Name: split green fluorescent protein system to visualize effectors delivered from bacteria during infection
Uploaded: 2018-05-24T14:30:00
Description: Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

Denne forskningen ble støttet av grunnleggende Science Research Program gjennom National Research Foundation av Korea (NRF) av departementet for vitenskap, IKT og fremtidig planlegging (NRF-2018R1A2A1A05019892) til DC og av et stipend fra anlegget molekylær formering Senter ( PMBC) av programmet neste generasjon Biogreen 21 av Rural Development administrasjon (PJ013201) til EP. Vi takker tenkelig midten av National instrumentering Center for miljøledelse å gi AC confocal mikroskop filmingen.

Merk: Alle trinnene utføres ved romtemperatur, ikke annet er angitt.
1. forberedelse av plantemateriale (4 uker)
<ol><li>Forberedelse for N. benthamiana planter<ol>
<li>Så 2 frø av N. benthamiana på overflaten av hver pott, dekke skuffen med en plast kuppel og tillate frø til å spire i en 25 ° C, 60% fuktighet vekst kammer med en 16/8-h lys/mørke fotoperiode syklus.</li>
		<li>Etter to uker, plukke ut og kaste den minste frøplante i hver pott og fortsette å vokse ...

<ol>
	<li>Melotto, M., Underwood, W., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+stomata+in+plant+innate+immunity+and+foliar+bacterial+diseases.">Role of stomata in plant innate immunity and foliar bacterial diseases.</a> Annu Rev Phytopathol. 46, 101-122 (2008).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126 (5), 969-980 (2006).</li><li>Toruno, T. Y., Stergiopoulos, I., Coaker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-Pathogen+Effectors:+Cellular+Probes+Interfering+with+Plant+Defenses+in+Spatial+and+Temporal+Manners.">Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners.</a> Annu Rev Phytopathol. 54, 419-441 (2016).</li><li>Buttner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behind+the+lines-actions+of+bacterial+type+III+effector+proteins+in+plant+cells.">Behind the lines-actions of bacterial type III effector proteins in plant cells.</a> FEMS Microbiol Rev. 40 (6), 894-937 (2016).</li><li>Dewoody, R. S., Merritt, P. M., Marketon, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+Yersinia+type+III+secretion+system:+traffic+control.">Regulation of the Yersinia type III secretion system: traffic control.</a> Front Cell Infect Microbiol. 3, 4 (2013).</li><li>Deng, W. L., Preston, G., Collmer, A., Chang, C. J., Huang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+hrpC+and+hrpRS+operons+of+Pseudomonas+syringae+pathovars+syringae,+tomato,+and+glycinea+and+analysis+of+the+ability+of+hrpF,+hrpG,+hrcC,+hrpT,+and+hrpV+mutants+to+elicit+the+hypersensitive+response+and+disease+in+plants.">Characterization of the hrpC and hrpRS operons of Pseudomonas syringae pathovars syringae, tomato, and glycinea and analysis of the ability of hrpF, hrpG, hrcC, hrpT, and hrpV mutants to elicit the hypersensitive response and disease in plants.</a> J Bacteriol. 180 (17), 4523-4531 (1998).</li><li>Lewis, J. D., Guttman, D. S., Desveaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+targeting+of+plant+cellular+systems+by+injected+type+III+effector+proteins.">The targeting of plant cellular systems by injected type III effector proteins.</a> Semin Cell Dev Biol. 20 (9), 1055-1063 (2009).</li><li>Alfano, J. R., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+III+secretion+system+effector+proteins:+double+agents+in+bacterial+disease+and+plant+defense.">Type III secretion system effector proteins: double agents in bacterial disease and plant defense.</a> Annu Rev Phytopathol. 42, 385-414 (2004).</li><li>Aung, K., Xin, X., Mecey, C., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Localization+of+Pseudomonas+syringae+pv.+tomato+Effector+Proteins+in+Plants.">Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.</a> Methods Mol Biol. 1531, 141-153 (2017).</li><li>Kay, S., Bonas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Xanthomonas+type+III+effectors+manipulate+the+host+plant.">How Xanthomonas type III effectors manipulate the host plant.</a> Curr Opin Microbiol. 12 (1), 37-43 (2009).</li><li>Bassham, D. C., Raikhel, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+cells+are+not+just+green+yeast.">Plant cells are not just green yeast.</a> Plant Physiol. 122 (4), 999-1001 (2000).</li><li>Courbot, 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=A+major+quantitative+trait+locus+for+cadmium+tolerance+in+Arabidopsis+halleri+colocalizes+with+HMA4,+a+gene+encoding+a+heavy+metal+ATPase.">A major quantitative trait locus for cadmium tolerance in Arabidopsis halleri colocalizes with HMA4, a gene encoding a heavy metal ATPase.</a> Plant Physiol. 144 (2), 1052-1065 (2007).</li><li>Geisler, M., Murphy, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+of+auxin+transport:+the+role+of+p-glycoproteins+in+plant+development.">The ABC of auxin transport: the role of p-glycoproteins in plant development.</a> FEBS Lett. 580 (4), 1094-1102 (2006).</li><li>Boucrot, E., Beuzon, C. R., Holden, D. W., Gorvel, J. P., Meresse, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+SifA+effector+protein+requires+its+membrane-anchoring+C-terminal+hexapeptide+for+its+biological+function.">Salmonella typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function.</a> J Biol Chem. 278 (16), 14196-14202 (2003).</li><li>Reinicke, A. 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+Salmonella+typhimurium+effector+protein+SifA+is+modified+by+host+cell+prenylation+and+S-acylation+machinery.">A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery.</a> J Biol Chem. 280 (15), 14620-14627 (2005).</li><li>Patel, J. C., Hueffer, K., Lam, T. T., Galan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversification+of+a+Salmonella+virulence+protein+function+by+ubiquitin-dependent+differential+localization.">Diversification of a Salmonella virulence protein function by ubiquitin-dependent differential localization.</a> Cell. 137 (2), 283-294 (2009).