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Immunologie et infection

Mønster-udløst oxidativ Burst og Seedling væksthæmning assays i Arabidopsis thaliana

Published: May 21, 2019
doi:
10.3791/59437
, , ,
1Department of Biology,Queen’s University

Summary

Dette papir beskriver to metoder til at kvantificere forsvars respons i Arabidopsis thaliana efter udsættelse for immun elicitorer: den forbigående oxidativ burst, og hæmning af frøning vækst.

Abstract

Planter har udviklet sig et robust immunsystem til at opfatte patogener og beskytte mod sygdom. Dette papir beskriver to assays, der kan bruges til at måle styrken af immun aktivering i Arabidopsis thaliana efter behandling med elicitor molekyler. Præsenteret først er en metode til at opfange den hurtigt induceret og dynamisk oxidativ burst, som kan overvåges ved hjælp af en luminol-baseret analyse. Præsenteret anden er en metode, der beskriver, hvordan man måler immun-induceret hæmning af frøning vækst. Disse protokoller er hurtige og pålidelige, kræver ikke specialiseret uddannelse eller udstyr, og er almindeligt anvendt til at forstå det genetiske grundlag for plante immunitet.

Introduction

For at opfatte og forsvare sig mod patogener har planter udviklet membranbundne mønster genkendelses receptorer (PRRs), som detekterer tolererer mikrobielle molekyler uden for cellen, der er kendt som Microbe-associerede molekylære mønstre (MAMPs)1. Binding af MAMPs til deres cognate PRRs initierer protein kinase-medieret immun signalering resulterer i bredspektret sygdomsresistens2. En af de tidligste svar efter PRR-aktivering er fosforylering og aktivering af Integral plasma membran RESPIRATORISKE BURST OXIDASE HOMOLOG (RBOH) proteiner, der katalyserer produktionen af ekstracellulære reaktive oxygenarter (ROS)3 , 4. ros spiller en vigtig rolle i at etablere sygdomsresistens, der virker både som sekundære budbringere til at udbrede immun signalering samt direkte antimikrobielle stoffer5. Den første observation af en immun-fremkaldt oxidativ burst blev beskrevet ved hjælp af kartoffelknolde af CV. Rishiri efter Phytophthora infestans inokulering6. ROS produktion er blevet evalueret i flere plantearter ved hjælp af blade skiver7, cellesuspension kulturer8, og protoplasts6. Beskrevet her er en simpel metode til bestemmelse mønster-udløst ros produktion i blade skiver Arabidopsis thaliana (Arabidopsis).

Som et svar på MAMP perception katalyserer aktiverede rboh-proteiner produktionen af superoxid radikaler (O2–), hydroxylradikaler (• Oh) og singlet oxygen (1O2), der omdannes til hydrogenperoxid (H2o 2) i det ekstracellulære rum9. H2O2 kan kvantificeres ved luminol-baseret chemiluminescens i nærværelse af det oxidationsmiddel peberrod PEROXIDASE (HRP)10. HRP oxiderer H2O2 genererer en hydroxid ion (Oh−) og oxygen gas (O2), som reagerer med i til at producere en ustabil mellemprodukt, der frigiver en photon af lys10. Foton emission kan kvantificeres som relative lysenheder (RLUs) ved hjælp af en mikropladen læser eller Imager stand til at detektere luminescens, som er blevet standard stykker af udstyr i de fleste molekylære laboratorier. Ved at måle lyset produceret over en 40-60-minutters interval, en forbigående oxidativ burst kan påvises så tidligt som 2-5 minutter efter elicitor behandling, toppede på 10-20 minutter, og vender tilbage til basal niveauer efter ~ 60 minutter11. Det kumulative lys, der produceres over dette tidsforløb, kan anvendes som et mål for immun styrken, svarende til aktivering af RBOH-proteiner12. Bekvemt, denne analyse kræver ikke specialiseret udstyr eller besværlige prøveforberedelse.

Toppede kort efter MAMP detektion, den oxidativ burst betragtes som en tidlig immunrespons, sammen med MAPK aktivering og ethylenproduktion5. Senere immunrespons omfatter transkriptional omprogrammering, tyde lukning, og callose deposition2,5. Langvarig udsættelse for MAMPs konstant aktiverer energisk-bekostelig immun signalering resulterer i hæmning af plantevækst, der indikerer en trade-off mellem udvikling og immunitet13. Mønster udløst væksthæmning (SGI) anvendes i vid udstrækning til at vurdere immun produktionen i Arabidopsis og har været en integreret del af identifikationen af flere nøglekomponenter i immun signalering, herunder PRRS14,15 ,16. Derfor præsenterer dette papir desuden en analyse for mønster udløst SGI i Arabidopsis, hvorved frøplanter dyrkes i multi-brønd plader, der indeholder standard medier eller medier suppleret med en immun elicitor i 8-12 dage og derefter vejes ved hjælp af en analytisk skala.

