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 th....

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....

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