</li><li>Fernandez-Alvarez, A., 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=Identification+of+O-mannosylated+virulence+factors+in+Ustilago+maydis.">Identification of O-mannosylated virulence factors in Ustilago maydis.</a> PLoS Pathog. 8 (3), e1002563 (2012).</li><li>Galan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+themes+in+the+design+and+function+of+bacterial+effectors.">Common themes in the design and function of bacterial effectors.</a> Cell Host Microbe. 5 (6), 571-579 (2009).</li><li>Rig&#243;, G., Ayaydin, F., Szabados, L., Koncz, C., Cs&#233;pl&#244;, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspension+protoplasts+as+useful+experimental+tool+to+study+localization+of+GFP-tagged+proteins+in+Arabidopsis+thaliana.">Suspension protoplasts as useful experimental tool to study localization of GFP-tagged proteins in Arabidopsis thaliana.</a> Proceedings of the 9th Hungarian Congress on Plant Biology. 52 (1), 59 (2008).</li><li>Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., Waldo, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+a+superfolder+green+fluorescent+protein.">Engineering and characterization of a superfolder green fluorescent protein.</a> Nat Biotechnol. 24 (1), 79-88 (2006).</li><li>Cabantous, S., Terwilliger, T. C., Waldo, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+tagging+and+detection+with+engineered+self-assembling+fragments+of+green+fluorescent+protein.">Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.</a> Nat Biotechnol. 23 (1), 102-107 (2005).</li><li>Cabantous, S., 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+new+protein-protein+interaction+sensor+based+on+tripartite+split-GFP+association.">A new protein-protein interaction sensor based on tripartite split-GFP association.</a> Sci Rep. 3, 2854 (2013).</li><li>Kamiyama, D., 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=Versatile+protein+tagging+in+cells+with+split+fluorescent+protein.">Versatile protein tagging in cells with split fluorescent protein.</a> Nat Commun. 7, 11046 (2016).</li><li>Van Engelenburg, S. B., Palmer, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+type-III+secretion+reveals+dynamics+and+spatial+segregation+of+Salmonella+effectors.">Imaging type-III secretion reveals dynamics and spatial segregation of Salmonella effectors.</a> Nat Methods. 7 (4), 325-330 (2010).</li><li>Park, E., Lee, H. Y., Woo, J., Choi, D., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+Monitoring+of+Pseudomonas+syringae+Effectors+via+Type+III+Secretion+Using+Split+Fluorescent+Protein+Fragments.">Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments.</a> Plant Cell. 29 (7), 1571-1584 (2017).</li><li>. Addgene Available from: <a target="_blank" href="https://www.addgene.org">https://www.addgene.org</a> (2018)</li><li>. Arabidopsis Biological Resource Center Available from: <a target="_blank" href="https://abrc.osu.edu">https://abrc.osu.edu</a> (2018)</li><li>Kvitko, B. H., 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=Deletions+in+the+repertoire+of+Pseudomonas+syringae+pv.+tomato+DC3000+type+III+secretion+effector+genes+reveal+functional+overlap+among+effectors.">Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors.</a> PLoS Pathog. 5 (4), e1000388 (2009).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Kovach, M. E., 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=Four+new+derivatives+of+the+broad-host-range+cloning+vector+pBBR1MCS,+carrying+different+antibiotic-resistance+cassettes.">Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.</a> Gene. 166 (1), 175-176 (1995).</li><li>Upadhyaya, N. 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=A+bacterial+type+III+secretion+assay+for+delivery+of+fungal+effector+proteins+into+wheat.">A bacterial type III secretion assay for delivery of fungal effector proteins into wheat.</a> Mol Plant Microbe Interact. 27 (3), 255-264 (2014).</li><li>Weigel, D., Glazebrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+agrobacterium+using+the+freeze-thaw+method.">Transformation of agrobacterium using the freeze-thaw method.</a> CSH Protoc. 2006 (7), (2006).</li><li>Boyes, D. C., Nam, J., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana+RPM1+disease+resistance+gene+product+is+a+peripheral+plasma+membrane+protein+that+is+degraded+coincident+with+the+hypersensitive+response.">The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response.</a> Proc Natl Acad Sci U S A. 95 (26), 15849-15854 (1998).</li><li>Nimchuk, Z., 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=Eukaryotic+fatty+acylation+drives+plasma+membrane+targeting+and+enhances+function+of+several+type+III+effector+proteins+from+Pseudomonas+syringae.">Eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type III effector proteins from Pseudomonas syringae.</a> Cell. 101 (4), 353-363 (2000).</li><li>Gao, Z., Chung, E. H., Eitas, T. K., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+intracellular+innate+immune+receptor+Resistance+to+Pseudomonas+syringae+pv.+maculicola+1+(RPM1)+is+activated+at,+and+functions+on,+the+plasma+membrane.">Plant intracellular innate immune receptor Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) is activated at, and functions on, the plasma membrane.</a> Proc Natl Acad Sci U S A. 108 (18), 7619-7624 (2011).</li><li>Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S., Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scalable+strategy+for+high-throughput+GFP+tagging+of+endogenous+human+proteins.">A scalable strategy for high-throughput GFP tagging of endogenous human proteins.</a> Proc Natl Acad Sci U S A. 113 (25), E3501-E3508 (2016).</li><li>Li, X., Yang, Q., Tu, H., Lim, Z., Pan, S. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+Agrobacterium-delivered+VirE2+in+recipient+cells.">Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.</a> Plant J. 77 (3), 487-495 (2014).</li><li>Tanaka, S., 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=Experimental+approaches+to+investigate+effector+translocation+into+host+cells+in+the+Ustilago+maydis/maize+pathosystem.">Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.</a> Eur J Cell Biol. 94 (7-9), 349-358 (2015).</li></ol>

