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

(A) The A. flavus wild-type (WT) strain was incubated on PDA amended with different concentrations of rapamycin at 37 °C for 5 days.

(B) Colony phenotype of 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 WT strain cultured in YES liquid media at 29°C for 6 days.

(D) Statistical analysis of the 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 rapamycinas as mentioned in (A).

(F) Quantitative analysis of sclerotium formation by the WT strain treated with different concentrations of rapamycin described in (C).

(G) Relative transcript levels of conidia-related genes in WT strain treated with 100 ng/mL rapamycin cultured for 48 h.

(H) Relative transcript levels of sclerotia-related genes in WT strain treated with 100 ng/mL rapamycin cultured for 48 h.

(I) Rative transcript levels of aflatoxin biosynthesis regulatory and structural genes in WT strain treated with 100 ng/mL rapamycin cultured for 48 h.

Disruption of FKBP3 significantly increases the resistance of A. flavus to rapamycin and FK506.

(A) Phenotype of A. flavus WT and all mutant strains (ΔFKBP1, ΔFKBP2, ΔFKBP3 and ΔFKBP4) grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.

(B) TLC assay 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 stress.

(D) AFB1 quantitative analysis of the WT and all mutant strains (ΔFKBP1, ΔFKBP2, ΔFKBP3 and ΔFKBP4) described in (B).

(E) Phenotype of 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 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 WT and all mutant strains (ΔFKBP3, K5A, K19A, K40A, K42A, K55A and K65A) as mentioned in (G).

Tor kinase plays important roles in A. flavus.

(A) The structure diagram of Tor kinase.

(B) Morphology of conidiophores of A. flavus WT, ΔTor1+/Tor2−, TORΔFRB and TORS1904L strains grown on PDA medium at 37°C for 5 days as observed by microscope.

(C) Phenotype of WT, ΔTor1+/Tor2−, TORΔFRB and TORS1904L strains grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.

(D) The growth inhibition rate of WT, ΔTor1+/Tor2−, TORΔFRB and TORS1904L strains under rapamycin stress.

(E) Relative transcript levels of conidia-related genes in WT, ΔTor1+/Tor2−, TORΔFRB strains.

(F) TLC assay of AFB1 production by the WT, ΔTor1+/Tor2−, TORΔFRB and TORS1904L strains cultured in YES liquid medium at 29°C for 6 days.

(G) Quantification analysis of AFB1 as in (F).

(H) Relative transcript levels of aflatoxin biosynthesis regulatory and structural genes in WT and ΔTor1+/Tor2−strains.

Sch9 is correlated with the HOG and TOR pathways.

(A) The structure diagram of Sch9 kinase.

(B) Phenotype of A. flavus WT, ΔSch9, Sch9ΔC2, Sch9ΔS_TKc, Sch9ΔS_TK_X and Sch9K340A strains grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.

(C) TLC assay of AFB1 production by the WT, ΔSch9, Sch9ΔC2, Sch9ΔS_TKc,Sch9ΔS_TK_Xand Sch9K340A strains cultured in YES liquid medium at 29°C for 6 days.

(D)Colonies of WT, ΔSch9, Sch9ΔC2, Sch9ΔS_TKc, Sch9ΔS_TK_Xand Sch9K340A strains on PDA media amended with 1 M NaCl and 1 M KCl for 3 days.

(E) The expressed levels of phospho-Hog1 were detected in WT and ΔSch9 strains.

(F) The growth inhibition rate of 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 promotion rate of WT, ΔSch9, Sch9ΔC2, Sch9ΔS_TKc, Sch9ΔS_TK_X and Sch9K340A strains under osmotic stress.

TapA and TipA regulate growth, sclerotia and aflatoxin biosynthesis in A. flavus.

(A) Phenotype of the A. flavus WT, ΔTapA1+/TapA2− and ΔTipA strains grown on PDA amended with 100 ng/mL rapamycin at 37°C for 5 days.

(B) Colony phenotype of the WT, ΔTapA1+/TapA2−and ΔTipA strains grown on CM medium at 37°C for 7 days.

(C) TLC assay of AFB1 production by the WT, ΔTapA1+/TapA2− and ΔTipA strains cultured in YES liquid medium at 29°C for 6 days.

(D) Fluorescent images of hyphal cells for the WT, ΔTapA1+/TapA2− and ΔTipA strains stained with calcofluor white (CFW).

(E) Amounts of sclerotia produced by the WT, ΔTapA1+/TapA2− and ΔTipA strains.

