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.
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.
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 laboratory utilizes the model smut fungus Ustilago maydis (U. maydis), which is the causal agent of the disease 'common smut of corn'. Mature U. maydis teliospores are characterized by growth arrest, reduced cellular metabolism, and low levels of cellular respiration3. In favorable environmental conditions (e.g., the presence of specific sugars), U. maydis teliospores germinate and complete meiosis, producing basidiospores which can initiate new rounds of infection. Germination is characterized by increased respiration, the return to metabolic activity, and the progression through observable morphological stages of germination4.
The initial stage of germination includes increased respiration and metabolic function, however, there are no morphological indications of change. The original measurements of respiratory change in U. maydis were carried out over 50 years ago, measuring oxygen consumption manometrically with a Warburg flask apparatus5. We have developed a new, simple method of studying precise changes in respiration during teliospore germination by measuring oxygen consumption over a time course of germination using a Clark-type microrespirometer. We previously used this method to study changes in respiratory rate between wild-type U. maydis haploid cells and mutants with defective mitochondria6, and have adapted the protocol here to study changes in teliospore respiration during germination. This provides a means of accurately identifying the timing of respiration change so that we can target teliospores at the appropriate time after the initiation of germination to investigate early molecular events. The progression of germination can be followed microscopically once the promycelia emerges from the teliospore, but the asynchronous nature inhibited the isolation of enough teliospores at a given stage for investigation. We developed a microdissection technique similar to those used for in vitro fertilization to physically collect teliospores at distinct morphological stages of germination.
1. Corn Cob Infection
2. Teliospore Harvesting
3. Teliospore Viability and Germination Test
4. Induction of Germination for Respiration Monitoring
5. Obtaining Oxygen Consumption Rate (OCR) Measurements
6. Data Analysis
7. Induction of Teliospore Germination to Isolate Teliospores at Distinct Stages of Germination
8. Preparation of Petri Dish and Micromanipulator
9. Isolation of Stage-specific Germinating Teliospores
10. Recovery of Collection Droplet
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 identified that teliospores that have been induced to germinate have a ~45 min delay in oxygen uptake (Figure 1B). This is indicated by the ~30 min delay in oxygen uptake (Figure 1B) in addition to the ~15 min delay between the induction of germination and start of oxygen measurements. This identifies a time point to begin assessing molecular changes in the germinating teliospores that are not visibly changing.
Subsequent changes during germination can be observed microscopically. Five stages of germination were determined. Stage I of germination represents teliospores that have been induced to germinate but remain indistinguishable from dormant teliospores. Stage II teliospores have an emerging promycelium with a length that is less than or equal to the diameter of the teliospore. Stage III teliospores have promycelia that are greater than the teliospore diameter. Stage IV of germination is the initial budding of basidiospores from the promycelia, and Stage V are the resulting haploid basidiospores that divide by budding (Figure 2). Using the microdissection technique that we have developed, we have successfully isolated 500 to 1,000 germinating teliospores for downstream applications such as RNA isolation for RT-PCR or RNA-Seq (Table 1). Figure 3 shows the general set up of the Petri dish for microdissection and the steps for microdissection using a micromanipulator.
Figure 1: Time course of oxygen consumption during teliospore germination. Dormant teliospores were induced to germinate, and oxygen levels were recorded continually for 6 h using a Clark-type microrespirometer. Un-induced dormant teliospores were used as a control, and all measurements were normalized to a blank sample. (A) Data represented as average OCR. (B) Raw data plotted to obtain respiration curves, permitting the detection of changes in OCR during the time course. Teliospores that have been induced to germinate consume oxygen at an average rate 2.4-fold faster than un-induced dormant teliospores (p <0.01; Student's t-test). PIG: post-induction of germination. Please click here to view a larger version of this figure.
Figure 2: Stages of teliospore germination. Stages I through V of teliospore germination are illustrated (A–E). Scale bar = 20 µm. Please click here to view a larger version of this figure.
