally, the phosphatase SitA and Ppg1 also affect pathogenicity and LD biogenesis (Fig. 7A, 9C, 9D). In addition, TapA/TipA and SitA/Ppg1 exhibits similar functions in sclerotia production and cell wall stress response. Based on these observations, we hypothesized that TapA-phosphatase complexes play a collaborative role in the regulation of the sclerotia formation and cell wall stress response in A. flavus.

After the deletion of the sitA and ppg1 genes, the expression level of genes related to cell wall synthesis has decreased significantly, resulting in damage to the cell wall and showing a higher sensitivity to cell wall integrity stress (Fig. 8C). Western blotting results showed that the phosphorylation level of MAPK kinase Slt2 has increased significantly in the ΔsitA and Δppg1 strains under cell wall stress, indicating that SitA and Ppg1 exert a negative regulatory effect on the CWI pathway (Fig. 8D). In M. oryzae, MoPpe1 and MoSap1 serve as an adaptor complex that connects the CWI and TOR signaling pathways. Activation of the TOR pathway results in the inhibition of the CWI pathway ^[39]. In addition, the phosphatase MoPpe1 interacts with the phosphatase-associated protein MoTip41, facilitating the exchange of information between the TOR and CWI signaling pathways ^[39]. These results suggest that the phosphatase Ppe1 engages in interactions with various proteins to effectively coordinate the TOR and CWI signaling pathways, thereby regulating the growth and pathogenicity of the rice blast fungus M. oryzae ^[39]. Taken together, we speculated that the phosphatase SitA and Ppg1 might exhibit differential regulation in the CWI and TOR pathways across species in order to fulfill their respective functions.

In F. graminearum, the phosphatase complex FgNem1/Spo7-FgPah1 cascade plays significant roles in fungal development, DON production, and lipid droplet (LD) biogenesis ^[24]. In this study, our results indicated that Nem1 and Spo7 are important for vegetative growth and secondary metabolism of A. flavus (Fig. 10A, 10B). We have also discovered that LD biogenesis is facilitated by the Nem1/Spo7 and SitA/Ppg1 pathways (Fig. 9C, 9D, 10E). These observations suggest that the PP2A phosphatases play a central role as core phosphatases in the LD biogenesis signaling network. However, it is likely that there are other kinases involved in the regulation of LD biogenesis. To gain a comprehensive understanding of the precise molecular mechanism, further research should focus on investigating the interplay between phosphatases and other kinases.

In conclusion, we identified the complex regulatory network that provides insight into the interplay between the TOR, HOG, and CWI signaling pathways, and how they collectively regulate the development, aflatoxin biosynthesis, and pathogenicity of A. flavus. By elucidating the functions of genes associated with the TOR signaling pathway, our research could establish a solid foundation for comprehending the molecular mechanism underlying the aflatoxin biosynthesis of A. flavus. This, in turn, can provide novel insights into the management of aflatoxin contamination and the mitigation of A. flavus infection.

Materials and methods

Strains and cultural conditions

The A. flavus strains in this study were shown in Table S3. All strains were cultivated on three different types of agar media: potato dextrose agar (PDA), yeast extract-sucrose agar (YES), and yeast extract-glucose agar (YGT). The growth and conidiation assays were conducted at 37℃. Additionally, aflatoxin production was assessed using YES liquid medium at 29℃.

Constructions of gene deletion and complemented mutants

The construction of gene deletion and complemented strains was carried out using the SOE-PCR strategy, following previously established protocols ^[63]. The primers utilized for the amplification of the flanking sequences of each gene can be found in Table S3. The putative gene deletion mutants were validated through PCR assays using the appropriate primers (Fig. S2), and their confirmation was subsequently obtained through sequencing analysis.

Detection of tor and tapA genes copy number in strains

Absolute quantification PCR has been used as a common method for molecular detection of gene copy numbers. According to previous reports, the sumo gene is a single-copy gene in A. flavus^[64]. Following previously established protocols^[65], the primers were used to design the amplification of the sumo reference gene and tor and tapA gene. Using sumo gene as reference, the tor and tapA gene copy number was calculated by standard curve.

