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.

1. Corn Cob Infection
<ol>
	<li>Grow Zea mays (cv. Golden Bantam) until cobs are formed and have started to silk (approximately 60 days).</li>
	<li>Culture compatible haploid U. maydis strains using standard protocols as previously described7.</li>
	<li>Infect corn cobs using standard protocols as previously described7.</li>
</ol>
2. Teliospore Harvesting
<ol>
	<li>Autoclave equipment (B&#252;chner funnels, B&#252;chner flasks, blenders, 250 mL centrifuge bottles, flat spatulas, and water) using a standard dry cycle with at least 30 min sterilization at 121 &#176;C (standard liquid cycle for water).</li>
	<li>Remove infected cobs from plants (approximately 28&#8211;35 days post infection) using a razor and set the cobs on a tray covered in bench protector.</li>
	<li>Remove the tumours from cobs with a razor blade and collect in a beaker.</li>
	<li>Fill a 250 mL laboratory blender cup with tumours until approximately 1/3 full and add autoclaved dH2O until the blender cup is approximately 3/4 full. Disrupt the tumours by pulsing the blender at low, until homogenized.</li>
	<li>Connect the vacuum pump to a water trap and then to a 1 L B&#252;chner flask.</li>
	<li>Insert large B&#252;chner funnel into the flask and line the bottom of the B&#252;chner Funnel with four layers of cheesecloth.</li>
	<li>Turn on the vacuum pump.</li>
	<li>Pour a portion of the homogenized tumours through the cheesecloth and scrape with a spatula.</li>
	<li>Pour some dH2O into the cheesecloth in order to flush the teliospores through.</li>
	<li>Repeat until the dH2O coming through the cheesecloth is clear.</li>
	<li>Wring out the cheesecloth containing the homogenized tumour material into the filter to ensure maximum teliospore recovery.</li>
	<li>When the 1 L B&#252;chner flask is getting close to full, empty it into a large Erlenmeyer flask and set it aside.</li>
	<li>Put a new piece of cheesecloth into the filter and repeat the steps (2.7&#8211;2.12) until all of the tumours have been disrupted and filtered.</li>
	<li>Pour the filtered teliospores into autoclaved 250 mL centrifuge bottles and centrifuge at 1,000 x g for 5 min, and decant the supernatant.</li>
	<li>Repeat step 2.14 until all of the filtered teliospores are pelleted by centrifugation.</li>
	<li>Suspend the pellets in a small amount of water and transfer to 50 mL centrifuge tubes.</li>
	<li>Centrifuge the tubes at 1,000 x g for 5 min, and decant the supernatant.</li>
	<li>Suspend the pellet in approximately 50 mL of dH2O, centrifuge the tubes at 1,000 x g for 5 min, and decant the supernatant. Gently scrape off the gray top layer with a spatula and dispose of it. Repeat until there is no longer a gray layer on top.</li>
	<li>Dry the samples overnight in a vacuum desiccator.</li>
	<li>Store dried teliospores at 4 &#176;C until use.</li>
	<li>If desired, treat the teliospores with copper sulphate8 before inducing to germinate. If not treated, then perform thorough microscopic analysis of the teliospores to confirm the sample represents pure teliospores and is devoid of bacterial or other contaminations.
	<ol>
		<li>Weigh out approximately 50 mg of teliospores in a 1.5 mL microcentrifuge tube.</li>
		<li>Add approximately 1.0 mL of 0.75% CuSO4 to the 1.5 mL microcentrifuge tube containing the teliospores. Pipette up and down to suspend the teliospores in the CuSO4 solution followed by agitating the sample for 3 h.</li>
		<li>Centrifuge the sample at 2,500 x g for 5 min and remove the supernatant. Resuspend the teliospore pellet with sterile water, repeat the centrifugation, and remove the supernatant.</li>
		<li>Repeat step 2.21.3 two more times.</li>
	</ol>
	</li>
</ol>
3. Teliospore Viability and Germination Test
<ol>
	<li>Weigh out approximately 10 mg of U. maydis teliospores in a 1.5 mL microcentrifuge tube to assess their viability and the timing of germination.</li>
	<li>In a biosafety cabinet, prepare potato dextrose broth (PDB, 24 g/L) supplemented with streptomycin sulfate (160 &#181;g/mL).</li>
	<li>Suspend the teliospores in 500 &#181;L of PDB. Gently pipette to mix and break up all clumps of teliospores.</li>
	<li>Transfer the teliospore suspension to an autoclaved 250 mL Erlenmeyer flask containing 10 mL of the PDB.</li>
	<li>Incubate the flask at 28 &#176;C shaking at 90 rpm for 12&#8211;16 h.</li>
	<li>In a biosafety cabinet, remove a 20 &#181;L sample of the teliospores induced to germinate and prepare a microscope slide.</li>
	<li>Using a microscope, visually assess stages of germination that are present and the presence of bacterial contamination.
	<ol>
		<li>Count the number of teliospores at stages I through V using a hemocytometer and determine the percent that have germinated.</li>
		<li>If only stage I teliospores are present, continue to incubate the flask for a total of 24 h before assessing teliospore germination. Continue incubation for a maximum of 48 h before deeming the sample non-viable.</li>
