3. Results
3.3. The role of the Activating Transcription Factor Atf1 in Fusarium graminearum
3.3.9. Fusarium graminearum Atf1 is involved in regulation of ROS metabolism and catalase
RESULTS
97 Figure 67. Pigmentation assay and corresponding gene expression analysis. A. Aurofusarin production on CM agar plates after 5 dpi. Aurofusarin biosynthesis was up-regulated in the ∆Fgatf1 mutant compared to the wild type (WT:PH1). B. Quantitative RT-PCR on genes involved in aurofusarin biosynthesis (primer list: Table. 5), gip1 (encoding a putative laccase), gip2 (transcription factor) and pks12 (polyketide synthase). Expression of gip1, gip2 and pks12 was up-regulated in the
∆Fgatf1 mutant compared to the wild type (expression level set at 1) in vitro. Error bars indicate the standard deviation. QRT-PCR was performed using two biological and three technical replicates.
Taken together these results underline the pivotal role of the FgOS-2/Fgatf1-signaling cascade in the regulation of secondary metabolite production.
3.3.9. Fusarium graminearum Atf1 is involved in regulation of ROS metabolism and
98 wild type, ΔFgOS-2 and Fgatf1 mutants under different stress conditions and in planta were also tested.
Detection of ROS using NBT revealed no remarkable coloration of ΔFgatf1 mutant colonies on CM containing 0.2 M NaCl compared to the wild type like it was detectable for the ∆FgOS-2 mutants (Fig. 68A). Hence, there was no accumulation of ROS under mild osmotic stress conditions. Interestingly, on high osmotic stress (0.8 M NaCl), ROS production in Fgatf1oe mutants was higher compared to ΔFgatf1 mutants but lower compared to the wild type (Fig.
68B). NBT staining also confirmed the previous result that the ΔFgOS-2 mutant showed a greater ROS production upon mild osmotic stress compared to the wild type. Fgatf1oe ::ΔFgOS-2mutants exhibited a lower accumulation in ROS production compared to ΔFgOS-2 mutants (Fig. 68A).
Figure 68. Nitroblue tetrazolium (NBT) staining for reactive oxygen species (ROS) production of the wild type (WT:PH1), ΔFgOS-2 mutant and the Fgatf1 mutants on CM and CM supplemented with 0.2 M NaCl (A) and 0.8 M NaCl (B) after 3 days of growth. The dark color indicates ROS production. On CM plates supplemented with 0.2 M NaCl, the FgOS-2 deletion strain showed a greater production of ROS upon osmotic stress compared to the wild type. The Fgatf1oe::ΔFgOS-2 mutant exhibited a lower ROS production on osmotic stress compared to the FgOS-2 deletion strain. No remarkable difference in ROS production was visible between the ΔFgatf1, Fgatf1oe and the wild type strains on this medium. On CM plates supplemented with 0.8 M NaCl, the wild type exhibited a higher production of ROS compared to the Fgatf1oe and the ΔFgatf1 mutants.
WT:PH1 ΔFgatf1 Fgatf1oe ΔFgOS-2 ΔFgOS-2: :Fgatf1oe
CMCM 0.2 M NaCl A
WT ΔFgatf1
CMCM 0.8 M NaCl
Fgatf1oe
B
99 In order to investigate the regulation of four putative catalase genes on the transcriptional level, mycelia of the wild type and ΔFgatf1 mutant strains were raised in liquid CM and subsequently shifted to induction media containing 0.8 M NaCl and 10 mM H2O2, respectively. Induction with 0.8 M NaCl led to a 2-3-fold up regulation of all catalase genes, except for cat2.1 which transcript level remained stable compared to the wild type (Fig. 69C and Table 22). Under oxidative stress condition the expression of all catalase genes was greater in ΔFgatf1 mutants compared to the wild type (Fig. 69B and Table 22). During infection of wheat heads (7dpi) transcript levels of cat1 and cat3 were nearly the same between the wild type and Fgatf1 deletion mutant strains. However, two catalase-encoding genes cat2.1 and cat2.2 were up-regulated approximately 1.5 times in the Fgatf1 deletion mutants compared to the wild type (Fig. 69A and Table 22). The influence of Fgatf1 on catalase expression levels under osmotic stress and oxidative stress conditions is also reflected by the results of an enzyme activity assay. As shown in Figure 69D, catalase activity was unremarkably increased in Fgatf1 deletion strains compared to the wild type under osmotic stress conditions. However, under oxidative stress conditions, catalase activity in ΔFgatf1 mutants was elevated compared to the wild type.
