Figure 4. DHS and DOHH overexpression affects infection structure formation and impairs DON mycotoxin production.

(A) Glumes inoculated with 5 μl of 2 × 10⁴ conidia per ml suspension of WT, DHSoex, DOHHoex or DHSoex/DOHHoex strains were incubated in a chamber with a photoperiod of 16 hours light at 16 °C. Complex infection structures were visualized 8 dpi. Maximum intensity projections of a z-stack of 20–30 pictures were produced with a Zeiss Axio Imager.Z1 microscope equipped with a Zeiss Apotome. The DHSoex mutant forms more complex infection structures than the wild type strain. In contrast, the DOHHoex mutant produced no infection structures. Hypha (H), complex infection structure (IC). Scale bar = 20 μm. Overexpression of DHS increases DON production as well as expression of the TRI genes in comparison to the wild type strain. On the contrary, DOHH overexpression suppresses production of DON mycotoxin and expression of the TRI4, TRI5 and TRI6 genes. DON mycotoxin production was measured in the overexpressing mutants grown in (B) wheat plants (cv. Nandu) for 21 days or (C) mycelia grown under inductive (5 mM (NH₄)₂SO₄ as the nitrogen source) and non-inductive (5 mM NaNO₃ as the nitrogen source) conditions at 28 °C, 150 rpm and 8 dpi. (D) RNA extracted from wheat plants infected with the wild type and overexpressing mutants 12 dpi was used to quantify the relative expression of TRI4, TRI5 and TRI6 genes. (E) Relative expression of TRI genes in culture was measured from mycelia grown under inductive conditions at 28 °C, 150 rpm and 3 dpi. Error bars indicate standard deviations calculated from data per triplicate samples and are representative of three independent experiments. Significance with respect to wild type: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (calculated with ANOVA-Bonferroni-Holm).