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Sexual communication between complementary mating partners in the fungal group of zygomycetes is mediated by the trisporoid pheromone system. A key enzyme towards biosynthesis of hormonally active trisporoids is 4-dihydromethyltrisporate dehydrogenase (TSP1), an enzyme occurring in all zygomycetous fungi. Trisporic acid and some of its precursor molecules serve as pheromones for recognizing complementary mating partners and for induction of the differentiation program towards sexual spore formation. In the parasitic zygomycete Parasitella parasitica, a biotrophic fusion parasite infecting many other zygomycetes, these substances have an additional function: They are also responsible for host-parasite recognition and the formation of the characteristic infection structures. Parasitic interactions are mating type dependent as well. In the Mucor-related mycoparasite P. parasitica we can study both types of communication in parallel. We were interested in protein structures of TSP1 from P. parasitica, the genome of which was recently sequenced by us, and especially in the mechanisms involved in the switch from sexual to parasitic communication. P. parasitica contains at least six genes coding for TSP1-like proteins. We created models of tertiary structures and performed protein-protein docking with the resulting protein structures to simulate dimerization and to provide support for probable regulatory mechanisms at the protein level. The resulting structure models show differences in putative activity and binding preferences between the different TSP1-like proteins. Two of them seem to be able to form solid binding pockets for substrate and cosubstrate after dimerization. The other four TSP1-like proteins are more likely to represent regulating subunits for the two active isoforms. The ability to form homodimers with enzymatic activity could be the crucial difference between sexual and parasitic communication pathways. TSP1 PARPA_07791 forms enzymatically inactive homodimers. The second TSP1, PARPA_04105, forms active homodimers and could be responsible for the parasitic pathway of communication. Both TSP1 proteins can form more or less active heterodimers with the additional TSP1-like proteins. TSP1 PARPA_07791 mediates the sexual pathway probably as in other zygomycetous fungi like Mucor mucedo. High sequence identities between this TSP1 isomer and TSP1 proteins from other zygomycetes substantiate its function. This bioinformatic study supports previous experimental findings of post-translational regulation of 4-dihydromethyltrisporate dehydrogenases in zygomycetes and, for the first time, provides a substantiated hypothesis of the underlying mechanism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The putative mating type locus of mucoralean fungi consists of a single high mobility group (HMG)-domain transcription factor gene, sexM or sexP, flanked by genes for an RNA helicase and a triosephosphate transporter. We used degenerate primers derived from the amino acid sequence of the RNA helicase to sequence a fragment of this gene from Mucor mucedo. This fragment was extended by inverse PCR to obtain the complete sequences of the sex loci from both mating types of M. mucedo. The sex loci in M. mucedo reflect the general picture obtained previously for Phycomyces blakesleeanus, presenting a single HMG-domain transcription factor gene, sexM and sexP in the minus and plus mating types, respectively. These are located next to a gene for RNA helicase. Transcriptional analysis by quantitative real-time PCR showed that only transcription of sexM is considerably stimulated by adding trisporoid pheromones, thus mimicking sexual stimulation, whereas sexP is only slightly affected. These differences in regulation between sexM and sexP are supported by the observation that the promoter sequences controlling these genes show no similarities. The protein structures themselves are considerably different. The SexM, but not the SexP protein harbours a nuclear localization sequence. The SexM protein is indeed transported to nuclei. This was shown by means of a GFP fusion construct that was used to study the localization of SexM in the yeast Saccharomyces cerevisiae. The fusion protein is highly enriched in nuclei.

The zygomycete Blakeslea trispora produces high amounts of the general zygomycete β-carotene-derived sexual signal compounds, the trisporoids. These can be isolated from the culture medium and purified by extraction with organic solvents followed by thin layer chromatography. Concentration is determined spectrophotometrically using specific extinction coefficients established for some members of this compound family. The effect of the extraction and activity of the isolated compounds is best tested physiologically, exploiting the ability of trisporoids to induce the formation of sexually committed hyphae, the zygophores, in other zygomycete species. Methods for B. trispora culture, trisporoid extraction, and further analyses of trisporoids are described in this chapter.

Recognition between mating partners, early sexual morphogenesis and development are regulated by a family of beta-carotene derived signal compounds, the trisporoids, in zygomycete fungi. Mating type-specific precursors are released from the hyphae and exert their physiological effects upon compatible mating partners. In a cooperative synthesis pathway, later intermediates and finally trisporic acid are formed. All trisporoids occur in a number of derivatives. Trisporic acid and some precursors directly influence the transcription of genes involved in sexual development. This has been demonstrated for TSP3, encoding the carotene oxygenase involved in sexually induced cleavage of beta-carotene. Species specificity of mating despite a common and commonly recognized signaling system is maintained by several factors. Specific distribution and recognition patterns of the trisporoid derivatives and the proposed divergence in trisporoid synthesis pathways in diverse species play a role. The derivatives elicit vastly differing, partially mating type-specific responses during early sexual development. Another specificity factor is the realization of different regulation levels for the trisporoid synthesis enzymes in different species. Enzymes in the trisporoid synthesis pathway show remarkable variations in mating type-specific activity and the exact activation time during sexual development. This allows for the observed complex network of possible interactions, but at the same time forbids successful mating between dissimilar partners because the necessary transcripts or gene products are not available at the appropriate developmental stage.