Sexual Crosses with the Mucoromycete Phycomyces blakesleeanus

Summary

Here we present a basic protocol for the induction of P. blakesleeanus mating.

Abstract

Phycomyces blakesleeanus, a filamentous fungus within the Mucoromycota phylum, is distinguished by its remarkable capacity for environmental perception and adaptive responses. While past work has shown that environmental stimuli, including gravity, light, moisture, and nutrient availability, influence its growth dynamics and reproductive strategies, the underlying mechanisms remain a focal area of research. Environmental cues trigger sexual or asexual reproduction. Sexual reproduction begins with pheromone signaling, which triggers hyphal chemoattraction, eventually leading to serial morphological transitions culminating in the formation of a zygospore.

In a laboratory setting, crosses of P. blakesleeanus result in complementary mycelia undergoing the sexual cycle at different stages. Our work aims to test if environmental cues can trigger mating across the mycelia of P. blakesleeanus. Crosses of P. blakesleeanus grown on nutrient-limited media will be subjected to nutrient-limited agar to trigger a sexual response in accordance with nutrient deprivation. Successful triggering of mating in P. blakesleeanus will facilitate future studies that require a large amount of sexually reproducing mycelia in specific stages. The outcome of this research will further enhance our understanding of how P. blakesleeanus reproductive mechanisms are influenced by environmental factors, contributing to the broader knowledge base on the sexual reproduction of filamentous fungi.

Introduction

The induction of mating in fungal systems in laboratory conditions has advanced the understanding of eukaryotic genetics, cell biology, evolutionary biology, and biotechnology. The dikaryan fungi, in particular the Ascomycota, feature the most extensive knowledge about mating induction in a laboratory setting¹. Interestingly, the first fungus in which sexual reproduction was proposed, Syzygites megalocarpus, is not a member of Ascomycota, but rather a member of the phylum Mucoromycota². The phylum Mucoromycota is an early-diverging group formerly classified as "Zygomycota" but now considered sister to the Dikaryan lineages^3,4. The Mucoromycota, along with Zoopagomycota, are evolutionarily significant as these phyla represent the transition of fungi to terrestrial ecosystems^4,5. Like other fungi, the Mucoromycetes use asexual and sexual reproductive strategies in response to their environment^3,6. Under nutrient-limited conditions, mucoromycetes will begin the sexual cycle⁶. Mucoromycetes exhibit both homothallic and heterothallic mating mechanisms, where compatibility is determined by the mating-type genes sexM and sexP, designated as (-) and (+) respectively^7,8,9.

Blakeslee¹⁰ emphasized the sensitivity of the mucoromycete sexual cycle to external conditions, noting that moisture is critical for their formation and that nutrient availability in the substrate plays a significant role. Complementary mating types begin the sexual cycle through cooperative synthesis of trisporic acids (TA) using beta-carotene as a precursor^11,12,13. Following the detection of TA, the responding vegetative hyphae thicken and become highly branched zygophores^14,15,16. The zygophores continue production of TA and mutually chemoattract. In P. blakesleeanus, zygophores differentiate within the substrate and are not readily visible on solid agar media¹⁷. Upon making contact, the zygophores intertwine and become aerial zygophores.

As the sexual cycle continues, the tips of the zygophores attach, and the middle of the cells push out to form a ring-like structure with coralloid swelling at the base, completing the transition to the progametangium. The tips of the cells forming the progametangium begin to fuse and develop into the gametangium. At the gametangia stage, the zygophores display thorn-like ornamentation. The cell wall at the tips dissolves and adventitious septa appear, delimiting the area where the zygospore will form, and the zygophores act as tong-like suspensors¹⁸. The zygospore will become pigmented as its cell wall thickens and it acquires additional thorn-like ornamentation^17,18. Once formed, the zygospore will enter a period of dormancy before restarting the growth cycle.

Phycomyces blakesleeanus is a heterothallic mucoromycete notable for its large cells and environmentally responsive sporangiophores^6,8,18. This organism is easily cultivated in the lab and the portions of the sexual cycle leading to the formation of zygospores can be observed in the span of 8-10 days. As a model, P. blakesleeanus has been examined for its capability to sense light in its environment^17,19. The ease of cultivation and the capacity to induce mating also made it an ideal model to study the mechanism behind its ability to perceive light²⁰; these findings also highlighted the evolutionary conservation of light-sensing mechanisms in fungi. In P. blakesleeanus, light has been shown to inhibit sexual reproduction via these conserved light-sensing proteins²¹. Recent evolutionary developmental studies have sought to understand what genes are contributing to cell differentiation during the P. blakesleeanus sexual cycle⁶. Correlating morphogenesis to specific genes would require that sufficient tissue of the same cell types be isolated to perform gene expression studies.

