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.

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

(A) Phenotype of the WT and all mutant strains (Δfkbp1, Δfkbp2, Δfkbp3, and Δfkbp4) 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 (Δfkbp1, Δfkbp2, Δfkbp3, and Δfkbp4) cultured in YES liquid medium at 29°C for 6 days.

(C) The growth inhibition rate of the WT and all mutant strains (Δfkbp1, Δfkbp2, Δfkbp3, and Δfkbp4) under rapamycin and FK506 stress.

(D) AFB1 quantitative analysis of the WT and all mutant strains (Δfkbp1, Δfkbp2, Δfkbp3, and Δfkbp4) as described in (B).

(E) Phenotype of the WT and all mutant strains (Δfkbp3, 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 (Δfkbp3, K5A, K19A, K40A, K42A, K55A, and K65A) under rapamycin stress.

(G) TLC assay of AFB1 production by the WT and all mutant strains (Δfkbp3, 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 (Δfkbp3, K5A, K19A, K40A, K42A, K55A, and K65A) as mentioned in (G).

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 torΔFRBstrains observed by microscope.

(C) Phenotype of the WT, tor+/-, and torΔFRBstrains 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 torΔFRBstrains cultured in YES liquid medium at 29°C for 6 days.

(E) The growth inhibition rate of the WT, tor+/-, and torΔFRBstrains under rapamycin stress.

(F) AFB1 quantitative analysis of the WT, tor+/-, and torΔFRBstrains described in (D).

The Sch9 kinase participates in aflatoxin biosynthesis and the HOG pathway

(A) The structure diagram of the Sch9 kinase.

(B) Phenotype of the WT, Δsch9, sch9ΔC2, sch9ΔS_TKc, sch9ΔS_TK_X, and sch9K340A 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, Δsch9, sch9ΔC2, sch9ΔS_TKc, sch9ΔS_TK_X, and sch9K340A strains cultured in YES liquid medium at 29°C for 6 days.

(D) The phenotypes of the WT, Δsch9, sch9ΔC2, sch9ΔS_TKc, sch9ΔS_TK_X, and sch9K340Astrains 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 Δsch9 strains were determined with or without osmotic stress.

(F) The growth inhibition rate of the WT, Δsch9, sch9ΔC2, sch9ΔS_TKc, sch9ΔS_TK_X, and sch9K340A strains under rapamycin stress.

(G) Quantitative analysis of AFB1 as shown in (C).

(H) The growth inhibition rate of the WT, Δsch9, sch9ΔC2, sch9ΔS_TKc, sch9ΔS_TK_X, and sch9K340A strains under osmotic stress.

TapA and TipA regulate sclerotia formation in A. flavus

(A) Phenotype of the WT, tapA +/−, and ΔtipA strains grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.

(B) Phenotypic characterization of the WT, tapA +/−, and ΔtipA strains grown on CM medium at 37°C for 7 days.

(C) Morphology of conidiophores in the WT, tapA +/−, and ΔtipA strains observed by microscope.

(D) TLC assay of AFB1 production by the WT, tapA +/−, and ΔtipA strains cultured in YES liquid medium at 29°C for 6 days.

(E) The growth inhibition rate of the WT, tapA +/−, and ΔtipA strains under rapamycin stress.

(F) Amount of sclerotia produced by the WT, tapA +/−, and ΔtipA strains.

(G) AFB1 quantitative analysis of the WT, tapA +/−, and ΔtipA strains described in (D).

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.

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.

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.

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 Δsch9 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 ΔsitA 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 Δppg1 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, Δnem1, and Δspo7 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, Δnem1, and Δspo7 strains cultured in YES liquid medium at 29°C for 6 days.

(C) Growth diameter of the WT, Δnem1, and Δspo7 strains on PDA media.

(D) Statistical analysis of the sporulation by the WT, Δnem1, and Δspo7 strains.

(E) Phenotype of lipid droplets accumulation in the mycelia of the WT, Δnem1, and Δspo7 strains treated with or without 100 ng/mL rapamycin for 6 h (Bar, 10 µm).

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.