Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Summary

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Abstract

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Introduction

The smut fungi (Ustilaginales) consist of over 1,600 species that infect grasses including the important cereal crops of corn, barley, and wheat, causing billions of dollars in crop losses annually¹. These fungi are characterized by the production of teliospores, which have darkly pigmented cell walls and are the dispersal agents. Teliospores function to shield genetic material during the stresses of dispersal between host plants, and can persist in a dormant state for years². As such, teliospores are an essential component of disease spread.

In order to study teliospore biology, our ....

Protocol

1. Corn Cob Infection

1. Grow Zea mays (cv. Golden Bantam) until cobs are formed and have started to silk (approximately 60 days).
2. Culture compatible haploid U. maydis strains using standard protocols as previously described⁷.
3. Infect corn cobs using standard protocols as previously described⁷.

2. Teliospore Harvesting

1. Autoclave equipment (Büchner funnels, Büchner flasks, blenders, 250 mL centrifuge bottles, flat spatulas, and water) using a standard dry cycle with at least 30 min sterilization at 121 °C (standard ....

Results

Using the Clark-type microrespirometer-based method of measuring changes in respiration during teliospore dormancy and germination, we confirmed that dormant teliospores exhibit a low level of respiration (~1,075 µmol/h/mg) compared to germinating teliospores (~2,614 µmol/h/mg; Figure 1A). This represents a ~2.4-fold change in average rate of respiration between dormant teliospores and teliospores that have been induced to germinate. In addition, we have iden.......

Discussion

Basidiomycete biotrophic plant pathogens cause billions of dollars in crop losses annually. The vast majority of these pathogens produce teliospores that are integral to fungal dispersal and sexual reproduction. Gaining knowledge of the development and germination of teliospores is critical to understanding the spread of the devastating diseases caused by these fungi. In order to identify molecular changes at key control points we have devised a method to identify the timing of physiological shifts and another to isolate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Streptomycin Sulfate	BioShop	STP101
Kanamycin Sulfate	BioShop	KAN201
Potato Dextrose Broth	BD Difco	254920
1 L Waring Laboratory blender	Waring	7011S
Cheesecloth	VWR	470150-438
Nalgene Polypropylene Desiccator with Stopcock	ThermoFisher Scientific	5310-0250
Unisense MicroRespiration system
MicroRespiration Sensor (O2)	Unisense	OX10
MicroOptode Meter Amplifier	Unisense	N/A
MR-Ch Small	Unisense	MR-Ch
SensorTrace Rate Software	Unisense	N/A
MicroRespiration Rack	Unisense	MR2-Rack
MicroRespiration Stirrer	Unisense	MR2-Co
Microdissection system
Axio Vert.A1 Inverted Light Microscope	Zeiss
Coarse Manipulator	Narishige	MMN-1
Three-axis Hanging Joystick Oil Hydraulic Micromanipulator	Narishige	MMO-202ND
Pneumatic Microinjector	Narishige	IM-11-2
TransferTip (ES)	Eppendorf	5175107004