3. Results
3.3. The role of the Activating Transcription Factor Atf1 in Fusarium graminearum
3.3.10. Fusarium graminearum Atf1 is involved in the regulation of light-dependent
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
103 addition, there are indications for a connection between the SAPK-signaling cascade and the light-dependent regulation. To check if –in F. graminearum– the light perception and the regulation of light dependent genes are influenced by Atf1, we conducted plate assays under different light conditions. First, CM plates were inoculated with the wild type and the mutants
∆FgOS-2, ∆Fgatf1, Fgatf1oe and Fgatf1oe::∆FgOS-2, respectively. Second, these plates were incubated at 28°C under permanent light, permanent darkness, and with a 6-hours-light/6-hours-darkness rhythm, respectively. Figure 72 shows the colonies morphology after 4 days of incubation. The wild type, ∆FgOS-2 and ∆Fgatf1 strains developed pericentric rings of yellowish and reddish regions when they were incubated in the light-darkness rhythm. In both Fgatf1 over-expressing mutants the red colour was evenly distributed in the centre of the colonies. The growing edge of colonies of all strains was uniformly yellowish. Under permanent darkness condition, the formation of rings was weaker in the wild type and ∆FgOS-2 mutant strains and absent in ∆Fgatf1 as well as Fgatf1oe mutants (Fig. 72). Finally, under permanent light condition, none of the strains produced coloured rings. Interestingly, ∆FgOS-2 mutants were strongly affected in the formation of aerial hyphae under permanent light condition.
Figure 72. Growth assay in different light conditions. The wild type (WT:PH1) and the mutants
∆FgOS-2, ∆Fgatf1, Fgatf1oe and Fgatf1oe::∆FgOS-2 were inoculated on CM plates and then incubated at 28°C for 4 days under permanent light, permanent darkness, and with a 6-hours-light/6-hours-darkness rhythm, respectively.
To proof if genes involved in the perception and transduction of light signals are influenced by Fgatf1 and FgOS-2, respectively, RT-PCR using cDNA obtained from liquid cultures raised under permanent darkness and permanent light conditions, respectively, were conducted. Three different putative green light receptors (opsins; FGSG_07554,
WT:PH1 ΔFgatf1 Fgatf1oe Fgatf1oe::∆FgOS-2 ΔFgOS-2
WT:PH1ΔFgatf1ΔFgOS-2Fgatf1oeFgatf1oe:: ∆FgOS-2
Tub Ops1 Ops2 Ops3 Frq1 Vvd1
FgOS-2
Fgatf1
Ops2 Ops3
Frq1 Vvd1 FgOS-2
Fgatf1
Ops2 Frq1
Vvd1
Perithecia formation:
candidates for light regulation 1. Ops3
2. Vvd1 3. Frq1
WT:PH1ΔFgatf1ΔFgOS-2Fgatf1oeFgatf1oe:: ∆FgOS-2
Tub Ops1 Ops2 Ops3 Frq1 Vvd1
FgOS-2
Fgatf1
Ops2 Ops3
Frq1 Vvd1 FgOS-2
Fgatf1 Frq1 Ops2
Vvd1
Perithecia formation:
candidates for light regulation 1. Ops3
2. Vvd1 3. Frq1
WT:PH1ΔFgatf1ΔFgOS-2Fgatf1oeFgatf1oe:: ∆FgOS-2
Tub Ops1 Ops2 Ops3 Frq1 Vvd1
FgOS-2
Fgatf1
Ops2 Ops3
Frq1 Vvd1 FgOS-2
Fgatf1
Ops2 Frq1
Vvd1
Perithecia formation:
candidates for light regulation 1. Ops3
2. Vvd1 3. Frq1
WT:PH1ΔFgatf1ΔFgOS-2Fgatf1oeFgatf1oe:: ∆FgOS-2
Tub Ops1 Ops2 Ops3 Frq1 Vvd1
FgOS-2
Fgatf1
Ops2 Ops3
Frq1 Vvd1 FgOS-2
Fgatf1 Frq1 Ops2
Vvd1
Perithecia formation:
candidates for light regulation 1. Ops3
2. Vvd1
3. Frq1
104 FGSG_01440 and FGSG_03064), the putative blue light receptor VIVID (Vvd1;
FGSG_08456) and the putative circadian clock regulator protein, Frequency (Frq;
FGSG_06454) were analyzed. In summary, Fgatf1 has a dominant influence on the expression of the ops2, frq1 and vvd1. Upon Fgatf1 deletion, the induction of ops2 expression in the light was absent. In the wild type, frq1 and vvd1 expression were induced in darkness and repressed under light conditions. This regulation pattern was inversed in ∆Fgatf1, Fgatf1oe and ∆FgOS-2 mutants. Interestingly, in Fgatf1oe::∆FgOS-2 mutants, frq1 and vvd1 transcript level were elevated both in darkness and in permanent light. In ∆FgOS-2 mutants under light conditions, ops2 and ops3 expression were strongly induced compared to the wild type. Ops1 expression was independent from light conditions and the genetic background (Fig. 73).
Figure 73. Expression analysis of genes encoding for putative light receptors and putative circadian clock receptor proteins in the wild type (WT:PH1) and the mutants ∆FgOS-2,
∆Fgatf1, Fgatf1oe and Fgatf1oe::∆FgOS-2. A RT-PCR (35 PCR cycles, primer list: Table. 4) using cDNA obtained from liquid cultures raised under permanent darkness and permanent light conditions, respectively. Expression analysis was performed using two biological and three technical replicates.
These results indicate that genes involved in the perception and transduction of light signals are influenced by Fgatf1 and FgOS-2.
Taken together, Fgatf1 is the main downstream target of FgOS-2. Fgatf1 plays important roles in stress adaptation, secondary metabolism including mycotoxin production, sexual reproduction and virulence towards wheat and maize.
WT:PH1ΔFgatf1ΔFgOS-2Fgatf1oeFgatf1oe:: ∆FgOS-2
Tub Ops1 Ops2 Ops3 Frq1 Vvd1
FgOS-2
Fgatf1
Ops2 Ops3
Frq1 Vvd1 FgOS-2
Fgatf1 Frq1 Ops2 Vvd1
Perithecia formation:
candidates for light regulation 1. Ops3
2. Vvd1 3. Frq1 WT:PH1∆Fgatf1∆FgOS-2Fgatf1oe:: ∆FgOS-2Fgatf1oe
105