Name: investigating teliospore germination using microrespiration analysis and microdissection
Uploaded: 2018-05-13T14:00:00
Description: Watch this Scientific Journal Video about Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection at JoVE.com

Vi vil gerne takke Dr. Paul Frost for brugen af hans microrespirometer, og Nicole Wagner og Alex Bell for teknisk bistand. Dette arbejde blev finansieret af et NSERC tilskud til B.J.S.

1. majs Cob infektion
<ol><li>Dyrke Zea mays (cv. Golden Bantam) indtil kolber er dannet og er begyndt at silke (ca. 60 dage).</li>
	<li>Kultur kompatibel haploide U. maydis stammer bruger standardprotokoller som tidligere beskrevet7.</li>
	<li>Inficere majs kolber bruger standardprotokoller som tidligere beskrevet7.</li>
</ol>2. teliospore høst
<ol><li>Autoklave udstyr (Büchner tragte, Büchner kolber, blendere, 250 mL centri...

<ol>
	<li>Saville, B. J., Donaldson, M. E., Doyle, C. E., Swan, 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> Meiosis - Molecular Mechanisms and Cytogenetic Diversity. , 411-460 (2012).</li><li>Christensen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Monograph Number 2. , (1963).</li><li>Caltrider, P. D., Gottlieb, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+activity+and+enzymes+for+glucose+catabolism+in+fungal+spores.">Respiratory activity and enzymes for glucose catabolism in fungal spores.</a> Phytopathology. 53, 1021-1030 (1963).</li><li>Allen, P. 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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=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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Basidiomycete biotrophic plante patogener forårsage milliarder af dollars i afgrøde tab årligt. Langt størstedelen af disse patogener producere teliospores, der er integreret til svampe spredning og seksuel reproduktion. At få viden om udviklingen og spiring af teliospores er afgørende for at forstå udbredelsen af de ødelæggende sygdomme forårsaget af disse svampe. For at identificere molekylære ændringer på vigtige kontrolpunkter har vi udtænkt en metode til at identificere timingen af fysiologiske ændrin...