Figure 69. Catalase expression and activity assay. A-C. Quantitative RT-PCR using cDNA obtained from inoculated wheat spikelets (7 dpi, A), H2O2 supplemented (10 mM, B) and NaCl-
cat1 cat2.1 cat2.2 cat3
0 1 2 3 4
cat1TRI4 cat2.1TRI5 cat2.2TRI6 cat3TRI10
Relative expression level
0,0 0,2 0,4 0,6 0,8 1,0 1,2
WT:PH1 DFgOS-2
0 1 2 3 4
TRI4 TRI5 TRI6 TRI10
Relative expression level
0,0 0,2 0,4 0,6 0,8 1,0 1,2
WT:PH1 DFgOS-2 WT:PH1
∆Fgatf1
CM H2O2 NaCl
0 200 400 600 800 1000
Specific enzyme activity [U mg-1]
CM 10 mM H2O2 0.8 M NaCl
TRI4 TRI5 TRI6 TRI10
Relative expression level
0,0 0,2 0,4 0,6 0,8 1,0 1,2
WT:PH1 DFgOS-2 WT:PH1
∆Fgatf1 0,0
0,5 1,0 1,5 2,0
cat1TRI4 cat2.1TRI5 cat2.2TRI6 cat3TRI10
Relative expression level
0,0 0,2 0,4 0,6 0,8 1,0 1,2
WT:PH1 DFgOS-2
TRI4 TRI5 TRI6 TRI10
Relative expression level
0,0 0,2 0,4 0,6 0,8 1,0 1,2
WT:PH1 DFgOS-2 WT:PH1
∆Fgatf1
A
X Data
cat1 cat2.1 cat2.2 cat3
0 10 20 30 60 70 80
cat1 cat2.1 cat2.2 cat3
0 10 20 30 60 70 80
TRI4 TRI5 TRI6 TRI10
Relative expression level
0,0 0,2 0,4 0,6 0,8 1,0 1,2
WT:PH1 DFgOS-2
TRI4 TRI5 TRI6 TRI10
Relative expression level
0,0 0,2 0,4 0,6 0,8 1,0 1,2
WT:PH1 DFgOS-2 WT:PH1
∆Fgatf1
B
C D
0 0.5 1.0 1.5 2.0
0 200 400 600 800 1000
ΔFgatf1
ΔFgatf1 ΔFgatf1
ΔFgatf1
100 Figure 69 continuance
supplemented samples (0.8 M, C). Expression of four different, putative catalase genes was assayed in the wild type and ∆Fgatf1 mutant strains. The wild type expression level was set at 1. Expression of all catalase genes except cat2.1 were up-regulated in the ΔFgatf1 mutant compared to the wild type under osmotic conditions (C). During infection of wheat heads (7dpi) in the ΔFgatf1 mutant, transcript levels of cat1 and cat3 were similar to the wild type. Cat2.1 and cat2.2 expression levels were up-regulated in the ΔFgatf1 mutant compared to the wild type. Under oxidative stress conditions gene expression of all catalase genes is drastically up in the ΔFgatf1 mutant compared to the wild type. Error bars indicate the standard deviation. QRT-PCR was performed twice in triplicate. D. Catalase activity assay using protein extract obtained from mycelium raised in CM with and without 2 h-induction by 0.8 M NaCl and 10 mM H2O2, respectively. Total catalase activity of the ΔFgatf1 mutant was slightly higher compared to the wild type under no-stress and osmotic-stress conditions and drastically elevated under oxidative stress conditions. The assay was performed using two biological and three technical replicates.
Table 22. Gene expression analysis of putative catalase genes. Quantitative real-time PCR results indicate up or down regulation in the ΔFgatf1 mutant compared to the wild type (WT:PH1) (set at 1).
Expression analysis was performed using two biological and three technical replicates. Gene expression was normalized against β-tubulin gene expression.
cat1 cat2.1 cat2.2 cat3
NaCl 3,2161±0,4806 1,2383±0,0555 1,8842±0,0534 2,0880±0,3238 H2O2 28,1526±5,4778 13,0572±1,4254 66,3641±3,9346 26,1159±5,3194 in planta 0,9721±0,1587 1,4482±0,1121 1,6498±0,2237 0,9012±0,0620
The expression analysis of four catalase genes in the mutants ΔFgOS-2, Fgatf1oe, Fgatf1oe::ΔFgOS-2 under osmotic stress conditions and in planta was also performed by qRT-PCR. Expression of cat1, cat2.1 and cat2.2 were up-regulated in the Fgatf1oe mutants compared to the wild type. Only cat3 expression level was down-regulated compared to the wild type after a 2-h induction in 0.8 M NaCl. The expression levels of the four catalase genes (in particular catalase 1 gene expression) were down-regulated in the FgOS-2-deletion strains compared to the wild type (Fig. 70 and Table 23).
