Sexual Crosses with the Mucoromycete Phycomyces blakesleeanus

Our lab is interested in exploring the evolution of developmental programs in filamentous fungi. We're curious about what genes participate in these serial morphological transitions that occurred during sexual reproduction.

Since fungal mycelia expands radially, different parts of the mycelium make contact at different times and experience asynchronous sexual reproduction. This can complicate gene expression studies that seek to tie gene expression with a particular sexual cell type. Our protocol allows us to limit the number of interacting cells and potentially provide clear gene expression signal.

Our protocol will facilitate the extraction of RNA from specific cells during sexual reproduction, allowing us to tie gene function to a specific morphological change.

[Narrator] To begin, obtain cultures of each Phycomyces blakesleeanus mating type. Using a scalpel, cut a portion of leading edge mycelia from the existing cultures and place the excised mycelium onto a fresh 100% cornmeal agar or potato dextrose agar plate. Incubate the pure cultures for one week at 27 degrees Celsius under a 12-hour light cycle. Once sporangiophores are present, flood a sporulating pure culture plate with 0.01% Tween 20 in sterilized deionized water using aseptic technique. Then using a P1000 micropipette, draw one milliliter of the Tween 20 spore mixture from the plate into a micro centrifuge tube. Centrifuge the micro centrifuge tube in a mini centrifuge for 30 seconds, and carefully decant the supernatant without disturbing the pellet. If more spores are needed, add another one milliliter of the Tween 20 spore mixture into the same micro centrifuge tube and repeat centrifugation. After obtaining a suitable amount of spores, decant the supernatant and replace it with 500 microliters of sterile deionized water. Use a hemocytometer to estimate the concentration of spores. Set up the crosses as two-way, four-way, or eight way crosses as appropriate. Alternatively, use a scalpel to inoculate the crosses directly with leading edge mycelia from the pure cultures. Then, using a sterilized razor blade or cork hole bore, excise a portion of the tissue and plate the excised inoculum immediately onto a fresh medium. For a four-way cross, place like mating types opposite each other while neighboring a complimentary mating type. Now place the spores or mycelia of the minus mating types opposite each other at least one centimeter away from the edge of a Petri dish containing potato dextrose agar or cornmeal agar. After inoculating, seal the plates with Parafilm. Place the plates into a secondary container. Incubate the plates at 22 degrees Celsius in the dark. Observe the plates daily for evidence of mating and take photographs from underneath the plate during culture growth. Trace the growing mycelia over time. Depending on the media type and formulation, if the mycelia begin to make contact, inspect the interacting complimentary mating types under a dissecting scope for evidence of mating. Take photographs of the interacting mycelia with a camera or smartphone mounted onto the dissecting scope. At one day post inoculation, strains grown on potato dextrose agar exhibited more yellow pigmentation compared to strains grown on cornmeal agar. At two days post inoculation, mating structures were observed on 100% potato dextrose agar, but not on cornmeal agar plates. At three days post inoculation, all formulations of potato dextrose agar supported mating with differing densities of differentiated cell types. By four days post inoculation, strains on potato dextrose agar plates exhibited both mating reactions and formation of asexual sporangiophores. Strains on cornmeal agar exhibited more diffuse mycelia and fewer sporangiophores compared to potato dextrose agar. On 100% potato dextrose agar, at four days post inoculation, aerial structures appeared as a mass of undifferentiated cells. On 25% potato dextrose agar, cells at the mating site appeared easily distinguishable and predominantly at similar developmental stages. On 50% potato dextrose agar, a mix of aerial zygospores and developing progametangia was observed.