Name: the plant infection test: spray and wound-mediated inoculation with the plant pathogen magnaporthe grisea
Uploaded: 2018-08-04T13:30:00
Description: Watch this Scientific Journal Video about The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea at JoVE.com

Dette arbeidet ble støttet av spesiell vitenskapelig prosjektet Beijing landbruk University (YQ201603) og vitenskapelige prosjektet i Beijing pedagogiske Committee (KM201610020005).

1. spray Inoculation med en suspensjon av M. grisea Conidia
<ol><li>
Fungal kultur for M.grisea
	<ol>
<li>Forberede havremel tomat agar (OTA) kultur medium fungal stammer.</li>
		<li>Veie 30-50 g av havremel, legge dette til 800 mL destillert/ikke-ionisert vann (ddH2O) og koke blandingen i 30 min i elektrisk potten.</li>
		<li>Filtrere kokt havregryn juice i begeret gjennom et stykke gasbind.</li>
		<li>Legge til 150 mL av tomatjuice og 20 g agar filtratet i begeret og legg...

<ol>
	<li>Li, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+natural+allele+of+a+transcription+factor+in+rice+confers+broad-spectrum+blast+resistance.">A natural allele of a transcription factor in rice confers broad-spectrum blast resistance.</a> Cell. 170 (1), 114-126 (2017).</li><li>Chi, M. H., Park, S. Y., Kim, S., Lee, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+pathogenicity+gene+is+required+in+the+rice+blast+fungus+to+suppress+the+basal+defenses+of+the+host.">A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host.</a> PLoS Pathogens. 5 (4), 1000401 (2009).</li><li>Jia, Y., Valent, B., Lee, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+host+responses+to+Magnaporthe+grisea.on+detached+rice+leaves+using+a+spot+inoculation+method.">Determination of host responses to Magnaporthe grisea.on detached rice leaves using a spot inoculation method.</a> Plant Disease. 87 (2), 129-133 (2003).</li><li>Ebbole, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnaporthe+as+a+model+for+understanding+host-pathogen+interactions.">Magnaporthe as a model for understanding host-pathogen interactions.</a> Annual Review of Phytopathology. 45, 437-456 (2007).</li><li>Hamer, J. E., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection-related+development+in+the+rice+blast+fungus+Magnaporthe+grisea.">Infection-related development in the rice blast fungus Magnaporthe grisea.</a> Current Opinion in Microbiology. 1 (6), 693-697 (1998).</li><li>Howard, R. J., Valent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+and+entering:+host+penetration+by+the+fungal+rice+blast+pathogen+Magnaporthe+grisea.">Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea.</a> Annual Review of Microbiology. 50, 491-512 (1996).</li><li>Chen, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Glycosylation+of+Effector+Proteins+by+an+&#945;-1,3-+Mannosyltransferase+Is+Required+for+the+Rice+Blast+Fungus+to+Evade+Host+Innate+Immunity.">N-Glycosylation of Effector Proteins by an &#945;-1,3- Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity.</a> The Plant Cell. 26 (3), 1360-1376 (2014).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.ARG1,+MoARG5,6+and+MoARG7+involved+in+arginine+biosynthesis+are+essential+for+growth,+conidiogenesis,+sexual+reproduction,+and+pathogenicity+in+Magnaporthe+oryzae.">M.ARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae.</a> Microbiological Research. 180, 11-22 (2015).</li><li>Du, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serine/threonine-protein+phosphatase+PP2A+catalytic+subunit+is+essential+for+asexual+development+and+plant+infection+in+Magnaporthe+oryzae.">A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae.</a> Current Genetics. 59 (1-2), 33-41 (2013).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protein+com1+is+required+for+normal+conidium+morphology+and+full+virulence+in+Magnaporthe+oryzae.">A novel protein com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae.</a> Molecular Plant-Microbe Interactions. 23 (1), 112-123 (2010).</li><li>Cao, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ash1-like+protein+MoKMT2H+null+mutant+is+delayed+for+conidium+germination+and+pathogenesis+in+Magnaporthe+oryzae.">An ash1-like protein MoKMT2H null mutant is delayed for conidium germination and pathogenesis in Magnaporthe oryzae.</a> BioMed Research International. 2016, 1575430 (2016).</li><li>Bryan, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+amino+acid+difference+distinguishes+resistant+and+susceptible+alleles+of+the+rice+blast+resistance+gene+Pi-ta.">A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.</a> The Plant Cell. 12 (11), 2033-2045 (2000).</li><li>Zhou, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+pathology:+a+life+and+death+struggle+in+rice+blast+disease.">Plant pathology: a life and death struggle in rice blast disease.</a> Current Biology. 26 (18), 843-845 (2016).</li><li>Guo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MoGrr1,+a+novel+F-box+protein,+is+involved+in+conidiogenesis+and+cell+wall+integrity+and+is+critical+for+the+full+virulence+of+Magnaporthe+oryzae.">MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae.</a> Applied Microbiology and Biotechnology. 99 (19), 8075-8088 (2015).</li><li>Talbot, N. 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N., McClung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Resistance+Spectra+of+the+Pi-ta+and+Pi-k+Genes+to+U.S.+Races+of+Magnaporthe+oryzae+Causing+Rice+Blast+in+a+Recombinant+Inbred+Line+Population.">Determination of Resistance Spectra of the Pi-ta and Pi-k Genes to U.S. Races of Magnaporthe oryzae Causing Rice Blast in a Recombinant Inbred Line Population.</a> Plant Disease. 93, 639-644 (2009).</li><li>Peng, Y. L., Shishiyama, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+sequence+of+cytological+events+in+rice+leaves+infected+with+Pyricularia+oryzae.">Temporal sequence of cytological events in rice leaves infected with Pyricularia oryzae.</a> Canadian Journal of Botany. 66 (4), 730-735 (1988).</li></ol>

