Proteins encoded by the xanthone (xpt) gene cluster are enriched in the

Hülle cells might produce specific secondary metabolites to protect the cleistothecia from fungivors. It is known that xanthones are secondary metabolites with antimicrobial activities (Chen et al., 2017, Noordin et al., 2016). To gain more insight into the production of an antimicrobial agent in Hülle cells the localization of enzymes encoded by the xanthone (xpt) secondary metabolite gene cluster was further investigated. Monodictyphenone represents a precursor for the production of xanthones. As an example of the 24 proteins that were common between Hülle cells and sexual mycelia the prenyltransferase XptB and the oxidoreductase XptC were found and their localization was investigated (Figure 25). The second prenyltransferase XptA was only found in a sexual mycelium and is listed in Supplementary table 4. The criteria concerning the localization investigation were the presence of the protein in both sexual mycelium and Hülle cells in the proteomics approach. Therefore, the localization of XptA is not shown.

The prenyltransferase XptB and the oxidoreductase XptC are involved in the conversion of monodictyphenone into xanthones, which are antimicrobial agents.

Localization of the prenytransferase XptB and the oxidoreductase XptC involved in the conversion of monodictyphenone into xanthones in Hülle cells was investigated and their presence could be confirmed (Figure 25). Hülle cells were enriched from sexual mycelium using the cleistothecia-rolling technique and the localization of XptB::GFP, XptC::GFP was observable in the cytoplasm of Hülle cells. The fusion proteins were mainly observed in the center of Hülle cells. XptB::GFP and XptC::GFP seem to be mainly equally distributed in the cytoplasm of Hülle cells. XptB::GFP and XptC::GFP was not clearly observable in the membrane. This suggests that proteins identified by the applied enrichment of Hülle cells correlate between the proteomic approach and the microscopic data in their fungal localization.

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Additionally, the localization of XptB and XptC was apparent during initial vegetative growth. Therefore, vegetative hyphae in submerged liquid cultures were used to visualize the presence of the fusion proteins. The localization of the fusion proteins XptB::GFP and XptC::GFP could be confirmed mainly in the cytoplasm of vegetative hyphae(Figure 26). This suggests that proteins encoded by the xanthone (xpt) gene cluster are found not only in Hülle cells but also in vegetative hyphae suggesting that these proteins are found during initial vegetative growth.

Figure 25. Enzymes encoded by the xanthone (xpt) gene cluster are localized in Hülle cells.

XptB::GFP and XptC::GFP are observable in the cytoplasm of Hülle cells. Fluorescence microscopy of Hülle cells: A) xptB::gfp;lysA;nkuA (AGB1086) B) xptC::gfp; nkuA (AGB1088) C) nkuA (AGB552) parental strain D) A strain expressing GFP constitutively (AGB596). Scale bar is 10 µm.

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The protein sequence of green fluorescent protein (GFP) consists of 239 amino acids with a predicted molecular mass of 26.9 kDa. The XptB protein consists of 463 amino acids with a predicted molecular mass of 52.4 kDa. The XptC protein consists of 622 amino acids with a predicted molecular mass of 67.8 kDa.

Western hybridization experiments showed that XptB::GFP is detectable as expected around 79 kDa and that XptC::GFP is detectable as expected around 95 kDa in three and five day old sexual mycelium with high amounts of Hülle cells plus a three day old vegetative mycelium (Figure 27). A strain expressing GFP constitutively (AGB596) served as a positive control where GFP was detectable around 27 kDa. The parental nkuA (ABG552) strain served as negative control where GFP was not observable. A proteolytic cleavage product of XptB::GFP was detectable around 27 kDa. This is most likely a degradation product of the full-length XptB::GFP fusion protein and represents the degraded GFP product. This result implies that the prenytransferase XptB and the oxidoreductase XptC is found in the cytoplasm of Hülle cells and that these proteins are present in vegetative and sexual mycelia.

Figure 26. Enzymes encoded by the xanthone (xpt) gene cluster are localized in vegetative mycelium.

XptB::GFP and XptC::GFP are localized in the cytoplasm of vegetative hyphae. Fluorescence microscopy of vegetative mycelium: A) xptB::gfp;lysA;nkuA B) xptC::gfp;nkuA C) nkuA E) A strain expressing GFP constitutively (AGB596). Scale bare is 20 µm

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3.2.7. Comparative proteomics revealed that the proteome of surface Hülle cells overlaps to other fungal tissues besides of a sexual mycelium

As shown in the Venn diagram (Figure 24) the proteome of surface Hülle cells overlapped to other fungal tissues such as asexual and vegetative mycelium besides sexual mycelium. Ten proteins were identified in Hülle cells and asexual mycelium (Table 8). These proteins were unidentified in Hülle cells grown in liquid media. This suggests that these proteins are only present during surface growth. Since these proteins were unidentified in Hülle cells grown in liquid media in the second proteomic

Figure 27. The fusion proteins XptB::GFP and XptC::GFP are found in sexual mycelium with high amounts of Hülle cells and a vegetative mycelium.

Western hybridization of vegetative and sexual mycelium of xptB::gfp; lysA; nkuA (AGB1086) and xptC::gfp; nkuA (AGB1088). Vegetative mycelium was harvested after 3 days. Sexual mycelium with high amounts of Hülle cells was harvested 3 and 5 days after inoculation. The XptB protein consists of 463 amino acids with a predicted molecular mass of 52.4 kDa. The XptC protein contains 622 amino acids with a predicted molecular mass of 67.8 kDa. In order to detect XptB::GFP (xptB::gfp; lysA

nkuA, AGB1086) and XptC::GFP (xptC::gfp; nkuA AGB1088 ) a primary -GFP antibody (sc-9996;

Santa Cruz) was used followed with an incubation of an -mouse secondary antibody (G21234, Invitrogen).The fusion protein XptB::GFP was detected as expected around 79 kDa and XptC::GFP was detected as expected around 95 kDa. A strain expressing GFP constitutively served as a control where GFP was detectable around 27 kDa. Proteolytic cleavage product of XptB::GFP was detectable around 27 kDa. This is most likely a degradation product of the full-length XptB::GFP fusion protein.

The parental strain nkuA served as a negative control where GFP and the fusion proteins were not detectable.

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approach the focus was not laid on these proteins. Proteins that were common in Hülle cells and vegetative mycelium are listed in Table 8. These proteins were identified in Hülle cells grown in liquid media and are discussed in the context of core proteome of both types of Hülle cells within the next chapter. Data reveal that the proteome of surface Hülle cells overlaps to that of other fungal tissues besides sexual mycelium.

Hülle cells & asexual mycelium

Table 8. Overlapping proteins of Hülle cells (HC), asexual (Asex.) and vegetative mycelium (Veg.).

The listed proteins are found in the red rectangle of the Venn diagram of Figure 16. Only proteins identified in two or more biological replicates and with two or more peptides per protein were considered for the analysis. Six proteins are listed and represent the proteins with the highest spectral counts. Numbers in brackets represent the average of spectral counts from three biological replicates. Six proteins out of ten are listed with the highest spectral counts, that were common in Hülle cells and asexual mycelium. The orange colour represents proteins found in Hülle cells and asexual mycelium. Six proteins out of eight are listed with the highest spectral counts, that are common in Hülle cells and vegetative mycelium. The green colour represents the proteins that were found in Hülle cells and vegetative mycelium.

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3.2.8. Functional annotation reveals that surface Hülle cells are