Growth, conidiation, and sclerotia analysis

To evaluate mycelial growth and conidia formation, 1 μL of 10⁶ spores/mL conidia of all strains was point inoculated onto the center of PDA medium, and then cultured at 37℃ for 5 days in the dark. The conidia were washed by 0.07% Tween-20 and counted using a hemocytometer and microscope ^[43]. For sclerotia formation analysis, each strain was inoculated and cultivated on CM agar medium at 37℃ in the dark for 7 days, and then 75% ethanol was used to wash away mycelia and conidia on the surface of the medium ^[64]. These assays were repeated at least three times.

Extraction and quantitative analysis of aflatoxin

To extract and quantify aflatoxins (AFs) production, 1 μL of 10⁶ spores/mL conidia of all strains was point inoculated onto liquid YES medium and cultures were then incubated at 29℃ in the dark for 6 days. AFs were extracted from the media and detected using thin layer chromatography (TLC) ^[65]. In addition, the standard AFB₁ was employed for visual comparison, and AFs were quantitatively analyzed using the Image J tool. The assays were repeated at least three times.

Stress assay

To determine the roles of the gene in A. flavus responsing to various environmental stresses, 1 μL of 10⁶ spores/mL conidia of all strains were point inoculated onto these PDA solid plates supplemented with various agents. These agents included 1M NaCl, 1M KCl, and 1M CaCl₂ for inducing osmotic stress, as well as 100 µg/mL SDS, 200 µg/mL Calcofluor white (CFW), and 200 µg/mL Congo red (CR) for inducing cell wall stress. After 5 days of incubation at 37℃ in the dark, the relative rates of inhibition were determined ^[43]. The assays were repeated at least three times.

Seeds infection assays

To measure the pathogenicity of A. flavus, all peanut and maize seeds were washed in 0.05% sodium hypochlorite, 75% ethanol, and sterile water, respectively. Subsequently, all peanut and maize seeds were immersed in sterile water containing 10⁶ spores/mL of all strains at 29℃ in the dark for 60 min. After the conidia adhesion, the seeds were cultured at 29℃ for 6 days in the dark ^[64].

Quantitative RT-PCR

For the real-time quantitative PCR (qPCR) assays, the A. flavus strains were cultivated in PDA medium at 29℃ for 3 days in the dark. Total RNA was extracted from the mycelia of all strains, and cDNA was synthesized with the First Strand cDNA Synthesis Kit, and then the cDNA was used as the template for qRT-PCR analysis with SYBR Green qPCR mix. The relative transcript level of each gene was calculated by the 2^-ΔΔCTmethod ^[66].

Western blot analysis

To extract the total protein, the mycelia of each strain were cultivated in YES liquid medium for 48 h, then the sample was grinded under liquid nitrogen to a fine powder and transferred to a 1.5 mL microcentrifuge tube added with RIPA lysis buffer and 1 mM PMSF protease inhibitor. Proteins were separated by 12% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. Phosphorylation levels of Hog1 and Slt2 were detected by Phospho-p38 MAPK antibody (Cell Signaling Technology, Boston, MA, USA) and Phospho-p44/42 MAPK (Erk 1/2) antibody (Cell signaling Technology, MA, USA), respectively. The experiment was conducted three times independently.

Statistical analysis

All data were measured with means ± standard deviation (SD) of three biological replicates. Graph Pad Prism 7 software was used for conducting statistical and significance analysis, and p-values < 0.05 was represented statistical significance. Student’s t-test was used to compare the two means for differences, whereas Tukey’s multiple comparisons were used for testing multiple comparisons.

Acknowledgements

The authors also thank Professor Zhenhong Zhuang, Jun Yuan, Yu Wang, Kunzhi Jia, Xiuna Wang, and Xinyi Nie for their help in the experiments. This work was supported by the National Natural Science Foundation of China (Grant No. 31972214).