Plante sykdom motstand genene spiller en viktig rolle i å forebygge infeksjoner av patogener, inkludert sopp patogener1,12. Ris blast er brukt som modell å forstå naturen av patogen befolkningen strukturer og identifisere anlegget motstand gener4. Derfor er det nødvendig å undersøke sykdom motstand genotype og avirulence genotyper av de viktigste variantene av agricultural planter i stor skala å identifisere sykdom-motstandsdyktig p...

Ris blast, forårsaket av M. grisea, er en av de mest alvorlige sykdommene for ris varianter verdensomspennende1,2. Prosessen som M. grisea infiserer Salix inkluderer conidia produksjon og overflate vedlegg, en conidia spiring og appressorium formasjon, en formasjon av penetrasjon pinne og smittsomme hypha differensiering, og en sykdom spre 3. alle disse stadiene er vanlig i mange andre plante patogene sopp, og, faktisk, en blokade av helst enkelt forhindrer infeksjon av Salix. Den økonomiske betydningen og genetisk tractability, M. grisea har dukket opp som en modell organisme for å studere mekanismer av plante-fungal patogen interaksjoner1,4. Derfor vil studere molekylære grunnlaget for disse utviklingsstadier i M. grisea bidra til å belyse molekylære mekanismer underliggende fungal virusets og identifikasjon av kandidat målet gener for screening og utforme roman soppmidler5.
Nylige rapporter om M. grisea infeksjon har fokusert på molekylære mekanismer av pre penetrasjon scenene, spesielt conidiation, appressorium dannelse, penetrasjon pinnene og smittsomme vekst3, 6. det er derfor viktig å utvikle en detaljert protokoll for å teste M. grisea infeksjon. Her presenterer vi en detaljert metode for en infeksjon test som benytter spray-mediert infeksjon analyser med en conidial suspensjon og inoculation av sår med mycelial pluggene til M. grisea. I denne rapporten fokuserer protokollen på kultur stammer, utarbeidelse av den conidiation løsningen for sprøyting og mycelial plug-mediert inoculation planter med M. grisea. Denne fremgangsmåten er beskrevet i detalj nedenfor, og en skjematisk visning viser hele arbeidsflyten metoden og en typisk lesjon er vist i figur 1 og 2, henholdsvis.