While protocols for inducing mating of P. blakesleeanus in a laboratory setting have been previously described, some only mention the type of medium to use and the relevant strains¹⁸. Some protocol descriptions do not include specific media formulation but do describe where to position presumptive complementary mating types on a plate¹⁰. More recent protocols allow for increased production of zygospores by either mixing spores of each mating-type and inoculating with the mixed spore suspension²¹ or by placing mating types some distance apart and letting the culture incubate for 20 days²². These approaches are useful for generating ample differentiated cells, specifically zygospores, but may not be suitable for observing a developmental time course or selecting sexual structures that form prior to zygospores for single-cell transcriptomics. Other work has addressed this by placing complementary mating types distance apart on solid media to allow the observation of serial morphological transitions as the sexual cycle begins^6,22,23. As is the case with other fungi, P. blakesleeanus mycelia expands radially^24,25. Therefore, when complementary mating types are grown on the same plate, different portions of their mycelia will come in contact at different times. Since contact between mycelia is among one of the first steps in P. blakesleeanus sexual cycle, this means that different portions of interacting mycelia will be at different stages in the sexual cycle. This asynchrony could impact the outcome of a gene expression study, such that if differentiated cell types are mixed and if they do have distinct gene expression programs, then it would be difficult to ascribe the role of a gene to one particular structure.

In addition to the value of P. blakesleeanus as a model for examining genes involved in morphogenesis during the sexual cycle, its vigorous growth and capacity to differentiate in the span of a few days make it an ideal fungus for training students interested in early-diverging filamentous fungi and for use in an undergraduate classroom setting to learn about the diversity of fungi and their developmental processes. The protocol presented here makes use of three concentrations of two different types of media to demonstrate the effect of nutrient availability on mycelium appearance, the induction of mating, and enrichment for particular sexual structures, either for quantifying, observation, or potential single-cell transcriptomics.

Protocol

1. Media preparation

1. Suspend powdered cornmeal agar (CMA) or potato dextrose agar (PDA) in deionized water. For 100% CMA or PDA, follow the manufacturer's instructions and suspend 17 g of CMA in 1 L of deionized water or 39 g of PDA in 1 L of deionized water. For N% CMA or PDA, suspend 1/N of the manufacturer-recommended amount and supplement with additional agar to achieve an agar concentration of 7.5% (w/v).
   NOTE: Optional step: Prior to autoclaving, add 25 µg/mL of chloramphenicol if bacterial contamination is a concern.
2. Sterilize media in an autoclave, at 121 °C for 15 min.
3. Cool the media to ~60 °C by placing it for 30 min in a water bath set to 60 °C.
   NOTE: If the warm bottle of media can be held for 6 s with little to no discomfort, then it is ready to pour.
4. Pour plates under aseptic conditions in a laminar flow hood or biological safety cabinet. Tilt the bottle of media over an open, empty Petri dish and pour only enough to completely cover the bottom.
   1. Alternatively, use a glass serological pipette and pipette 20 mL of media into each plate.
      NOTE: This approach increases the time that the media bottle is at a lower temperature and raises the risk of prematurely solidifying the agar.
5. Allow the media to resolidify. Store the plates at 4 °C if not used immediately.