Author contributions

Guoqi Li and Xiaohong Cao designed the experiments. Guoqi Li, Xiaohong Cao, and Qianhua Zeng performed the experiments. Guoqi Li, Xiaohong Cao, and Shihua Wang analyzed the data. Guoqi Li, Elisabeth Tumukunde, and Shihua Wang wrote the manuscript. The manuscript was discussed and revised by all the authors.

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Article and author information

Author information

Guoqi Li
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
ORCID iD: 0009-0005-6512-0892
Xiaohong Cao
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
Elisabeth Tumukunde
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
Qianhua Zeng
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
Shihua Wang
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
ORCID iD: 0000-0002-1544-5972
- Corresponding author: Name: Shihua Wang Professor, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China, Phone/Fax: +86-591-83789312, E-mail: wshyyl@sina.com

Author Notes

These authors contributed equally to this work.

Version history

Sent for peer review: May 23, 2023
Preprint posted: June 18, 2023
Reviewed Preprint version 1: July 19, 2023
Reviewed Preprint version 2: January 5, 2024
Reviewed Preprint version 3: March 8, 2024
Reviewed Preprint version 4: June 11, 2024
Version of Record published: July 11, 2024

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers.

Reviewing Editor
Gustavo Goldman
Universidade de Sao Paulo, Sao Paulo, Brazil
Senior Editor
Jonathan Cooper
Fred Hutchinson Cancer Research Center, Seattle, United States of America

Reviewer #1 (Public Review):

Their absolute quantification PCR results with the sumo reference gene led the authors to conclude that A. flavus has two copies of tor and tapA in its genome. However, the the genomic location of the additional copies of tor and tapA are unknown.

I have concerns about the conclusion for the following reasons:

First, the authors should provide more convincing data showing that tor and tapA genes are indeed duplicated genes in A. flavus. The authors appeared to use the A. flavus PTS strain as a parental strain for constructing the tor and tapA mutants. If so, the A. flavus CA14 strain (Hua et al., 2007) should be the parental wild-type strain for the A. flavus PTS strain. I did a BLAST search in NCBI for the torA (AFLA_044350) and tapA (AFLA_092770) genes using the most recent CA14 genome assembly sequence (GCA_014784225.2) and only found one allele for each gene: torA on chromosome 7 and tapA on chromosome 3. I could not find any other parts with similar sequences. Even in another popular A. flavus wild-type strain, NRRL3357, both torA and tapA exist as a single allele. Based on the published genome assembly data for A. flavus, there is no evidence to support the idea that tor and tapA exist as copies of each other. Therefore, the authors could perform a Southern blot analysis to further verify their claim. If torA and tapA indeed exist as duplicate copies in different chromosomal locations, Southern blot data could provide supporting results.

If the tor and tapA genes indeed exist as dual copies, do the duplicate genes have identical DNA and protein sequences? If they have different DNA or protein sequences, they should be named differently as paralogs, such as torA and torB or tapA and tapB.

Second, the authors should consider the possibility of aneuploidy for their constructed mutants. When an essential gene is targeted for deletion, aneuploidy often occurs even in a fungal strain without the "ku" mutation, which results in seemingly dual copies of the gene. As the authors appear to use the A. flavus PTS strain having the "ku" mutation, the parental strain has increased genome instability, which may result in enhanced chromosomal rearrangements. So, it will be necessary to Illumina-sequence their tor and tapA mutants to make sure that they are not aneuploidy.

Furthermore, the genetic nomenclature +/- and -/- should be reserved for heterozygous and homozygous mutants in a diploid strain. As A. flavus is not a diploid strain, this type of description could cause confusion for the readers.

https://doi.org/10.7554/eLife.89478.2.sa2

Reviewer #2 (Public Review):

In this study, authors identified the complex TOR, HOG and CWI signaling networks-involved genes that relatively modulate the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation.