Figure 3: Microdissection to isolate distinct morphological stages of germinating teliospores. The general set up of a Petri dish for microdissection and the steps for isolating teliospore at stage III of germination are illustrated. (A) Illustration of a Petri dish set up with rows of droplets containing either sterile water, RNA stabilization solution, or germinating teliospores. Following germination induction, teliospores were isolated at specific stages of germination using microdissection. (B) A germination droplet containing induced to germinate teliospores in Stages I through III. (C) A prepared microcapillary was brought up to a Stage III teliospore for collection through aspiration. (D) The Stage III teliospore in a glass microcapillary is removed from the germination droplet and moved to a collection droplet. (E) The microcapillary was inserted into the collection droplet containing RNA stabilization solution and the Stage III teliospore was injected into the droplet. (F) A collection of Stage III teliospores in the RNA stabilization solution. Scale bar = 20 µm (A-D, F) and 50 µm (E). Please click here to view a larger version of this figure.
Germination Stage | Number of germinating teliospores isolated |
Stage I | 1,000 |
Stage II | 500 |
Stage III | 650 |
Table 1: Number of germinating teliospores successfully isolated for each germination stage in a standard isolation experiment. The table illustrates average numbers of teliospores that have been isolated using microdissection for Stages I through III before collection for downstream applications.
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 teliospores at distinct stages of germination. Seto et al. (unpublished) noted five stages of teliospore germination by light microscopy (Figure 2). In order to investigate physiological activation during Stage I and to assess respiration rate during germination, we used a Clark-type microrespirometer to precisely measure changes in oxygen consumption. Our sample data indicate that our method is precise and highly reproducible. Our findings confirm that germinating U. maydis teliospores exhibit a drastic increase in cellular respiration compared to un-induced dormant teliospores. For the first time, we have identified that U. maydis teliospores that have been induced to germinate exhibit a ~45 min delay in oxygen uptake. This suggests that U. maydis teliospores may require some time to process germination signals (e.g., the presence of sugars) before responding, that increased oxygen uptake is not among the very immediate responses to germination signals or that our assay was not sensitive enough to detect the minimal change in initial oxygen uptake.
Previous studies examining respiration rates of smut teliospores3 relied on a Warburg flask apparatus to measure oxygen levels manometrically5. Briefly, this method measures oxygen consumption and CO2 production by detecting changes in pressure in an enclosed flask through the direct observation of fluid level changes in the manometer arm. The experiments can be difficult to set up, and measurements can be imprecise. The apparatus must be attended throughout the period of measurement and, extensive calculations are required to estimate OCR. Our protocol makes use of technological advances, eliminating the requirement for the user to remain by the apparatus for the duration of the experiment, take measurements by eye, and use extensive mathematical formulas. Others have used early Clark-type respirometers to measure OCR of Neurospora crassa9 and Botryodpilodia theobromae10 spores, however, these early instruments permitted continuous measurements for a maximum of 20 min. This limitation would not have allowed the identification of the ~45 min delay in oxygen uptake we observed with the newer model respirometer. Our protocol has made data interpretation simpler, as the readout is the concentration of oxygen remaining in the chamber, which can be directly graphed without any calculations or data manipulation. In addition, it is possible to take continuous measurements (every 2 s) for an indefinite amount of time until available oxygen is completely depleted. This permits the identification of small changes in respiration over a long period of time. Therefore, we have improved upon earlier techniques and developed a simple, precise, and reproducible method to measure oxygen consumption of fungal spores. To our knowledge, this is the first study to use a modern Clark-type respirometer to study respiration of dormant versus germinating teliospores of smut fungi.
Despite the ease and simplicity of this protocol, optimization is required and there are biological realities that limited the analysis. First, appropriate sample sizes must be identified to achieve reasonable OCRs. Too much sample can lead to premature crashing of oxygen levels, and too little sample can result in the inability to observe meaningful changes in oxygen consumption. Second, it is imperative to allow the probe time to stabilize (~3 min) to provide accurate initial data. Lastly, it is important to supplement germination medium with antibacterial agents (e.g., streptomycin sulfate) in order to ensure bacterial contamination does not alter OCR readings. The biological limitations we faced were a low germination rate over the time course of measurement, (~1%) as determined by observing visual morphological changes. Determining spore viability would allow this rate determination to be converted to a rate per spore number and isolating teliospores with higher rates of germination would lead to higher OCRs. The asynchronous germination of U. maydis teliospores11 is a reality that must be accounted for and may have contributed to an inability to detect oxygen consumption earlier in germination.
