The mdp/xpt clusters derived metabolites are localized in Hülle cells

The mdp/xpt clusters are especially expressed during fungal sexual development in A. nidulans, and four out of 15 encoded enzymes were confirmed to be localized in sexual mycelia and Hülle cells (Bayram et al., 2016, Dirnberger, 2018). A. nidulans wild type produced the final products shamixanthone (2) and epishamixanthone (3) derived by the mdp/xpt gene clusters. Deletion strains lacking different genes of mdp/xpt clusters did not produce the final products. These mutant strains, however, accumulated various intermediates including emodin, chrysophanol and monodictyphenone and their derivatives (Fig. 12-13). These compounds are yellow in color when they are in powder form (Sanchez et al., 2011).

To further characterize the roles of these products, their localizations were investigated during asexual and sexual development in A. nidulans. A. nidulans prefers asexual development in the light and forms conidiophores with green spores but preferentially undergoes sexual development in the dark with low oxygenlevels and produces mainly sexual fruiting bodies surrounded by Hülle cells. As seen in Figure 15A, after three days of growth in the light, the wild type formed a colony with an overall green conidiation. Overexpressing the gene mdpG and deleting the genes of mdp/xpt clusters had no obvious conidiation difference but deleting the genes mdpC and mdpL resulted in a yellowish colony in the light. After three days of growth in the dark, the wild type formed an overall yellow colony with less green conidiation. The deletion strains ∆mdpG and ∆mdpF showed overall less yellow color compared to wild type, while the mutant strains ∆mdpC and ∆mdpL showed more overall yellow color compared to wild type (Fig. 15A). OEmdpG and the deletion strains ∆mdpD, ∆xptA ∆xptB and ∆xptC showed the same colony phenotype with wild type in the dark. Moreover, an accumulation of reddish pigments was observed on the back side of the colony of ∆mdpC and ∆mdpL strains in the light and dark.

When zooming in, the color of single Hülle cell could be determined (Fig. 15B). The color of the colony was derived from the Hüllecells “nest”. Hüllecells of wt, OEmdpG, ∆mdpD, ∆xptA,

∆xptB and ∆xptC had a similar color. Hüllecells of ∆mdpG and ∆mdpF were pale, and ∆mdpC and ∆mdpL were reddish. This finding indicates that secondary metabolites derived by the

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mdp/xpt clusters are localized in Hüllecells. Deletion of genes of the mdp/xpt clusters changed the metabolites production, which led to the accumulation of different metabolites in Hüllecells.

Figure 15. Secondary metabolites produced by mdp/xpt clusters were accumulated in Hülle cells.

A) Phenotypical analysis of mdp/xpt mutant strains of A. nidulans. Approximately 1000 conidiospores of each strain were point-inoculated on minimal medium (MM) and grown for three days in light (upper panel) and dark (lower panel) at 37°C. B) Deletion of genes of the mdp/xpt clusters changed the color of Hülle cells. Photomicrographs of Hülle cells after five days of sexual growth. White bars = 50 µm.

The backbone gene mdpG encodes the PKS protein as the first enzyme in the biosynthetic pathway of epi-/shamixanthone. Deletion of the gene mdpG resulted in the abolishment of all products of mdp/xpt clusters (Fig. 11-13) and the formation of colorless Hülle cells (Fig. 15B).

In order to verify the role of the gene mdpG in the secondary metabolites production and coloring of Hülle cells, the reintroduction of mdpG in its deletion strain was performed. The

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phenotype of mdpG complementation can be restored to wild type with yellow Hülle cells (Fig.

16A) and its secondary metabolites production can be restored completely with the production of emericellin (1), shamixanthone (2), epishamixanthone (3) and arugosin A (18), which were present in wild type but lost in the ∆mdpG strain (Fig. 16B).

Figure 16. The complementation of mdpG was restored to wild type.

A) Phenotypical analysis of mdpG strains. Approximately 1000 conidiospores of each strain were point-inoculated on minimal medium (MM) grown for three days in light (upper panel) and dark (lower panel) at 37°C. B) EICs (extracted ion chromatogram) of the accumulated compounds in SM_3d of mdpG strains in the dark. wt = wild type, ∆ = deletion, ^com = complementation strain. ID: compound number used in this study. m/z of 1, 2 and 3 were detected in positive mode, 18 was detected in negative mode.

1) emericellin; 2) shamixanthone; 3) epishamixanthone; 18) arugosin A.

The gene mdpC encodes a ketoreductase converting emodin and its derivatives to downstream products in epi-/shamixanthone biosynthetic pathway. Deletion of the gene mdpC resulted in the accumulation of 2, ω-dihydroxyemodin (4), ω-hydroxyemodin (5) and emodin (6) in Hülle cells and further reddish pigmented colony in comparison to wild type. In order to verify the role of the gene mdpC in the secondary metabolites production and coloring of Hülle cells, the reintroduction of mdpC in its deletion strain was performed. The phenotype of mdpC complementation was restored to wild type with yellow Hülle cells (Fig. 17A) and its secondary metabolites production was partly restored (Fig. 17B). The complementation strain produced the final shamixanthone (2), epishamixanthone (3), and emericellin (1) and arugosin A (18) presented in wild type and lost the accumulation of emodins (4, 5, 6), but all of its peaks were

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higher than that presented in wild type including austinol (20), dehydroaustinol (21) (Szewczyk et al., 2008), and an unidentified compound 22 (C18H35NO) (Table 6) as well.

Figure 17. The SM production of the complementation of mdpC was partially restored to wild type.

A) Phenotypical analysis of mdpC strains. Approximately 1000 conidiospores of each strain point-inoculated on minimal medium (MM) and grown for three days in light (upper panel) and dark (lower panel) at 37°C. B) EICs (extracted ion chromatogram) of the accumulated compounds in SM_3d of mdpC strains in the dark. wt = wild type, ∆ = deletion, ^com = complementation strain. ID: compound number used in this study. m/z of 1, 2, 3, 20, 21 and 22 were detected in positive mode, 4, 5, 6 and 18 were detected in negative mode. 1) emericellin; 2) shamixanthone; 3) epishamixanthone; 4) 2, ω-dihydroxyemodin; 5) ω-hydroxyemodin; 6) emodin; 18) arugosin A; 20) austinol; 21) dehydroaustinol;

22) C¹⁸H³⁵NO (unidentified).

In summary, the mdp/xpt gene clusters are expressed during fungal sexual development to produce epi-/shamixanthone, arugosin A and emericellin. The products are localized in the special cell type Hülle cells. Deletion of genes of the mdp/xpt clusters interrupted the epi-/shamixanthone biosynthetic pathway and resulted in the accumulation of various intermediates

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in Hülle cells, which led to the color changes of Hülle cells and phenotypical changes of colonies.

3.2 Precursors of epi-/shamixanthone repress sexual but not asexual development initially