Functional Fgatf1 is necessary for proper vegetative growth and interacts with

To test the influence of Fgatf1 on vegetative growth, plate assays on different media were conducted. When grown on CM agar plates, Fgatf1 deletion strains were slightly reduced in growth compared to the wild type (Fig. 52). On agar plates supplemented with osmotic agents the growth reduction was more pronounced. However, in contrast to FgOS-2 deletion strains, growth is still possible on plates containing 0.8 M NaCl and KCl, respectively or 1.2 M sorbitol (Fig. 52). Fgatf1^oe mutants also showed a slightly reduced growth rate on the osmotic stress media but grew better than the deletion strains. Interestingly, Fgatf1^oe::∆FgOS-2 mutants, grew much better than ∆FgOS-2 mutants on medium conferring osmotic stress (Fig. 52).

Figure 52. Colony morphology of the wild type (WT:PH1) and the mutants ΔFgatf1, Fgatf1^oe, ΔFgOS-2 and Fgatf1::ΔFgOS-2 after 3 days of growth on the osmotic stress media. Complete

WT:PH1

ΔFgatf1

CM CM

1.2 M sorbitol

CM 0.8 M KCl

CM 0.8 M NaCl

Fgatf1^oe::∆FgOS-2 Fgatf1^oe

∆FgOS-2

82 Figure 52 continuance

medium (CM) inoculated with mycelial plugs from 3-day-old cultures. In order to test growth behavior, this medium was supplemented with the osmotic agents: Sorbitol (1.2 M), NaCl (0.8 M) and KCl (0.8 M). Growth of the ΔFgatf1 mutant was slightly retarded on the CM without osmotic stress agent and drastically reduced on osmotic media. In contrast to FgOS-2 deletion strain, growth of the Fgatf1^oe::ΔFgOS-2 mutant was still possible on plates containing 0.8 M NaCl and KCl, respectively or 1.2 M sorbitol. The Fgatf1^oe mutant also showed a slightly reduced growth rate on the osmotic stress medium but grew better than the Fgatf1 deletion strain.

On medium conferring mild osmotic stress, overexpression of Fgatf1 led to an almost full restoration of growth in the ∆FgOS-2 mutant background (Fig. 53).

Figure 53. Colony morphology of the wild type (WT:PH1), ΔFgOS-2 and Fgatf1^oe::∆FgOS-2 strains after 3 days of growth on the mild osmotic stress medium. The basic medium used was complete medium (CM) inoculated with mycelial plugs from 3-day-old cultures. To test sensitivity towards osmotic stress, medium was supplemented with 0.3 M NaCl. Constitutive expression of Fgatf1 led to a pronounced complementation of the growth defect caused by the FgOS-2 deficiency.

The germination of the wild type and the mutant´s conidia in non-osmotic and osmotic media was tested. In the presence of 0.8 M NaCl, germ tubes usually emerged at one apical compartment of ∆Fgatf1-conidia. The wild type in contrast usually germinated at two places.

Moreover, on medium containing 0.8 M NaCl, the germ tubes of Fgatf1 deletion strains were delayed in growth compared to the wild type after 16 hours of incubation. Under non-stress conditions conidial germination was similar between the strains (Fig. 54).

CM CM

0.3 M NaCl

WT:PH1

Fgatf1^oe::∆FgOS-2

∆FgOS-2

Fgatf1^oe::∆FgOS-2

∆FgOS-2 WT:PH1

CM CM

0.3 M NaCl

WT:PH1

∆FgOS-2

Fgatf1^oe::∆FgOS-2

CM CM

0.3 M NaCl

WT:PH1

∆FgOS-2

Fgatf1^oe::∆FgOS-2

RESULTS

83 Figure 54. Bright-field microscopy of germinating conidia of the wild type and ∆Fgatf1 mutant strains in liquid CM and liquid CM supplemented with 0.8 M NaCl after 16 hours of incubation. In non-osmotic medium, the wild-type and ΔFgatf1 mutant conidia formed multiple normally shaped germ tubes. In osmotic medium, the wild type conidia germination was similar to non-osmotic medium. The ∆Fgatf1 germ tubes generally emerged at one apical compartment of

∆Fgatf1-conidia and were delayed in the development compared to the wild type. The wild type and ΔFgatf1 mutant strains showed comparable conidial germination rates in both osmotic and non-osmotic media. Scale bar: 10 µm.

