Figure 1.
Primary and second – tier screening of putative Fusarium oxysporum strain 11C transformants.
Box and Whisker plot depicting the distribution of putative Fusarium oxysporum strain 11C transformants (n =1,563) screened for altered ethanol tolerance during primary screening at 48 hours (A) and 96 hours (B) and transformants (n =402) screened for altered ethanol tolerance during secondary screening (C). Fungi were grown at 25°C in minimal medium [37] with five different treatments; no ethanol control; hygromycinB 60 µg ml-1 control; 0.5% (vv-1) ethanol; 6% (vv-1) ethanol; 10% (vv-1) ethanol) (Primary-tier) (A, B) or six treatments; no alcohol control; 6% (vv-1) ethanol; 7% (vv-1) ethanol; 8% (vv-1) ethanol; 9% (vv-1) ethanol; 10% (vv-1) ethanol (Second-tier) (C). Fungal growth (OD600nm) was measured after 96 hours using a spectrophotometer (Safire2, Tecan, Austria).
Figure 2.
Tertiary screening of Fusarium oxysporum strain 11C transformants (n=29).
Fungi were grown in minimal medium [37] supplemented with; no alcohol (A); 2% (vv-1) ethanol (ethyl alcohol; Sigma, UK) (B), 4% (vv-1) ethanol (C), 6% (vv-1) ethanol (D) or 0.25% (vv-1) butanol (n-butanol; Sigma, UK) (E), 0.5% (vv-1) butanol (F) or 0.75% (vv-1) butanol (G) and incubated at 25°C for 7 days. Percentage increase or decrease in fungal growth relative to the positive control (wild-type strain 11C) was determined. Results represent the mean of three independent experiments and bars indicate SEM. Transformants significantly different from the positive control (wild-type strain 11C) are highlighted with an asterisk (level of significance: <0.050 *, <0.01**, <0.001***).
Figure 3.
Principal component analysis (PCA) of tertiary screen.
Principal component analysis (PCA) was used to reduce the dimension of the tertiary dataset comprising of several interrelated variables (no alcohol, ethanol and butanol treatments on all 29 transformants) whilst retaining the variation present within the dataset. The dataset was transformed into variables (F1-F7) through component analysis using XLSTAT (Addinsoft) v2013.1 software. Scree plot of PCA analysis with the first three Eigenvalues (F1-F3) corresponding to the highest percentage of variance (A) Observation plot (built on Scree plot) in the factor space according to the first and second Principal components F1 and F2 (B) Observation plot (built on Scree plot) in the factor space according to the first and second Principal components F1 and F3 (C).
Figure 4.
Temporal analysis of alcohol tolerance in F. oxysporum
Fungal conidial inoculum was produced in Mung bean broth [32] and resuspended in minimal medium [37] at a concentration of 106 ml-1. A volume 100 µl conidia (106 ml-1) was added to microtiter (96-well; Sarstedt, Germany) plates with either no alcohol (A), ethanol (Ethyl absolute, Sigma, UK) at a concentration of 4% (vv-1) (B) or butanol (n-Butanol Butyl alcohol, Sigma, UK) at a concentration of 0.75% (vv-1) (C). Fungi were maintained at 25°C for 168 hours and growth (OD600nm) was measured every 24 hours. Fungal growth (OD600nm) was measured using a spectrophotometer (Spectra Max 340 PC 96-well plate reader, Molecular Devices, USA). Bars indicate SEM. Growth of Tr. 259 significantly different from 11C is highlighted with an asterisk (level of significance: *<0.05, ** < 0.01).
Figure 5.
RT-PCR analysis of putative hexose transporter under alcohol stress.
Temporal analysis of putative hexose transporter transcript (FOXG_09625) during shake flask growth of Fusarium oxysporum strain 11C and transformant Tr. 259 under normal conditions (A), ethanol stress (B) and butanol stress (C). F. oxysporum (2ml 106 ml-1) was aerobically cultured in Erlenmeyer flasks (100 ml) in 48 ml of minimal medium [37] for 12 hours shaking at 150 rpm at 25°C. At 12 hours, flasks were amended with 2ml of water, ethanol or butanol to a final concentration of 0%, 4% and 0.75% (vv-1), respectively. RT-PCR was conducted on RNA samples harvested at 0.5, 2, 12 hours respectively post – inoculation. Putative HXT accumulation was quantified relative to the housekeeping gene β-tubulin (FOXG_06228) [38] Results are based on three experiments (no alcohol, ethanol) or two experiments (butanol), each with three replicates per time point per strain tested. RT-PCR was conducted twice on each experiment. Bars indicate SEM. Tr. 259 FOXG_09625 mRNA accumulation significantly different from strain 11C is highlighted with an asterisk (level of significance: *<0.05, **≤ 0.01, ***< 0.001).