They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, especially found that the conserved site K19 of FKBP3 plays a key role in regulating the aflatoxin biosynthesis. In general, there is heavy workload task carried in this study and the findings are interesting and important for understanding or controlling the aflatoxin biosynthesis. However, findings have not been deeply explored and conclusions are mostly are based on parallel phenotypic observations. In addition, there are some concerns for the conclusions.

https://doi.org/10.7554/eLife.89478.2.sa1

Reviewer #3 (Public Review):

The paper by Li et al. describes the role of the TOR pathway in Aspergillus flavus. The authors tested the effect of rapamycin in WT and different deletion strains. This paper is based on a lot of experiments and work but remains rather descriptive and confirms the results obtained in other fungi. It shows that the TOR pathway is involved in conidiation, aflatoxin production, pathogenicity, and hyphal growth. This is inferred from rapamycin treatment and TOR1/2 deletions. Rapamycin treatment also causes lipid accumulation in hyphae. The phenotypes are not surprising as they have been shown already for several fungi. In addition, one caveat is in my opinion that the strains grow very slowly and this could cause many downstream effects. Several kinases and phosphatases are involved in the TOR pathway. They were known from S. cerevisiae or filamentous fungi. The authors characterized them as well with knock-out approaches.

https://doi.org/10.7554/eLife.89478.2.sa0

Author Response

The following is the authors’ response to the original reviews.

Response to Reviewer 1 Comments (Public Review):

Point 1: While the authors provided a large amount of data regarding the genes involved in the TOR pathway, it is highly descriptive and mostly confirmative data, as numerous papers have already shown that the TOR pathway plays essential roles in a myriad of biological processes in multiple fungi.

Response 1: Thank you for your comment. The target of rapamycin (TOR) signal pathway plays critical roles in various eukaryotic organisms. However, its specific role in controlling the development and virulence of opportunistic pathogenic fungi like A. flavus has remained unclear. Additionally, the underlying mechanism of the TOR pathway remains elusive in the A. flavus. As such, our study provides a useful contribution, as it is the first to comprehensively investigate the majority of genes in the conserved TOR signaling pathway in A. flavus.

Point 2: The authors seemed to perform a series of parallel studies in several genes involved in the TOR pathway in other fungi. However, their data are not properly interconnected to understand the TOR signaling pathway in this fungal pathogen. The authors frequently drew premature conclusions from basic phenotypic observations. For instance, based on their finding that sch9 mutant showed high calcium stress sensitivity, they concluded that Sch9 is the element of the calcineurin-CrzA pathway. Furthermore, based on their finding that the sch9 mutant show weak rapamycin sensitivity and increased Hog1 phosphorylation, they concluded that Sch9 is involved in TOR and HOG pathways. To make such conclusions, the authors should provide more detailed mechanistic data.

Response 2: Yes, we agree with the reviewer's comment. We have carefully reviewed the manuscript and made necessary revisions to eliminate arbitrary conclusions. For example, we have removed the statement that "Sch9 is the element of the calcineurin-CrzA pathway". Furthermore, we have rephrased our conclusions to better reflect our findings. "these results reflected that Sch9 regulates osmotic stress response via the HOG pathway in A. flavus"(Lines 279-280, page 13). We appreciate the reviewer's input, which has contributed to the clarity and accuracy of our work.

Point 3: In the section "Tor kinase plays important roles in A. flavus", some parts of their data are confusing. The authors said they identified a single Tor kinase ortholog, which is orthologous to S. cerevisiae Tor2. And then, they said failed to obtain a null mutant, but constructed a single copy deletion strain delta Tor1+/Tor2-. What does this mean? Does this mean A. flavus diploid strain? So is this heterozygous TOR/tor mutant? Otherwise, does the haploid A. flavus strain they used contain multiple copies of the TOR gene within its genome? What is the real name of A. flavus Tor kinase (Tor1 or Tor2?). "tor1+/tor2-" is the wrong genetic nomenclature. What is the identity of detalTor1+/Tor2-? Please provide detailed information on how all these mutants were generated. A similar issue was found in the analysis of TapA, which is speculated to be essential (what is the deltaTapA1+/TapA2-?). I couldn't find any detailed information even in Materials and Methods. The authors should provide southern blot data to validate all their mutants.