		<li>If bacterial contamination is present, supplement the PDB with kanamycin sulfate (50 &#181;g/mL) as well as streptomycin sulfate (160 &#181;g/mL) and then repeat steps 3.1 to 3.7. If bacterial contamination persists, treat teliospores with copper sulfate and repeat steps 3.1 to 3.7.</li>
	</ol>
	</li>
</ol>
4. Induction of Germination for Respiration Monitoring
<ol>
	<li>Weigh equal amount (e.g., 50 mg) of teliospores for each respiration experiment.</li>
	<li>In a biosafety cabinet, add teliospores to an autoclaved respiration chamber.</li>
	<li>Fill the chamber with PDB (24 g/L) supplemented with streptomycin sulfate (160 &#181;g/mL) and kanamycin sulfate (50 &#181;g/mL).</li>
	<li>Pipette up and down to create a teliospore suspension.</li>
	<li>Place the chamber lid in the chamber to create air tight seal.</li>
</ol>
5. Obtaining Oxygen Consumption Rate (OCR) Measurements
<ol>
	<li>Place the chamber in the chamber rack inside a water bath (preheated to 28 &#176;C).</li>
	<li>Place the O2 probe inside the opening of the chamber.</li>
	<li>Monitor the data points appearing in real time on the &#34;SensorTrace Rate&#34; program, and let the probe stabilize (~3 min after the probe is placed in the chamber).</li>
	<li>Click &#34;Measure&#34; to measure O2 levels continuously for 6 h with measurements recorded at 2 s intervals.</li>
	<li>Stop the measurement, and repeat steps 4.1&#8211;5.4 for each sample to be analyzed.</li>
	<li>Export the data to Microsoft Excel by clicking &#34;File | Export | Save as .xls&#34;.</li>
</ol>
6. Data Analysis
<ol>
	<li>Calculate OCR
	<ol>
		<li>In the exported Excel file under the &#34;Within_Rates&#34; tab, record the &#34;Rate&#34; for each chamber measurement (nmol/h).</li>
		<li>For each experimental sample, subtract the &#34;Rate&#34; of the blank chamber from the &#34;Rate&#34; of the experimental sample chamber to obtain a corrected OCR value, and take the absolute value of this number.</li>
		<li>Calculate the total OCR per mg of teliospores by dividing the corrected absolute OCR value by the cellular mass used.</li>
		<li>Average replicate &#34;OCR per mg of teliospores&#34; values for each strain.</li>
	</ol>
	</li>
	<li>Analyze the data using appropriate statistical method (e.g., student&#39;s t-test, analysis of variance) using Microsoft Excel of other statistical software.</li>
	<li>To graph raw data, calculate the percentage of oxygen remaining for each time-point you wish to graph. Divide first reading by itself and multiply by 100 (100% oxygen remaining), then divide each subsequent reading by the first reading, and multiply by 100 to obtain the percent of oxygen remaining in the chamber.</li>
</ol>
7. Induction of Teliospore Germination to Isolate Teliospores at Distinct Stages of Germination
<ol>
	<li>Prepare PDB (24 g/L) supplemented with streptomycin sulfate (160 &#181;g/mL) in a biosafety cabinet.</li>
	<li>Place approximately 10 mg of U. maydis teliospores into a 1.5 mL microcentrifuge tube.</li>
	<li>Suspend the teliospores in 500 &#181;L of PDB. Gently pipette to mix until there are no clumps of teliospores in the medium.</li>
	<li>Transfer the teliospore suspension to an autoclaved 250 mL Erlenmeyer flask containing PDB supplemented with streptomycin sulfate.</li>
	<li>Incubate the flask overnight at 28 &#176;C shaking at 90 rpm.</li>
</ol>
8. Preparation of Petri Dish and Micromanipulator
<ol>
	<li>Prepare a Petri dish (57 cm2) by pipetting rows of droplets for microcapillary preparation, and sample collection.
	<ol>
		<li>Pipette 5 &#181;L (x4) dH2O droplets across the top of the Petri dish.</li>
		<li>Pipette 2 &#181;L (x3) of RNA stabilization solution on the Petri dish to be used for sample collection.</li>
		<li>Pipette 5 &#181;L (x30) droplets of germinating teliospores on the Petri dish.</li>
	</ol>
	</li>
	<li>Add 15 mL of mineral oil to the petri dish. Ensure that all droplets are covered by oil before proceeding.</li>
	<li>Prepare a microcapillary with a 15 &#181;m inner diameter, 1 mm flange, 55 mm length, and a 20&#176; tip angle by placing it in the microcapillary holder and submerging it in the mineral oil where capillary action will allow the mineral oil to enter the microcapillary. Release the pressure in the microcapillary before bringing it to the water droplet. Aspirate to prepare the microcapillary with water.</li>
</ol>
9. Isolation of Stage-specific Germinating Teliospores
<ol>
	<li>Using the controls of the micromanipulator, move the prepared microcapillary to one of the germination droplets. Penetrate the droplet, lower the microcapillary, and bring the mouth of the microcapillary up to a germinating teliospore at the stage of germination of interest.</li>
	<li>Slowly aspirate to capture the germinating teliospore. Stop aspirating once the teliospore has entered the microcapillary. Repeat until there are approximately five teliospores in the microcapillary.</li>
	<li>Raise the microcapillary with the micromanipulator and bring it to the collection droplet of RNA stabilization solution. Penetrate the droplet and inject the teliospores into the droplet.</li>
