DNA damage repair and oxidative stress response is affected by

Figure 13: Mitochondrial localization of C-terminal Gfp-tagged Thi4 in Verticillium dahliae. Localization of C-terminal Gfp-tagged VdThi4 was detected by fluorescence microscopy. The Gfp signal is shown in green;

mitochondria were stained with 50 nM MitoTracker^® and are visible in red (white arrow). (a) The VdThi4 - Gfp strain visualizes the localization of V. dahliae Thi4 in the mitochondria. The green Gfp signals of VdThi4 - Gfp merges with the stained mitochondria red signals, visualized in yellow color. (b) V.dahliae Gfp control strain with Gfp fluorescent cytosol and stained mitochondria. Liquid cultures were grown for 5 days at 25°C.

3.1.3 DNA damage repair and oxidative stress response is affected by Verticillium Thi4 Verticillium Thi4 was shown to be involved in thiamine pathway (Figure 11). Homologues of the gene in other organisms are important for more than their role in the thiamine pathway.

Yeast Thi4p for example is proposed to play a dual role. Besides its role in thiamine biosynthesis it is involved in maintenance of mitochondrial genome stability (Machado et al., 1997). In V. dahliae it was shown that Thi4 is localized in the mitochondria (Figure 13), based on this fact experiments for a likewise involvement in mitochondrial genome stability were performed to estimate a further function of the protein. The repair of UV damaged DNA was estimated by survival experiments after UV-B light treatment. 1000 cells per plate were inoculated on minimal medium and treated with UV-light. Subsequently the survival rate was estimated by counting fungal colonies (Figure 14).

Figure 14: UV-illumination assay. Survival rate in percent of Yeast and Verticillium growing colonies after treatment with UV-light (302 nm) for 5, 10, 15 and 20 s. 3 plates per condition were used, 0 s accords to 100%

(a) More colonies (12-15%) of yeast ∆thi4 strain complemented with Verticillium DNA were growing compared to ∆thi4 strain (3%). Growth difference is highly significant at 5 sec time point. Verticillium DNA containing yeast ∆thi4 mutants growing better after UV-treatment than wild type yeast strain BY4741. Colonies were counted after 3 days incubation at 30°C. (n=3) (b) Growing V. dahliae colonies after treatment with UV-light (302 nm) for 5, 10, 15 and 20 s. More colonies of ∆THI4 complementation strains were growing compared to

∆THI4 mutant. The growth difference is significant at 5 s time point. Colonies were counted after 4 days incubation at 25°C ( n=3). P-value 0.001 = highly significant *** or +++, 0.01 = significant ** or ++ and 0.05 = less significant * or + (significance: * to Vd wt; + to ∆THI4).

Survival after UV-treatment, affected by the existence or absence of Vd or VlTHI4, was tested in S. cerevisiae and V. dahliae. The tests revealed that Verticillium THI4 compensates the lethal effect caused by UV-light in the Vd∆THI4 and the yeast ∆thi4 strains.

Only 3% of the UV exposed colonies of the yeast ∆thi4 strain survived (Figure 14 a) after 5 s of treatment (0 s UV-exposure accords to 100% growth). In contrast, 12 to 15% colonies of the Verticillium THI4 complemented strains were surviving under these conditions. The effect on UV-light treated V. dahliae strains was comparable to yeast wild tpye. 24% of the Vd wild type colonies were surviving in comparison to mutant strains (7% surviving colonies) and the complemented strains (26-33% surviving colonies) (Figure 14 b). The colonies on plate were counted after four days but plates were incubated up to seven days. By this it was excluded that the growth difference was an effect caused by slower growing of the THI4 deletion strain.

Thiamine plays an important role in oxidative stress response (Ahn et al., 2005; Rapala-Kozik et al., 2008; Rapala-Kozik et al., 2012; Tunc-Ozdemir et al., 2009). In previous studies on cellular lines it was shown that oxidative stress can lead to the degradation of mtDNA and the inhibition of base excision repair (Shokolenko et al., 2009). In order to screen for oxidative stress-response of the ∆THI4 mutant, growth tests on oxidative stress inducing agents were performed (Figure 15). In the cell oxidative stress always leads to stress in the mitochondria likewise, where Verticillium Thi4 is localized (Figure 13).

For the F. oxysporum homolog ∆sti35 mutant it was shown, that it has a reduced growth on 0.001% menadione containing plates (Ruiz-Roldan et al., 2008). A similar growth defect was monitored for Vd∆THI4 mutant strains in comparison to V. dahliae wild type during menadione-mediated oxidative stress (Figure 15 marked in red).

Furthermore, the antibiotic 2,4-Diacetylphloroglucinol (2,4-DAPG) produced by Pseudomonas fluorescens was tested in this study for putative inducing oxidative stress on the Vd∆THI4 strain. In S. cerevisiae it was shown that this affects multiple basic cellular processes, including membrane function, reactive oxygen regulation and cell homeostasis (Kwak et al., 2011). We could reveal that the oxidative stress activating P. fluorescens antibiotic 2,4-DAPG affected fungal growth of both strains, the mutant and the wild type in same extent (Figure 15 marked in red). No growth difference or defect occurs on complete medium for both oxidative activating reagents.

Figure 15: Oxidative stress tests on gradient plates. Under oxidative stress conditions (0.001% menadione) the deletion strain Vd∆THI4 exhibits reduced growth in comparison to Vd wt on CDM agar (indicated by red rectangle). The stress inducing antibiotic 2,4-DAPG induces growth reduction of both, Vd wt and Vd∆THI4 mutant strain on synthetic CDM medium (indicated by red rectangle). The stressors menadione and 2,4-DAPG induced no growth reduction phenotype on PDA medium. The experiments were performed with two different Vd∆THI4 transformants with the same effect. Gradient plates were used with low concentration (0%, µg left) and high concentration (0,001%, 20µg right). Spore amount: 10⁴ spores. Plates were incubated at 25°C for 7 days.

The response of the fungus to reactive oxygen species is important for the plant-pathogen interaction. Hence, testing the Thi4 lacking strain with reduced tolerance to DNA damage agents (UV or menadione) in a plant infection assay in vivo inside the plant in the xylem-sap was the following experiment in this study.