(F) Relative transcript levels of sclerotia-related genes in WT and ΔTapA1+/TapA2−strains.

(G) Relative quantification of AFB1 production as in (C).

(H) Relative transcript levels of aflatoxin biosynthesis regulatory and structural genes in WT and ΔTapA1+/TapA2−strains.

Phosphatases SitA and Ppg1 are involved in growth, conidiation and aflatoxin biosynthesis in A. flavus.

(A) The colony morphology of A. flavus WT, single knockout and double knockout strains of SitA and Ppg1 cultured on PDA medium amended with 100 ng/mL rapamycin at 37℃ for 5 days.

(B) colony morphology of A. flavus WT, single knockout strain and double knockout strain of SitA and Ppg1 cultured on WKM medium at 37℃ for 7 days.

(C) TLC detection of WT, single knockout and double knockout strains of SitA and Ppg1 cultured in YES liquid medium at 29℃ for 6 days.

(D) Comparison of conidial production among WT, single knockout and double knockout strains on PDA media.

(E) Sclerotia statistics of WT, single knockout and double knockout strains.

(F) Quantitative analysis of AFB1 of WT, single knockout and double knockout strains.

(G) Relative transcript levels of conidia synthesis genes in WT, single knockout and double knockout strains.

(H) Relative transcript levels of sclerotia synthesis genes in WT, single knockout and double knockout strains.

(I) Relative transcript levels of aflatoxin biosynthesis genes in WT, single knockout and double knockout strains.

Phosphatase SitA and Ppg1 are important for pathogenicity in A. flavus.

(A) Phenotypes of peanut and maize infected by A. flavus WT, single and double knockout strains of SitA and Ppg1.

(B) TLC detection of toxin from peanut and maize infected by WT, single knockout and double knockout strains.

(C) Spores statistics from peanut and maize infected by WT, single and double knockout strains.

(D) Quantitative analysis of AFB1 from peanut and maize infected by WT, single knockout and double knockout st

Phosphatase SitA and Ppg1 are involved in regulating cell wall integrity.

(A) Morphology of A. flavus WT, single knockout and double knockout strains of SitA and Ppg1 grown on PDA 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) Expression levels of cell wall synthesis genes (chsA, chsD, chsE and β-1,6) in WT, single knockout and double knockout strains.

(D) Phosphorylation level of Slt2 in WT, single knockout and double knockout strains was detected by Western blotting.

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 A. flavus WT strain and observed under a fluorescence microscopy. The Phenotype of lipid droplets accumulation in the mycelia of A. flavus WT strain treated with or without 100 ng/mL rapamycin cultured for 6 h. Bar, 10 µm.

(B) The phenotype of lipid droplets accumulation in the mycelia of ΔSch9 strain treated with or without 100 ng/mL rapamycin cultured for 6 h.Bar, 10 µm.

(C) The phenotype of lipid droplets accumulation in the mycelia of ΔSitA strain treated with or without 100 ng/mL rapamycin cultured for 6 h.Bar, 10 µm.

(D) The phenotype of lipid droplets accumulation in the mycelia of ΔPpg1 strain treated with or without 100 ng/mL rapamycin cultured for 6 h. Bar, 10 µm.

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

(A) Phenotype of the A. flavus 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 by the A. flavus WT, ΔNem1 and ΔSpo7 strains cultured in YES liquid medium at 29°C for 6 days.

(C) Statistical analysis of the diameter and sporulation by the A. flavus WT, ΔNem1 and ΔSpo7 strains.

(D)The Phenotype of lipid droplets accumulation in the mycelia of the A. flavus WT, ΔNem1 and ΔSpo7 strains treated with or without 100 ng/mL rapamycin cultured for 6 h. Bar, 10 µm.

The proposed model of target of the rapamycin (TOR) pathway in A. flavus.

Based on the above results, we hypothesized the TOR signaling pathway model in A. flavus. Rapamycin forms a complex with FKBP3, and this complex can bind to the Tor kinase to inhibit its normal function. Sch9 as a downstream component of the Tor kinase, regulates sclerotia formation, aflatoxin biosynthesis, the TOR and HOG signaling pathways. In addition, We speculate that the Sch9 as a element for the calcineurin-CrzA pathway in response to calcium stress. As another important target of the Tor kinase, Tap42-phosphatase complex is involved in the regulation of growth, sporulation, sclerotia, aflatoxin production, CWI signaling pathways, lipid droplet synthesis and other processes. In conclusion, the TOR signaling pathway plays an important role in the vegetative development, stress responses, aflatoxin biosynthesis and pathogenicity in A. flavus.