Like FgOS-2 deletion strains, also ∆Fgatf1 mutants exhibited a partial resistance against the phenylpyrrolic fungicide fludioxonil (Fig. 55). These results indicate that Fgatf1 is a downstream target of FgOS-2 under osmotic and fungicide stress conditions and that activation of Fgatf1 is necessary for proper vegetative growth. Surprisingly, Fgatf1^oe::∆FgOS-2 mutants exhibited the highest resistance against fludioxonil.

Fgatf1 deletion strains, but not the overexpression mutants, grew much better on medium supplemented with H₂O₂ compared to the wild type (Fig. 55). I could demonstrate that deletion of FgOS-2 also led to an increased resistance towards oxidative stress. This observation substantiates the assumption that the FgOS-2/Fgatf1-cascade is the central trigger of ROS metabolism in F. graminearum (see also 3.2.3 and 3.2.7).

ΔFgatf1 WT:PH1

CM 0.8 M NaCl

84 Figure 55. Colony morphology of the wild type (WT:PH1) and the mutants ∆FgOS-2, ∆Fgatf1, Fgatf1^oe and Fgatf1^oe::ΔFgOS-2 after 4 days of growth on oxidative stress and fungicide agar plates. The basic medium was complete medium (CM) inoculated with mycelial plugs from 3-day-old cultures. Media were supplemented with 10, 15 and 20 mM H2O2 or 0.05 mg l^-1 fludioxonil. No differences in growth performance between the wild type and FgOS-2 deletion mutant strains were observed on agar plates supplemented with 10, 15 mM H2O2. On plates supplemented with 20 mM H2O2, the ΔFgOS-2 mutant grew even better than the wild type after 4 days post-incubation. The Fgatf1 deletion strain, but not Fgatf1^oe and Fgatf1^oe::ΔFgOS-2 mutants, grew much better on medium supplemented with H2O2 compared to the wild type. On plates containing 0.05 mg l^-1 fludioxonil, growth of the FgOS-2 deletion and Fgatf1^oe::ΔFgOS-2 mutant strains were similar to CM control plates, while growth of the wild type and Fgatf1^oe mutant strains nearly ceased. The ∆Fgatf1 mutant displayed a partial resistance against the phenylpyrrolic fungicide fludioxonil.

To further proof the interaction of FgOS-2 and Fgat1 under stress conditions I performed a bimolecular fluorescence complementation assay (BiFC; Hoff and Kück, 2005). For this purpose, the open reading frame (ORF) of FgOS-2 was fused to the N-terminus of the yellow fluorescent protein (YFP) and the ORF of Fgatf1 was fused to the C-terminus of YFP. A strain expressing a histon-1-mCherry fusion which facilitates detection of nuclei (A. L.

Martínez-Rocha, unpublished results), was used as recipient for both plasmids. Strains obtained from co-transformation of both plasmids were initially analysed by PCR. Mutants positive for both plasmids were subsequently analysed by confocal laser scanning microscopy

WT:PH1

ΔFgatf1

CM CM CM 0.05 mg l^-1 10 mM H₂O₂ 15 mM H₂O₂ 20 mM H₂O₂ Fludioxonil

∆FgOS-2

Fgatf1^oe::∆FgOS-2 Fgatf1^oe

85 (see material and methods). Unstressed mycelia of the mutant and the wild type strains did not emit YFP-fluorescence when excited with a 514 nm laser line. Application of 1.2 M NaCl, mutants evoked fluorescence inside the nucleus of fungal hyphae within 30 minutes (Fig. 56).

This result provides further evidence for an interaction of Fgatf1 and FgOS-2 under osmotic stress conditions.

Figure 56. Bimolecular fluorescence complementation assay. FgOS-2 was fused to the N-terminus and Fgatf1 to the C-N-terminus of YFP, respectively. Both construct were co-transformed into F. graminearum wild type. 24h after germination of conidia osmotic stress mediated by 1.2 M NaCl was applied and hyphae were subsequently imaged using confocal laser scanning microscopy. Scale bar: 10 µm.

3.3.6. Overexpression of Fgatf1 restores sexual reproduction in FgOS-2 deletion strains