In order to improve the accuracy and precision of measuring changes in teliospore respiration, future adaptations to this method could include measuring OCR on a single cell-basis. Micromanipulation techniques could be used to isolate a single teliospore, which can then be induced to germinate, and its respiration rate can be monitored. This could improve resolution, providing information regarding the OCR during the dormancy-germination shift per teliospore, rather than per mg of teliospores. In addition, this would solve the confounding issue of asynchronous germination.
For later stages of germination, we developed a micromanipulation method to isolate teliospores at common stages of germination. This allowed the creation of relatively synchronous teliospore populations for analysis. Various methods for isolating single microorganisms have been described and have been improved upon over the years12. These methods include the dilution of spore suspensions to obtain single microorganisms, semi-mechanical methods with the use of microcapillaries to obtain spores that are transferred to medium for culturing, and mechanical methods which use micromanipulators. Previous methods that we used to obtain teliospores at the same stage of germination include counterflow centrifugal elutriation and filtering germinating teliospores through a nylon membrane with a specific pore size. Using these methods allowed us to enrich for germinating teliospores, however, our samples still contained teliospores in various stages of germination13. Current technology for micromanipulation of single microorganisms has improved with the introduction of higher magnification and instruments for fine control of capillary needles, aspiration, and transfer of microorganisms. Previous micromanipulation techniques have focused on isolating single cells for culturing or for use in single cell PCR applications14. The use of micromanipulators to isolate single fungal spores has not previously been established. A previous method for isolating single fungal spores involved the use of fine forceps or needles to pick small pieces of solid medium containing germinating spores15. Micromanipulation with the use of micromanipulators is widely used in yeast studies where clusters of ascospores can be separated following sporulation in culture on agar medium for meiotic genetic analysis16. We have developed a method which combines the micromanipulation technique for bacterial cells14 and in vitro fertilization methods for isolating germinating teliospores. We have shown that hundreds of common germination stage teliospores can be obtained with this technique. These samples can be used for downstream expression studies using techniques such as RT-qPCR or RNA-seq. Obtaining a population of teliospores in which germination is synchronized permits the analysis of specific changes in gene expression that occurs during early, mid and later stages of teliospore germination.
Microdissection of stage specific germinating teliospores may require experience in set up and recognizing the different stages of germination, however, this experience can be obtained quickly through practice. There are several steps that must be followed for successful microdissection followed by RNA isolation. First, germination medium must be supplemented with antibiotics (e.g., streptomycin sulfate) to suppress the growth of bacterial contamination when germination is initiated as well as during collection of germinating teliospores. Second, it is important to use a stabilization solution to stabilize and protect RNA for isolation. The RNA stabilization solution also prevents collected teliospores from progressing to the next germination stage while collecting additional teliospores. Thirdly, it is important to remove the mineral oil once the collection droplet has been recovered to ensure successful RNA extraction. Lastly, we have noticed some loss of RNA quality if isolated teliospores are stored in RNA stabilization solution for an extended period of time; therefore, it is recommended that RNA is isolated immediately following collection of germination stage specific teliospores. A limitation of the method is that the Stage I teliospores collected could contain dormant, dead, and induced to germinate teliospores as these three stages are morphologically indistinguishable. In addition, when collecting Stage III teliospores, a mixture of teliospores in meiosis I or meiosis II could be obtained. One way to aid in distinguishing between truly dormant and dead teliospores could be to determine the viability of the sample. A method for assessing fungal spore viability using live/dead cell viability assays may be able to assess percentage of viable teliospores from which more informative germination rates could be determined17. In addition, nucleus staining with DAPI, for example, could be used to visualize the events of meiosis that are occurring during Stage III and the transition to Stage IV in order to further characterize teliospores morphologically at Stage III. This would aid in the collection of teliospores in only one stage of germination when using our microdissection method.
In conclusion, we have developed a simple, precise and reproducible method of measuring the changes in cellular respiration that occur during the dormancy-germination shift of Ustilago maydis teliospores. In addition, we have developed a method for collecting specific stages of germinating teliospores that could be used for downstream applications, such as RNA-seq. Our methods can be adapted to accommodate various cell-types and species. We anticipate that improvements to our techniques will facilitate the detection of respiratory changes on a single spore level as well as further defining the events that are occurring in the later stages of germination.
The authors have nothing to disclose.
We would like to thank Dr. Paul Frost for use of his microrespirometer, and Nicole Wagner and Alex Bell for technical assistance. This work was funded by an NSERC grant to B.J.S.
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 |