Response 3: Thank you for your comments. We acknowledge the confusion in our presentation and will ensure that accurate genetic nomenclature is used consistently throughout the paper.

In response to your queries, we have included a section in the Materials and Methods, titled "Detection of tor and tapA genes copy number in strains" (Lines 615-621, page 29), to provide details on how we determined the copy numbers of the tor and tapA genes in the strains. Our findings revealed that both the tor and tapA genes are present in double copies in our strains, which guided our decision to construct single-copy deletion strains using homologous recombination. We have verified these copy numbers using absolute quantification PCR (Table S1).

The use of the abbreviation '+/-' for the single copy knockout strains, such as tor+/- and tapA+/-, is consistent with common fungal literature practice. We apologize for any confusion caused by this nomenclature.

Although we did not employ southern blot data for validation, we conducted PCR and gene sequencing to confirm the mutants. We appreciate your comments to improve the clarity and accuracy of our manuscript.

Point 4: How were the FRB domain deletion mutants constructed? If the FKBP12-rapamycin binding (FRB) domain is specifically deleted in the Tor kinase allele, should it be insensitive and resistant to rapamycin? However, the authors showed that the FRB domain deleted TOR allele was indeed non-functional.

Response 4: We appreciate the reviewer's attention to the construction of the Fkbp12-rapamycin binding (FRB) domain deletion mutants and the discrepancy between the expected and observed results.

For the knockout of the FRB domain, we used the homologous recombination method, but because tor genes are double-copy genes, there are also double copies in the FRB domain. Despite our efforts, we encountered challenges in precisely determining the location of the other copy of the tor gene.

We speculate the common expectation that the deletion of the FRB domain should result in insensitivity and resistance to rapamycin, as it disrupts the binding site for Fkbp-rapamycin. However, we observed that the FRB domain-deleted mutant was more sensitive to rapamycin. This intriguing result suggests that there are additional factors or complexities involved in TOR signaling pathway regulation in A. flavus. We hypothesize that this result is related to the double copy of the tor gene. The reviewer's keen observation and comment have contributed to our efforts to better understand and explain this intriguing result.

Point 5: In Figure 4C, the authors should monitor Hog1 phosphorylation patterns under stressed conditions, such as NaCl treatment, and provide quantitative measurements. Similar issues were found in the western blot analysis of Slt2 (Fig. 8D).

Response 5: We agree with the reviewer that we should monitor Hog1 phosphorylation patterns under stressed conditions. In response to this valuable suggestion, we conducted additional experiments to examine Hog1 phosphorylation patterns under NaCl treatment for 30 minutes. The quantitative measurements of Hog1 phosphorylation levels under stress have been added to Figure 4E in the revised manuscript. Similarly, we have addressed the issue raised regarding Slt2 in Figure 8D.

Point 6: For all the deletion mutants generated in this study, the authors should generate complemented strains to validate their data.

Response 6: We appreciate the reviewer's suggestion to generate complemented strains for all the deletion mutants in our study to validate our data. However, due to the extensive number of genes involved in this research, it is hard to create complemented strains for each individual deletion mutant. As suggested by the reviewer, we have constructed complemented strains for several key deletion mutants, such as ΔsitA-C and Δppg1-C.

Response to Reviewer 1 Comments (Recommendations For The Authors):

Point 1: Overall, this manuscript was very poorly organized and not presented logically. It requires extensive English language editing.

Response 1: We appreciate the reviewer's feedback regarding the organization and language quality of our manuscript. To address these concerns, we have restructured the manuscript to improve its logical flow and coherence. We thank the reviewer for their constructive criticism, which has been instrumental in the manuscript's refinement.