	<li>Repeat steps 8.1 to 8.3 until approximately 1,000 teliospores have been captured.</li>
</ol>
10. Recovery of Collection Droplet
<ol>
	<li>Pipette up the collection droplet and transfer it to the lid of an RNase/DNase-free 2.0 mL microcentrifuge tube. Carefully remove the mineral oil with a pipette without disturbing the collection droplet.</li>
	<li>Use the teliospores for downstream applications such as RNA isolation.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+aspects+of+spore+germination+in+fungi.">Metabolic aspects of spore germination in fungi.</a> Ann. Rev. Phytopathol. 3, 313-342 (1965).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+tumours.">Metabolism of tumours.</a> Biochemische Zeitschrift. 142, 317-333 (1923).</li><li>Ostrowski, L. A., Saville, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+antisense+transcripts+are+linked+to+the+modulation+of+mitochondrial+function+and+teliospore+dormancy+in+Ustilago+maydis.">Natural antisense transcripts are linked to the modulation of mitochondrial function and teliospore dormancy in Ustilago maydis.</a> Mol Microbiol. 103 (5), 745-763 (2017).</li><li>Morrison, E. N., Donaldson, M. E., Saville, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+analysis+of+genes+expressed+in+the+Ustilago+maydis+dikaryon:+uncovering+a+novel+class+of+pathogenesis+genes.">Identification and analysis of genes expressed in the Ustilago maydis dikaryon: uncovering a novel class of pathogenesis genes.</a> Canadian Journal of Plant Pathology-Revue Canadienne De Phytopathologie. 34 (3), 417-435 (2012).</li><li>Doyle, C. E., Cheung, H. Y. K., Spence, K. L., Saville, B. 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T., Saville, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+and+EST+analyses+of+Ustilago+maydis+germinating+teliospores.">Gene expression and EST analyses of Ustilago maydis germinating teliospores.</a> Fungal Genet Biol. 40 (1), 47-64 (2003).</li><li>Hilderbrand, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+the+isolation+of+single+microorganisms.">Techniques for the isolation of single microorganisms.</a> Botanical Review. 4 (12), 38 (1938).</li><li>Seto, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of gene transcripts during Ustilago maydis teliospore dormancy and germination. , (2013).</li><li>Fr&#246;hlich, J., K&#246;nig, H., K&#246;nig, H., Varma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soil Biology - Intestinal Microorganisms of Termites and Other Invertebrates. , 425-437 (2006).</li><li>Choi, Y., Hyde, K., Ho, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+spore+isolation+of+fungi.">Single spore isolation of fungi.</a> Fungal Diversity. 3, 11 (1999).</li><li>Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+started+with+yeast.">Getting started with yeast.</a> Guide to Yeast Genetics and Molecular and Cell Biology, Pt B. 350, 3-41 (2002).</li><li>Chen, Y., Seguin-Swartz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+assessing+the+viability+of+fungal+spores.">A rapid method for assessing the viability of fungal spores.</a> Canadian Journal of Plant Pathology-Revue Canadienne De Phytopathologie. 24 (2), 230-232 (2002).</li></ol>

The authors have no competing financial interests or other conflicts of interest to disclose.

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...

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 &#39;common smut of corn&#39;. 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.

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

investigating teliospore germination using microrespiration analysis and microdissection

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.

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 &#181;mol/h/mg) compared to germinating teliospores (~2,614 &#181;mol/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...

Watch this Scientific Journal Video about Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection at JoVE.com

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

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 ...

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7.2K Views.  Trent University. Smut schimmels veroorzaken vele verwoestende landbouw ziekten. Zij worden verspreid als slapende teliospores die in antwoord op het milieu signalen ontkiemen. Duidelijk naar voren komt twee methoden voor het onderzoek naar moleculaire veranderingen tijdens kieming: meten van verhoging van de ademhaling voor het detecteren van metabole activering en beoordelen van moleculaire gebeurtenissen wijzigen door het isoleren van teliospores in verschillende morfologische stadia.

Video: Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Immunology and Infection

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Bioengineering

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

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