Fig 1.
Multiple sequence alignment and phylogenetic analysis of RmtA and other fungal homologs.
(A) Sequences aligned using Muscle (http://www.ebi.ac.uk/Tools/msa/muscle/). Alignment was visualized with BoxShade v3.21 (http://ch.embnet.org/software/BOX_form.html). (B) Phylogenetic tree of RmtA homologs from different fungal species. Phylogenetic trees constructed using MEGA v6.0. Trees were generated with Maximum-Likelihood model with a bootstrap value of 1000.
Fig 2.
Construction of the rmtA deletion and complementation strains.
(A) Diagram showing the gene replacement strategy using the selection marker pyrG from A. fumigatus and Southern blot analysis. Recombination events between the flanking regions are indicated with crosses. KpnI sites are indicated in both the wild-type and modified loci. A 1.3 kb fragment was used as probe as indicated. On the right, Southern blot image confirming of the deletion of rmtA. Genomic DNA from the wild type (WT) and from a selected deletion mutant ΔrmtA was digested with KpnI. Expected 5.0 kb and 3.2 kb bands are shown for WT and ΔrmtA respectively. (B) Linearized representation of the plasmid containing the rmtA wild-type allele. The niaD gene from A. fumigatus was used as selection marker for fungal transformation. Primers OE_RMTA_F and OE_RMTA_R (S1 Table), used for confirmation of the strain, are labeled in this figure as F and R respectively. On the right, gel electrophoresis results showing the presence of a 1.8 kb PCR product, confirming the presence of the wild-type allele in the complementation strain. Wild type and ΔrmtA were used as positive and negative control, respectively. (C) Expression analysis of rmtA by qRT-PCR. Strains were inoculated in PDB (106 spores/ml), and cultures were grown for 48 h at 30°C. The error bars represent standard errors. Values were normalized to the expression levels in the wild type, considered as 1. Different letters on the columns indicate values that are statistically different (p < 0.05).
Fig 3.
Confirmation of the OErmtA (OE).
(A) Linearized representation of the over-expression plasmid. The pyrG gene from A. fumigatus was used as a selection marker for fungal transformation. (B) Gel electrophoresis results showing the presence of a 1.8 kb PCR product, confirming the presence of the over-expression cassette. Plasmid pTRS.1rmtAOE and genomic DNA from the wild type were used as positive and negative control respectively. F and R represent primers OE_RMTA_F and OE_RMTA_R respectively. (C) Expression analysis of rmtA by qRT-PCR. Strains were inoculated in PDB (106 spores/ml), and cultures were grown for 48 h at 30°C. The error bars represent standard errors. Values were normalized to the expression levels in the wild type, considered as 1. Different letters on the columns indicate values that are statistically different (p < 0.05).
Fig 4.
Effect of rmtA on conidiation.
(A) Quantification of conidia. Aspergillus flavus wild type (WT), ΔrmtA, complementation (com) and OErmtA (OE) strains were grown on PDA medium for up to 7 days at 30°C. Seven millimeter cores were taken from each culture. Conidia were counted using a hemocytometer. Values represent the average of 3 replicates. Error bars represent standard error. (B, C, D) qRT-PCR expression analysis of brlA. abaA and wetA, respectively. Strains were inoculated in PDB stationary cultures (106 spores/ml), and were grown for 72 h at 30°C. The error bars represent standard errors. Values were normalized to the expression levels in the wild type, considered as 1. Different letters on the columns indicate values that are statistically different (p < 0.05).
Fig 5.
Effect of rmtA on sclerotial production on PDA.
(A) A. flavus wild type (WT), ΔrmtA, complementation (com) and OErmtA (OE) strains were point-inoculated on PDA medium and incubated for 9 days in the dark at 30°C. Photographs of cultures were taken before and after spraying 70% ethanol to remove conidia in order to improve visualization of sclerotia. Micrographs were obtained approximately 1.5 cm away from the center of the plate using a Leica MZ75 dissecting microscope at 12.5X magnification. (B) Quantification of sclerotia. A. flavus wild type, (WT), ΔrmtA, complementation (com) and OE rmtA strains grown on PDA medium for 24 days at 30°C. Sixteen millimeter cores were collected 1 cm away from the center. Number of sclerotia in each core were counted under a Leica MZ75 dissecting microscope. The experiment included 3 replicates. Error bars represent standard error. (C) qRT-PCR expression analysis of veA. A. flavus wild type (WT), ΔrmtA, complementation (com) and OErmtA (OE) strains were inoculated in PDB stationary cultures (106 spores/ml), and were grown for 48 and 72 h at 30°C. Error bars represent standard errors. Values were normalized to the expression levels in the wild type, considered as 1. Different letters on the columns indicate values that are statistically different (p < 0.05).
Fig 6.
rmtA positively regulates Aflatoxin B1 production.
(A) TLC analysis of aflatoxin B1 produced by A. flavus wild type (WT), ΔrmtA, complementation (com) and OErmtA (OE) strains growing at 30°C on PDA top-agar inoculated cultures for 48 h and 72 h. Aflatoxin standard (AFB1) was also included on either side of the plate. Arrow indicates an unknown metabolite whose synthesis is also affected by rmtA. Densitometry of aflatoxin performed using GelQuantNET software is shown. (B) Northern Blot analysis of ver1. All strains were grown in PDB stationary cultures (106 spores/ml) for 72 h at 30°C. 18S rRNA was used as loading control. Densitometry of Northern blot results is shown. (C & D) Expression analysis of aflR and aflJ by qRT-PCR respectively. The error bars represent standard errors. Different letters on the columns indicate values that are statistically different (p < 0.05).
Fig 7.
Role of rmtA in oxidative stress tolerance.
A. flavus wild type (WT), ΔrmtA, complementation (com) and OErmtA (OE) strains were point-inoculated on PDA containing a range of menadione concentrations (5 mM, 7.5 mM, 10 mM, 12.5 mM, and 15 mM). Cultures were incubated for 48 h at 30°C.