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	<tbody>
		<tr height="77">
			<td height="77" style="height:77px;width:216px;">&#160;Agar</td>
			<td style="width:177px;">AOBOX Biotechnology&#65288;China&#65289;</td>
			<td style="width:207px;">01-023</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Filter paper</td>
			<td style="width:177px;">GE Healthcare brand(Sweden)&#160;&#160;</td>
			<td>10311387</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50-mL tube</td>
			<td style="width:177px;">CORNING(Amercia)</td>
			<td style="width:207px;">430290</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifuge</td>
			<td style="width:177px;">Eppendorf(Amercia)</td>
			<td style="width:207px;">5804R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Tween-20</td>
			<td style="width:177px;">Coolaber&#65288;China&#65289;</td>
			<td style="width:207px;">CT11551-100ml</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Culture dish</td>
			<td style="width:177px;">Thermofisher(Amercia)</td>
			<td>150326</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">0.5-5 mL pipette</td>
			<td>Eppendorf</td>
			<td style="width:207px;">&#160;4920000105</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">100-1000uL pipette</td>
			<td>Eppendorf</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Vacuum pump</td>
			<td style="width:177px;">Leybold</td>
			<td style="width:207px;">D25B</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Dissection needle</td>
			<td style="width:177px;">FST</td>
			<td style="width:207px;">26000-35</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Incubator</td>
			<td style="width:177px;">MEMMERT</td>
			<td style="width:207px;">PYX313</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Inoculation ring</td>
			<td style="width:177px;">Greiner Bio One</td>
			<td style="width:207px;">731175</td>
			<td></td>
		</tr>
	</tbody>
</table>

the plant infection test: spray and wound-mediated inoculation with the plant pathogen magnaporthe grisea

Planter har et kraftig system for å forsvare seg mot potensielle trusler av patogene sopp. For agriculturally viktig planter, men gjeldende tiltak for å bekjempe slike patogener bevise for konservative og dermed ikke tilstrekkelig effektiv, og de kan potensielt utgjøre miljørisiko. Derfor er det svært nødvendig å identifisere vert-motstand faktorene å hjelpe inne kontrollerende anlegget sykdommer naturlig gjennom identifikasjon av motstandsdyktig germplasm, isolering og karakterisering av motstand gener og molekylære oppdrett av motstandsdyktig kultivarer. I denne forbindelse, det er behov å etablere en nøyaktig, rask og omfattende inoculation metode å avle og utvikle anlegget motstand gener. Ris sprenge fungal patogen Magnaporthe grisea forårsaker alvorlig sykdomssymptomer og yield tap. M. grisea har nylig dukket opp som en modell organisme for å studere mekanismer av plante-fungal patogen interaksjoner. Derfor rapportere vi utviklingen av en plante virulens test metoden som gjelder M. grisea. Denne metoden gir både spray inoculation med en conidial suspensjon og såret inoculation med mycel kuber eller dråper av conidial suspensjon. Det viktigste trinnet i såret inoculation metoden for frittstående ris blader er å lage sår på anlegget blader, som unngår forstyrrelser forårsaket av verten gjennomtrenging motstand. Dette spray/såret protokollen bidrar til rask, nøyaktig og omfattende screening av pathotypes av M. grisea isolerer. Dette integrert og systematisk anlegget infeksjon metoden vil tjene som et utgangspunkt for å få et bredt perspektiv av problemene i anlegget patologi.

Hele arbeidsflyten for teknikken er vist i figur 1. Anlegget infeksjon analyser ble utført på 14 dager gamle utsatt ris planter (O. sativa cv CO-39) eller utsatt 7 dager gamle bygg (H. vulgare cv Golden lover)7,8,9. For å teste for en infeksjon på ris bladene, en conidial suspensjon (1.0 x 105 sporer/mL) av M. grisea vill-typ...

Watch this Scientific Journal Video about The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea at JoVE.com

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Anlegget infeksjon testen: Spray og såret-mediert Inoculation med anlegget patogen Magnaporthe Grisea

Her presenterer vi en protokoll for å teste anlegg virulens med anlegget patogen Magnaporthe grisea. Denne rapporten vil bidra til storskala screening av pathotypes av sopp isolerer og tjene som et utgangspunkt for å forstå motstandsdyktig mekanismer for planter under molekylær avl.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

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