Figure 1.
Differentially regulated genes in comparisons of veA deletion, overexpression and complementation strains against the wild-type strain.
For each gene, the log2 rRPKM is reported along the x-axis and the negative log10 of the Bonferroni-corrected Fisher’s Exact p-value is reported along the y-axis. P-values with an infinite log10 value (p-values equal to zero) are reported as 400. Genes with a log2 rRPKM greater than 2 and a Bonferroni-corrected p-value less than 0.05 are considered differentially regulated and are represented by red dots, whereas genes below these thresholds are not considered to be differentially regulated and are represented by blue dots. (A) Comparison between the strain that carries a deletion of the veA gene (∆veA) and the wild-type (WT) strain; (B) Comparison between the veA over-expression strain (OEveA) and the WT strain; (C) Comparison between the veA complementation strain (com) and the WT strain.
Figure 2.
The genome-wide distribution of de novo identified gene clusters that appear to be regulated by veA.
De novo identification of gene clusters differentially regulated by the deletion versus wild-type (shown above each chromosome) and overexpression versus wild-type (shown below each chromosome) comparisons. Upregulated gene clusters are represented by red boxes and downregulated gene clusters are represented by blue boxes. Gene clusters identified in both comparisons whose boundaries overlap are denoted by bracket symbols. Previously reported secondary metabolism gene clusters [60,71] are boxed and numbered. 1: This cluster contains a conidial pigment biosynthesis cluster from Afu2g17530 – Afu2g17600 [82]; 2. This cluster contains the Fumigaclavine C cluster from Afu2g17960 – Afu2g18060 [83]; 3. Cluster of unknown function; 4. Cluster of unknown function; 5. This cluster contains an endocrocin secondary metabolism cluster from Afu4g00210 – Afu4g00230 [84]; 6. This cluster contains the pathway responsible for helvolic acid biosynthesis from Afu4g14770 – Afu4g14850 [85]; 7. Cluster of unknown function; 8. Cluster of unknown function; 9. This cluster contains the gliotoxin cluster from Afu6g09630 – Afu6g 09740 [86]; 10. This cluster contains the fumiquinazoline biosynthetic cluster from Afu6g12040 – Afu6g12110 [87]; 11. Cluster of unknown function; 12. Cluster of unknown function; 13. This cluster contains the fumitremorgin G cluster from Afu8g00260 – Afu8g00170 [76]; 14. This cluster contains the fumagillin gene cluster from Afu8g00370 – Afu8g00520 [53]; and the pseurotin A cluster from Afu8g00530 – Afu8g00570 [88].
Figure 3.
Expression patterns of the fumagillin (A), fumitremorgin G (B), and fumigaclavine C (C) gene clusters in the ΔveA vs WT and OEveA vs WT comparisons.
Genes labeled with numbers correspond to locus tags without the chromosomal prefix or trailing zeros (i.e., the gene labeled 380 in the fumagillin cluster corresponds to Afu8g00380). The sole exception is fumR (Afu8g00420), the name given to the regulatory gene for the fumagillin cluster characterized in this study. Genes are color-coded by differential expression; blue indicates downregulation, red indicates upregulation, and white indicates no differential regulation.
Figure 4.
veA regulates the production of fumagillin, fumitremorgin G, fumigaclavine C and Glionitrin A.
Secondary metabolites were extracted from 120 h old Czapek-dox stationary liquid cultures of wild type (WT), ΔveA, complementation and OEveA strains. Extracts were analyzed with Shimadzu 2010 EV LC-MS as described in the materials and methods section. The predicted m/z [M+H]+ ratio was (A) fumagillin (m/z=459), (B) fumitremorgin G (m/z = 433) (C) fumigaclavine C (m/z = 299) and (D) Glionitrin-A (m/z = 354). The bars represent the mean of three samples and error bars represent standard error.
Figure 5.
fumR is necessary for the expression of the genes in the fumagillin cluster.
The transcriptional pattern of the genes in the fumagillin cluster (Afu8g00370, Afu8g00520, Afu8g00370, Afu8g00510, Afu8g00500, Afu8g00480, Afu8g00470, Afu8g00440, Afu8g00430) was evaluated by qRT-PCR in the wild type (WT, TSD51.1) and αfumR strains. Total RNA was extracted from Czapek-Dox stationary liquid cultures incubated for 48 h and 72 h. The relative expression was calculated using 2-ΔΔCT as described by Schmittgen and Livak [89]. 18S gene expression was used as internal reference. Means of three replicates is shown. Values were normalized to WT expression at 48 h considered as 1. Error bar represents standard error.
Figure 6.
fumR regulates fumagillin biosynthesis.
Aspergillus fumigatus wild type (WT TSD51.1) and ∆fumR strains were grown on Czapek-Dox as stationary liquid cultures for 72 h and 120 h. Culture supernatants were extracted with chloroform and analyzed by LC-MS (A) as described in materials and methods. (B) Quantitative data of the LC-MS analysis. The experiment was done with three replicates. Error bars represent standard error. No fumagillin was detected (n.d.) in ∆fumR mutants. A standard curve was obtained using commercial fumagillin (Sigma).
Figure 7.
Expression of fumR and PKS370 is regulated by laeA.
Total RNA was extracted from WT and ∆laeA strains, grown as stationary liquid Czapek-Dox cultures for 48 h and 72 h. Relative expression of fumR and PKS gene Afu8g00370 was calculated using 2-ΔΔCt method as described by Schmittgen and Livak [89]. Primers used for the expression analysis are listed in Table S2. The bar represents the mean of three replicates and error bars represent standard error. Expression of 18S was used as internal reference. Values were normalized to expression levels of wild-type levels considered as 1.