Molecular aspects of sex and trisporoid signalling in mucoralean fungi
A haploid asexual phase predominates life cycle in Mucorales, but adverse extrinsic factors along with darkness promote “sexual communication”. Little is known about the physiological functions of the enzymes and products involved in the sexual phase. We hypothesise that each of the intermediate trisporoid has a specific regulatory function upon the genes linked to sex pheromone production. Real-time quantitative PCR kinetics conducted in Blakeslea trispora (Choanephoraceae), displayed a high transcriptional upregulation during sexual phase for carotenoid cleavage dioxygenase gene tsp3. In Mucor circinelloides f. lusitanicus (Mucoraceae), two putative carotenoid cleavage oxygenases (CarS and AcaA) were identified based on phylogeny and amino acid sequence similarity. Unlike Blakeslea, during sex, Mucor had no significant transcriptional up regulation for carotenoid cleavage oxygenases. CrgA, a repressor of carotenogenesis, has no regulatory function on carotenoid cleavage as transcript levels of carS or acaA were on par among wild type Mucor (-) and its crgA mutant. Heterologous co-expression of TSP3 with β-carotene overproducing plasmid through in vivo enzyme assay and LC-MS analysis led to the identification of β-apo-12’- carotenal, as first apocarotenoid in Blakeslea. Trisporin C stimulated gene transcription in Mucor (-) and had no impact upon Blakeslea. Supplementation of the first C18 trisporoid, β-apo-13-carotenone (D’orenone), entailed a positive feedback regulation on carRP/carRA and acaA/tsp3 gene expression in Mucor (-) and Blakeslea (+). Our data supports the hypothesis that mode of action of trisporoids varies among genotypes and growth phases which in turn lead to the concept of “specific chemical dialect”. We successfully detected sex hormone and its highly abundant precursor β-carotene in the gametangial phase in M. mucedo (Mucoraceae) by a non-invasive, novel approach of Coherent anti- Stokes Raman Spectroscopy (CARS).