For at demonstrere, hvordan ROS og SGI-assays kan anvendes til at overvåge PRR-medieret signalering, blev tre genotyper, der repræsenterer varierende immun output, valgt: (1) Wild type Arabidopsis tiltrædelsen Columbia (Col-0), (2) den dominerende-negative bak1-5 mutant, hvor den multi-funktionelle PRR Co-receptor brassinosteroid ufølsom 1-associeret kinase 1 (BAK1) er ikke-funktionel i immun signalering17,18og (3) den recessiv cpk28-1 mutant, som mangler den regulatoriske protein calcium-afhængig protein kinase 28 (CPK28) og viser forhøjet immun-udløste respons19,20. ROS og SGI assays er præsenteret som reaktion på en syntetisk produceret elf18 peptid epitop af bakteriel forlængelse faktor Tu (EF-Tu), anerkendt i Arabidopsis af PRR EF-Tu receptor (EFR)15. Disse protokoller kan anvendes med andre immun elicitorer såsom bakteriel motilitet protein flagellin14 eller endogene plante Elicitor proteiner (AtPeps)16, dog skal det bemærkes, at anlæggets reaktionsevne varierer afhængigt af elicitor21. I fællesskab kan ROS og SGI-assays anvendes til hurtig og kvantitativ vurdering af tidlige og sene PRR-medierede responser.

Protocol

1. påvisning af ros burst i Arabidopsis blade skiver efter immun udløsning Plantevækst og vedligeholdelse. For at synkronisere spiring, stratificere Arabidopsis frø ved at suspendere ca 50 frø i 1 mL steril 0,1% agar [w/v] og opbevares ved 4 °c (ingen lys) for 3-4 dage.Bemærk: stratificere en vildtype baggrundskontrol (f. eks. Col-0) og genotyper med høje og lave immun udgange (for eksempel cpk28-1 og bak1-5 ) for at fungere som interne kontroller. …

Representative Results

Mutant cpk28-119,25 og bak1-517,18 planter blev brugt til at påvise forventede udfald for genotyper med henholdsvis høj og lav immunrespons, i oxidativ Burst og SGI analyser i forhold til en Wild-type baggrundskontrol (Col-0). For at vurdere dosisafhængige effekter blev der anvendt en 10-fold peptidfortyndingsserie (1-1000 nM) af elf18. Som forventet h…

Discussion

Dette papir beskriver to metoder til bestemmelse mønster-udløst immunrespons i Arabidopsis, der tilbyder kvantitative tilgange til evaluering af immun produktionen uden brug af specialiseret udstyr. I kombination, mønster-udløst ros og SGI kan bruges til at vurdere tidlige og sene svar på mikrobe perception, hhv.

Den største begrænsning af den oxidativ burst-analyse er variabilitet. Af grunde, der ikke er helt forstået, absolutte RLUs ofte afviger med en størrelsesorden melle…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Arbejde i vores laboratorium er finansieret gennem Natural Resources og Engineering Research Council of Canada (NSERC) Discovery program, det canadiske fundament for innovation John R. Evans Leaders fund, og Queen’s University. KS og IS er understøttet af tandem Ontario Graduate stipendier og NSERC Canada Graduate stipendier til master’s Students (CGS-M).

Materials

20-20-20 Fertilizer Plant Prod 10529 Mix 1g/L in water and apply to plants every 2 weeks for optimal growth.
4 mm Biopsy Punch Medical Mart 232-33-34-P A cork borer set with a 0.125 cm^2 surface area can also be used.
48-Well Sterile Plates with Lid Sigma-Aldrich CLS3548
Analytical Scale with Draft Sheid VWR VWR-225AC Any standard analytical scale can be used for growth inibition assays, however, a direct computer output is optimal.
BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL) Sartorius 725240 Any multichannel pipette can be used, as can a single pipetter if necessary.
elf18 (Ac-SKEKFERTKPHVNVGTIG) EZ Biolab cp7211 Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C.
Forceps Fisher Scientific 22-327379
Horseradish Peroxidase Sigma-Aldrich P6782 Dissolve in pure water. Store at -20C and away from light.
Luminol Sigma-Aldrich A8511 Dissolve in DMSO. Store at -20C and away from light.
Murisage and Skoog Basal Salts Cedarlane Labs MSP09-100LT Store at 4C.
Soil SunGrow Horticulture Sunshine Mix #1 Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots.
SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module Molecular Devices Must request a quote Any plate reader capable of detecting luminescence can be used for these assays.
Sucrose Sigma-Aldrich S0389-1KG Store at room temperature.
White Polystyrene 96-Well Plates Fisher Scientific 07-200-589
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Tags

Keywords: Arabidopsis ThalianaPattern-triggeredOxidative BurstSeedling Growth InhibitionImmune OutputsPlant GenotypesLaboratory EquipmentLeaf DiscsElicitorsReactive Oxygen SpeciesMicroplate ReaderMS MediumSeedling GrowthGrowth Inhibition
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Bredow, M., Sementchoukova, I., Siegel, K., Monaghan, J. Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana. J. Vis. Exp. (147), e59437, doi:10.3791/59437 (2019).
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