Point 2: The authors did not present their figures in the order of description. For example, the authors suddenly described Figure 9A data in lines 128-130 in the middle of describing Figure 1. Furthermore, Figures 1D and 1F were described earlier than Figures 1B and 1C. In addition, Figure S2 was shown earlier than Figure S1. Please check this throughout the manuscript.

Response 2: We thank the reviewer for their insightful observation. We acknowledge the importance of a logical and coherent figure sequence for reader comprehension. After careful review, we have rearranged the text and images throughout the entire document to enhance the reading experience. The revised manuscript now presents figures in a consistent and logical order, following the sequence of descriptions. We believe this improvement will enhance the overall readability and comprehension of our research.

Point 3: The authors should follow the standard genetic nomenclature rules.

Response 3: Thank you for your suggestion. We have revised our manuscript to ensure that we are following the standard genetic nomenclature rules throughout. This includes the correct naming of genes, proteins, and mutations, as well as the use of appropriate italicization and formatting. We follow the rules: gene symbols are typically composed of three lowercase italicized letters, while protein symbols are not italicized, with an initial capital letter followed by lowercase letters.

Point 4: These are just a few examples. Besides the ones that I mentioned, I found numerous grammatically wrong or awkward sentences throughout the manuscript. So this manuscript requires extensive English proofreading.

Response 4: We apologize for the problem of our manuscript. We have asked an English native speaker to enhance the overall language quality and readability of the text. We believe that these improvements will significantly enhance the manuscript's overall quality and make it more accessible to a broader audience.

Response to Reviewer 2 Comments (Public Review):

Point 1: However, findings have not been deeply explored and conclusions mostly are based on parallel phenotypic observations. In addition, there are some concerns that exist surrounding the conclusions.

Response 1: We are grateful for the suggestion. We conduct additional experiments and analyses to delve more deeply into our findings and ensure a more robust basis for our conclusions.

Response to Reviewer 2 Comments (Recommendations For The Authors):

Point 1: Verification for mutants: a single copy deletion strain ΔTor1+/Tor2(containing one copy of the Tor gene), however, in the table of strain list, it seems like null mutants. There are no further verifications for relative genes' expression and no complementary strains.

A. Flavus ΔTor: Δku70; ΔniaD; ΔTor::pyrG

A. Flavus ΔTapA Δku70; ΔniaD; ΔTapA::pyrG

As described in pp208, "While we failed to obtained a null mutant, we constructed a single copy deletion strain ΔTor1+/Tor2- (containing one copy of the Tor gene) constructed by homologous recombination)"? But the authors think there was only one Tor kinase ortholog (AFLA_044350). It is hard to understand for this mutant What is the evidence to verify phenotypes of the ΔTor1+/Tor2- strain resulted from deletion of Tor2, no detail for how to make ΔTor1+/Tor2- strain.

Response 1: Thank you for your important comments and suggestion. We apologize for the confusion caused by genetic nomenclature. We make the necessary corrections in the table of strain lists to accurately reflect the genotypes of the strains (Table S3).

Multicopy variation of genes has not been explored in detail in fungi, especially in A. flavus, but is a commonly known phenomenon in mammalian genomes[1-2]. In yeast, the presence of two tor genes, tor1 and tor2, whereas in higher eukaryotes such as plants, animals, and filamentous fungi, there is only one tor gene[3-4]. The homology comparison results show that the genome of A. flavus contains only one tor gene. However, the tor gene in A. flavus exhibited varying copy numbers, as was confirmed by absolute quantification PCR at the genome level (Table S1).

In this study, we constructed a single copy deletion strain, tor+/-, through homologous recombination. This strain contains one copy of the tor gene. We provide a more detailed and explicit description of the methods used to detect of the genes copy number in strains (Lines 615-621, page 29). We thank the reviewer for pointing out these important issues.