Intriguingly, overexpression of Fgatf1 partially restored the expression levels of the four catalases in the ∆FgOS-2 mutant background. Catalase1 expression level in Fgatf1oe::ΔFgOS-2 mutants was 15-fold up-regulated compared to the ΔFgOS-2 mutants (Fig. 70 and Table 23).
101 Figure 70. Catalase gene expression analysis. Quantitative RT-PCR using cDNA obtained from induction medium containing 0.8 M NaCl after a 2-h induction. Expression of four different, putative catalase genes was assayed in the wild type and the mutants ∆FgOS-2, Fgatf1oe::ΔFgOS-2 and Fgatf1oe mutants. The wild-type expression level was set at 1. The Fgatf1oe mutant showed an increase in cat1, cat2.1 and cat2.2 expression levels. Cat3 expression level was down-regulated compared to the wild type. All catalase genes were down-regulated in the FgOS-2-deletion strain compared to the wild type.
Overexpression of Fgatf1 led to a partially restoration of the expression levels of all catalases in the
∆FgOS-2 mutant background. Error bars indicate the standard deviation. QRT-PCR was performed twice with triplicate each.
Table 23. Gene expression analysis of putative catalase genes in osmotic stress medium (0.8 M NaCl). Quantitative real-time PCR results indicate up or down regulation in the mutants ΔFgOS-2, Fgatf1oe:: ΔFgOS-2 and Fgatf1oe compared to the wild type (WT:PH1) (set at 1). Expression analysis was performed using two biological and three technical replicates. Gene expression was normalized against β-tubulin gene expression.
cat1 cat2.1 cat2.2 cat3
ΔFgOS-2 0,0296±0,00109 0,7236±0,032 0,4286±0,0098 0,4426±0,0582 Fgatf1oe
::ΔFgOS-2 0,4471±0,0451 0,7785±0,0,0576 0,5844±0,0231 0,634±0,1206 Fgatf1oe 1,1399±0,1077 3,42±0,153 1,6786±0,079 0,6674±0,1006
In planta, the expression levels of cat2.1 and cat2.2 were up-regulated in Fgatf1oe mutants as well as Fgatf1oe::ΔFgOS-2 mutants compared to the wild type, respectively. On the other hand, expression levels of cat1 and cat3 were down-regulated. However, the expression levels of cat1
WT:PH1 ΔFgOS-2
Fgatf1oe
Fgatf1oe : :ΔFgOS-2
102 and cat3 genes in Fgatf1oe as well as Fgatf1oe::ΔFgOS-2 were about 10 and 5 times higher compared to FgOS-2 deletion strains, respectively (Fig. 71 and Table 24).
Figure 71. Catalase gene expression analysis. Quantitative RT-PCR using cDNA obtained from inoculated wheat spikelets (7 dpi). Expression of catalase genes was assayed in the wild type (WT:PH1) and the mutants ∆FgOS-2, Fgatf1oe and Fgatf1oe::ΔFgOS-2. The wild-type expression level was set at 1.
Expression analysis was performed using two biological and three technical replicates. Gene expression was normalized against β-tubulin gene expression.
Table 24. Gene expression analysis of putative catalase genes of F. graminearum during wheat infection. Quantitative real-time PCR results indicate up or down regulation in the mutants Fgatf1oe, ΔFgOS-2 and Fgatf1oe::ΔFgOS-2 compared to the wild type (WT:PH1) (set at 1). Expression analysis was performed using two biological and three technical replicates. Gene expression was normalized against β-tubulin gene expression.
cat1 cat2.1 cat2.2 cat3
ΔFgOS-2 0,0171±0,00066 0,4623±0,019 0,457±0,0182 0,0523±0,0051 Fgatf1oe
::ΔFgOS-2 0,109±0,00985 1,3426±0,0498 2,0377±0,1138 0,2227±0,0302 Fgatf1oe 0,104±0,01339 1,664±0,0593 2,75±0,0764 0,253±0,0418
These results substantiate the assumption that the FgOS-2/Fgatf1-cascade is the central trigger of ROS metabolism via catalase gene expression in F. graminearum.
3.3.10. Fusarium graminearum Atf1 is involved in the regulation of light-dependent