2. Preparing fungal spores or tissue

1. Obtain cultures of each P. blakesleeanus mating type (e.g., NRRL 1555 (-), NRRL 1554 (+), NRRL 1464 (-), and NRRL 1465 (+)). To inoculate crosses with spores, first grow each mating type in pure culture on 100% CMA or PDA
2. Cut a portion of leading-edge mycelia from existing cultures and place the excised mycelium on a 100% CMA/PDA plate. Incubate pure cultures for 1 week at 27 °C under a 12 h light cycle.
3. Once sporangiophores are present, flood a sporulating pure culture with 0.01% Tween 20 in sterilized, deionized water using aseptic technique. With a P1000 micropipette, draw 1.0 mL of the Tween 20-spore mixture from the plate and into a microcentrifuge tube.
4. Centrifuge in a mini centrifuge for 30 s, then decant the supernatant.
   1. If more spores are needed, continue to add 1.0 mL of the Tween 20 spore mixture into the same microcentrifuge tube and repeat step 2.4. After obtaining a suitable amount of spores, decant the supernatant and replace it with 500 µL of sterile deionized water.
   2. Optional: Use a hemacytometer to estimate the concentration of spores, which will inform whether more water needs to be added or if the spores need to be re-centrifuged.
5. Alternatively, inoculate the crosses with leading-edge mycelia from the pure cultures.
   1. Use a sterilized razor blade or cork hole borer to excise the tissue and plate the inoculum immediately.
6. Set up the crosses as 2-way crosses, 4-way crosses, or 8-way crosses (Figure 1).
   NOTE: If crosses are being set up for transcriptomic work, it is recommended to set up 1-2 additional plates per condition/media to serve as indicator plates. Indicator plates allow investigators to limit light exposure to experimental plates, as light inhibits sexual reproduction in P. blakesleeanus.
   1. For a 2-way cross, place complementary mating types opposite each other (Figure 1A).
      1. Place the mycelia of the (-) mating-type, NRRL 1555 or NRRL 1464, at least 1 cm away from the edge of a Petri dish on PDA or CMA.
      2. On the same plate, opposite of where the (-) mating-type was placed and 1 cm away from the edge of the Petri dish, place the (+) mating-type, NRRL 1554 or NRRL1465 (Figure 1A).
         NOTE: The distance between complementary mating types will influence the size of the tissue participating in the sexual cycle. Mating types further apart allow the mycelia to expand more, which will increase the number of sexual interactions between the two partners.
   2. For a 4-way cross, place like mating types opposite each other but neighboring a complementary mating type (Figure 1B).
      1. Place the spores or mycelia of the (-) mating types, NRRL 1555 and NRRL 1464, opposite each other at least 1 cm away from the edge of a Petri dish on PDA or CMA.
      2. On the same plate, choose a location between the two (-) mating types and at least 1 cm away from the edge of the plate and inoculate the site with spores or mycelia of the (+) mating type, NRRL 1554 or 1465.
      3. On the same plate, opposite where the first (+) mating type was inoculated and at least 1 cm away from the plate, place the other (+) mating type.
   3. For an 8-way cross, alternate mating types along the periphery of the Petri dish, featuring four of each mating type (Figure 1C).
7. After inoculating, seal the plates with parafilm or (leave them unsealed) and place them in a secondary container prior to incubating at 22 °C in the dark. Observe the plates daily for evidence of mating during which time, take photographs from underneath the plate and trace the mycelia as the cultures grow.
   NOTE: After 24 h post inoculation, expect to see mycelia expanding on all media types but individuals are not likely to be in contact at this stage.
8. Depending on the media type (PDA or CMA) and the formulation (25%, 50%, 100%), if the mycelia begin to make contact, inspect mycelia of complementary mating types that are in contact under a dissecting scope for evidence of mating. Take photos of interacting mycelia with a camera or a smartphone mounted to the dissecting scope.
   1. Acquire images of mycelia and stitch them together (e.g., using Panorama Stitcher application). Use ImageJ to estimate the area of the mycelia (see online tutorials).

Results

Following a 4-way cross, each strain varied slightly in its growth rate as determined by the change in area of the mycelium (Figure 2). While not statistically significant, NRRL 1555 had a faster change in area when plated on 25% CMA, 25% PDA, or 100% PDA. Similarly, NRRL 1465 had a higher change in area when grown on 50% PDA or CMA. NRRL 1464 had the quickest increase in area when grown on 100% CMA. After 24 h (1 DPI), strains growing on PDA appeared more yellow relative to the strains grow...

Discussion

A simple protocol for inducing sexual reproduction in P. blakesleeanus in a laboratory setting is presented here. One of the most critical considerations for this protocol is nutrient limitation. It is hypothesized that fungi co-opted sexual reproduction as a response to harsh environmental conditions such as nutrient limitation^{6,28,29,30,31,}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar powder	ThermoFisher	3453PK
chloramphenicol	FisherSci	BP904-100
Cornmeal agar	Carolina	742460
Panorama Stitcher	Apple App Store
Phycomyces blakesleeanus (-) NRRL 1555	United States Department of Agriculture - Agricultural Research Service	1555	USDA-ARS will only ship to academic researchers; see below for alternatives
Phycomyces blakesleeanus (-) NRRL 1564	United States Department of Agriculture - Agricultural Research Service	1564	USDA-ARS will only ship to academic researchers; see below for alternatives
Phycomyces blakesleeanus (-) Tube Culture	Carolina Biological Supply	156183	Education-grade culture available for purchase; NRRL 1555
Phycomyces blakesleeanus (+) NRRL 1554	United States Department of Agriculture - Agricultural Research Service	1554	USDA-ARS will only ship to academic researchers; see below for alternatives
Phycomyces blakesleeanus (+) NRRL 1565	United States Department of Agriculture - Agricultural Research Service	1565	USDA-ARS will only ship to academic researchers; see below for alternatives
Phycomyces blakesleeanus (+) Tube Culture	Carolina	156182	Education-grade culture available for purchase; NRRL 1554
Potato dextrose agar	FisherSci	DF0013-17-6
Sprout Plus Mini Centrifuge	Heathrow Scientific	SKU 120610
Tween 20	Sigma-Aldrich	P6585-10ML