Point 2: For a point mutant strain TORS1904L, they found that the sensitivity to rapamycin is consistent with the WT strain, it could not tell anything. It should be moved to Suppl.

Response 2: Thanks for your important comments. We acknowledge that these results may not provide significant insights. In response to this suggestion, we delete the data related to the TORS1904L point mutant strain and its sensitivity to rapamycin to ensure that the main manuscript focuses on the most pertinent and informative findings. Corresponding modifications have been made in the revised manuscript.

Point 3: For subtitle "Sch9 is correlate with the HOG and TOR pathways "What is the meaning for "correlate" similarly?

Response 3: Thank you for this comment. We apologize for the unclear wording. To enhance clarity, we revise the subtitle to more explicitly convey this conclusion, for example, "The Sch9 kinase is involved in aflatoxin biosynthesis and the HOG pathway". (Lines 242, page 12).

Point 4:for the ΔTapA 1+/TapA 2- strain (containing one copy of the TapA gene). It should have the complementary strain to verify the specific role of TapA. In FigS1B, ΔTOR and ΔTapA it could not tell TOR gene has been edited. Did you test mRNA of TOR gene?

Response 4: Thanks for your important comments. Due to the large number of genes involved, we did not perform a complementation experiment. However, we used PCR and sequencing to verify the editing of our gene. Additionally, we conducted copy number and mRNA analyses to verify its function. The transcriptional level of the tor gene in the tor+/- mutant was downregulated compared to the level in the wild-type strain (Fig. S6).

Response to Reviewer 3 Comments (Public Review):

Point 1: As for many results, I miss the re-complementation of the created mutants throughout the manuscript. This is standard praxis.

Response 1: Thanks for your suggestions. We acknowledge that re-complementation is a standard practice for validating the effects of gene deletions. However, due to the large number of genes involved in our study, we have performed supplementary experiments on a selection of them, such as ΔsitA-C and Δppg1-C. We are grateful to the reviewer for your understanding of this practical consideration.

Point 2: Fig. 1: cultures were grown for 48 h before measuring the transcript level. The authors show that brlA, abaA, and some sexual regulators are less expressed. In my opinion, this does not allow the conclusion that there is a direct control through rapamycin. Since the colonies grow very slowly in the presence of rapamycin, the authors should add rapamycin and follow gene expression after 15, 30, 60, 90 min. The figure legend needs to be more detailed. Which type of cultures were used, liquid, solid medium? Etc.

Response 2: We deeply appreciate the reviewer’s suggestion. Since we found that there were no significant differences in gene expression changes following shorter treatment times, we extended the treatment duration. We conduct additional experiments to examine the gene expression levels at longer time intervals (3, 6, and 9 h) after the addition of rapamycin (Figure 1H-1J). These time points allow us to capture the dynamic changes in gene expression in response to rapamycin more effectively. Additionally, we enhance the figure legend to provide a more comprehensive description that specifies the type of cultures used in the experiments.

Point 3: Why in chapter one Fig. 9 is already cited? Those data should then be included in Fig. 1 for the general phenotype.

Response 3: Thank you for the suggestion. We have reordered the figures in the updated version of the manuscript to ensure that the data for consistent and clarity.

Point 4: The authors wrote that radial growth and conidiation were gradually reduced with increasing rapamycin concentrations. This is not true. There is no gradient! However, it should be tested if there is a gradient if lower concentrations are used. The current data imply that there is a threshold concentration, so either there is 100 % growth or a reduction to 25 %. This looks strange.

Response 4: Thank you for underlining this deficiency. We agree that a threshold concentration versus a gradient is an important distinction that needs to be clarified. Our results show that the addition of excessive quantities of rapamycin does not increase the inhibition of A. flavus growth. As the concentration of the FK506 drug increases, there is a gradual decrease in the growth and cell production of A. flavus. This phenomenon could potentially be attributed to varying mechanisms of action exhibited by the drugs. Therefore, we have revised these confused sentences. ( Lines 120-121, Page 5)

Point 1: There are many wrong spellings:

Fig. 1. Before washed, before washing; RelaTEtive gene expERSion should read relative gene expression. Sclerotial should be sclerotia. See also Fig. 5 F, H, Fig. 6 E. 6D colon diameter should be colony diameter.