Smut svampe (Ustilaginales) består af mere end 1.600 arter, der inficerer græsser herunder vigtige kornafgrøder af majs, byg, hvede, forårsager milliarder af dollars i afgrøde tab årligt1. Disse svampe er karakteriseret ved produktion af teliospores, der har mørkt pigmenterede cellevægge og spredning agenter. Teliospores funktion til at beskytte genetisk materiale under belastninger af spredning mellem værtsplanter, og kan vare ved i en sovende tilstand for år2. Teliospores er en væsentlig bestanddel af sygdom spredes.
For at studere teliospore biologi, udnytter vores laboratorium model sjofelheder svamp Ustilago maydis (U. maydis), som er den kausale sygdomsagensen 'fælles sjofelheder majs'. Modne U. maydis teliospores er kendetegnet ved vækst anholdelse, reduceret cellulære metabolisme og lave niveauer af cellulære respiration3. Under gunstige forhold (fx., tilstedeværelsen af specifikke sukker), U. maydis teliospores spire og komplet meiose, producerer basidiospores, som kan indlede nye runder af infektion. Spireevnen er karakteriseret ved øget respiration, den tilbagevenden til metaboliske aktivitet og progression gennem observerbare morfologiske stadier af spiring4.
Den indledende fase af spiring omfatter øget respiration og metabolisk funktion, men der er ingen morfologiske angivelser af ændringen. De oprindelige målinger af respiratoriske ændring i U. maydis blev foretaget over 50 år siden, måle iltforbruget manometrically med en Warburg kolbe apparat5. Vi har udviklet en ny og enkel metode til at studere præcise ændringer i respiration under teliospore spiring ved at måle iltforbruget over et tidsforløb af spiring ved hjælp af en Clark-type microrespirometer. Vi har tidligere brugt denne metode til at studere ændringer i respirationsfrekvens mellem vildtype U. maydis haploide celler og mutanter med defekte mitokondrier6, og har tilpasset protokol her for at studere ændringer i teliospore åndedræt under spiring. Dette giver mulighed for præcist at identificere timingen af respiration ændring, således at vi kan målrette teliospores på et passende tidspunkt efter indledningen af spiring at undersøge tidlig molekylære begivenheder. Progression af spiring kan følges mikroskopisk, når promycelia fremgår af teliospore, men den asynkrone natur hæmmede isolation af nok teliospores på et givet tidspunkt for undersøgelsen. Vi udviklede en microdissection teknik svarende til dem, der anvendes til in vitro- befrugtning til fysisk indsamle teliospores på forskellige morfologiske stadier af spireevnen.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:437px;">Streptomycin Sulfate</td>
			<td style="width:75px;">BioShop</td>
			<td style="width:124px;">STP101</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin Sulfate</td>
			<td>BioShop</td>
			<td>KAN201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potato Dextrose Broth</td>
			<td>BD Difco</td>
			<td>254920</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L Waring Laboratory blender</td>
			<td>Waring</td>
			<td>7011S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cheesecloth</td>
			<td>VWR</td>
			<td>470150-438</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nalgene Polypropylene Desiccator with Stopcock</td>
			<td>ThermoFisher Scientific</td>
			<td>5310-0250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unisense MicroRespiration system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">MicroRespiration Sensor (O2)</td>
			<td>Unisense</td>
			<td>OX10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroOptode Meter Amplifier</td>
			<td>Unisense</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MR-Ch Small</td>
			<td>Unisense</td>
			<td>MR-Ch</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SensorTrace Rate Software</td>
			<td>Unisense</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroRespiration Rack</td>
			<td>Unisense</td>
			<td>MR2-Rack</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroRespiration Stirrer</td>
			<td>Unisense</td>
			<td>MR2-Co</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microdissection system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axio Vert.A1 Inverted Light Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coarse Manipulator</td>
			<td>Narishige</td>
			<td>MMN-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Three-axis Hanging Joystick Oil Hydraulic Micromanipulator</td>
			<td>Narishige</td>
			<td>MMO-202ND</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumatic Microinjector</td>
			<td>Narishige</td>
			<td>IM-11-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TransferTip (ES)</td>
			<td>Eppendorf</td>
			<td>5175107004</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating teliospore germination using microrespiration analysis and microdissection

Smut svampe er de ætiologiske agenter af flere ødelæggende landbrugs sygdomme. De er kendetegnet ved produktion af teliospores, som er tykvæggede spredning agenter. Teliospores kan forblive sovende i årtier. Vækstdvale er kendetegnet ved lavt stofskifte, standsede makromolekylære biosyntese og stærkt reduceret niveau af respiration. Når vi modtager den nødvendige miljømæssige signaler, spire teliospores til at producere haploide celler, som kan indlede nye runder af infektion. Teliospore spiring er karakteriseret ved genoptagelsen af makromolekylære biosyntesen, øget respiration og dramatiske morfologiske ændringer. For at netop måle ændringer i cellulære respiration i de tidlige stadier af spiring, har vi udviklet en enkel protokol beskæftiger en Clark-type respirometer. De senere faser af spireevnen er kendetegnet ved bestemte morfologiske ændringer, men spireevnen er asynkron. Vi udviklede en microdissection teknik, der gør det muligt for os at samle teliospores på forskellige spiring stadier.

Ved hjælp af Clark-type microrespirometer-baseret metode til måling af ændringer i respiration under teliospore vækstdvale og spiring, bekræftede vi, at hvilende teliospores udviser en lav grad af respiration (~ 1,075 µmol/h/mg) i forhold til at spire teliospores (~ 2,614 µmol/h/mg; Figur 1A). Dette repræsenterer en ~2.4-fold ændring i gennemsnitlige sats for respiration mellem hvilende teliospores og teliospores, der har været tilskyndet til at spi...

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 svampe forårsager mange ødelæggende landbrugs sygdomme. De er spredt som hvilende teliospores, at spirer i reaktion på miljømæssige stikord. Vi redegøre for to metoder til at undersøge molekylære ændringer under spiring: måling respiration stigning for at opdage metabolisk aktivering og vurdering af skiftende molekylære begivenheder ved at isolere teliospores på forskellige morfologiske stadier.

Undersøge Teliospore spiring ved hjælp af Microrespiration analyse og Microdissection

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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