Fig. 4E. The expressED level... should read Expression level..... (also without article) Also in A, F, H.

Fig. 6C. TLC detection of WT.... The authors mean AF detection in extracts of WT..... AF was extracted and analyzed by TLC.....

Labelling of axes in one figure should be uniform.

Response 1: Thank you for your reminder. We apologize for the oversights, and we carefully address and correct all the mentioned spelling issues to ensure the accuracy and clarity of the manuscript.

Point 2: If the authors refer to the genes, I think they should be in small letters and italics, if it is the protein, the first letter should be capitalised tap1 (italics) and Tap1.

Response 2: We appreciate this suggestion. We have carefully checked the entire manuscript and revised follow the standard genetic nomenclature rules. We follow the naming conventions for microbial genes and proteins, where gene symbols are typically composed of three lowercase italicized letters, and protein symbols are not italicized, with an initial capital letter followed by lowercase letters.

Point 3: Very often articles are used where I would not use them.

Response 3: Thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the articles harmonized within the whole manuscript. We value the reviewer's feedback, which will contribute to the overall quality of our writing.

References:

[1] Handsaker R, Van Doren, V, Berman, J. et al. Large multiallelic copy number variations in humans. Nat Genet 47, 296–303 (2015).

[2] Wang Y, Wang S, Nie X. et al. Molecular and structural basis of nucleoside diphosphate kinase-mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus. J Biol Chem. 2019 Aug 16;294(33):12415-12431.

[3] Kim DH, Sarbassov DD, Ali SM, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002; 110(2): 163-75.

[4] Fu L, Liu Y, Qin G, et al. The TOR-EIN2 axis mediates nuclear signalling to modulate plant growth. Nature. 2021; 591(7849): 288-292.

https://doi.org/10.7554/eLife.89478.2.sa4

Significance of findings

Strength of evidence

Abstract

Introduction

Results

Rapamycin exhibits inhibitory effects on the growth, sporulation, sclerotia formation, and aflatoxin production in A. flavus

Impacts of rapamycin on the growth, sporulation, sclerotia formation, and aflatoxin production of Aspergillus flavus

The impact of Fkbp3 and its lysine succinylation on AFB1 biosynthesis and rapamycin resistance in A. flavus

Disruption of fkbp3 significantly increases the resistance of A. flavus to rapamycin and FK506

The Tor kinase plays a critical role in A. flavus

The Tor kinase plays a vital role in A. flavus

The Sch9 kinase participates in aflatoxin biosynthesis and the HOG pathway

TapA and TipA regulate sclerotia formation in A. flavus

The Sch9 kinase is involved in aflatoxin biosynthesis and the HOG pathway

TapA and TipA participate in regulation of sclerotia development and cell wall stress response

Phosphatase SitA and Ppg1 are involved in growth, conidiation, and sclerotial formation

The impact of the phosphatases SitA and Ppg1 on the growth, conidiation, and aflatoxin biosynthesis in A. flavus

SitA and Ppg1 play crucial roles in aflatoxin biosynthesis and pathogenicity

Pathogenicity analysis of the phosphatase SitA and Ppg1 in A. flavus

SitA and Ppg1 are involved in regulating cell wall integrity

Sensitivity of phosphatase SitA and Ppg1 to cell wall damaging agents in A. flavus

The phosphatase complex Nem1/Spo7 are important for growth, aflatoxin biosynthesis, and LD biogenesis

Phosphatase SitA and Ppg1 are involved in regulating lipid droplet biogenesis

Phosphatase complex Nem1/Spo7 play significant role in growth, conidiation, aflatoxin, and lipid droplet biogenesis