Protokollen beskrives her brukes for overvåking av nøyaktig lokalisering av effektor proteiner injiseres av bakteriell T3SS verten anlegget celle på infeksjon. Tidligere delt GFP systemet ble brukt som et verktøy til å studere subcellular lokalisering av pattedyr proteiner23,36, Salmonella T3E lokalisering, og Agrobacterium VirE2 levering gjennom T4SS i den Plant celler37. Hvis du vil bruke dette systemet i anlegget ...

Planter er fastsittende organismer som møter mange invaderende patogener inkludert bakterier, sopp, virus, insekter og nematoder gjennom hele livssyklusen. Blant phytopathogens, gram-negative bakteriell patogener som Pseudomonas spp. og Ralstonia spp., infisere sine Salix ved å angi gjennom sår eller naturlig åpninger, som stomata og hydathode1. For å lykkes kolonisere Salix, bakteriell patogener utviklet seg for å utvikle en rekke virulens faktorer2. Når bakterier invaderer næringsplante, de injiserer en rekke virulens proteiner, kjent som effektor-direkte inn i anlegget celler å fremme deres virusets. Disse effektor undertrykke eller modulerer anlegget medfødt immunitet og manipulere vert cellulære prosesser for å føre til bakteriell overlevelse3.
Patogene bakterier bruker hovedsakelig en T3SS for å levere effektor proteiner direkte til verten cellene4. T3SS ligner en molekylær sprøyte med en nål som kanal koble fra en stillaset proteinstruktur over indre og ytre bakteriell membraner til injeksjonsstedet verten celle5. Denne T3SS-mediert effektor (T3E) sekresjon mekanismen er godt bevart i ulike gram-negative bakteriell patogener av anlegget samt menneskelige. En representant anlegget patogener, P. syringae pv. Tomat DC3000 hrcC mutant som vanligvis har en defekt T3SS, har begrenset vekst i planter sannsynlig på grunn av manglende evne av denne mutant å fullstendig undertrykke anlegget immunitet (ved å injisere effektor proteiner)6. På translokasjon inn i vertsceller målrette effektor ulike vert proteiner som er viktig for celle vertssystemet, inkludert anlegget forsvar svar, gene transkripsjon, celledød, proteasome, vesicle smugling og hormon trasé7 , 8 , 9 , 10. sporing av mobilnettet lokalisering av effektor proteinene i verten cellene er derfor et attraktivt mål å forstå deres funksjoner med hensyn til modulering av anlegget immunitet.
De fleste av lokalisering studier av T3Es har ansatt Agrobacterium -mediert overuttrykte med en stor fluorescens protein i verten anlegget9. Metoden heterologous uttrykk for gener som er innført i andre arter har imidlertid vist seg å være mis lokaliserte eller noen ganger ikke-fungerende11,12,13. I tillegg viste flere studier at bakteriell effektor gjennomgå modifikasjon for riktig målgruppe i de vert celler14,15,16,17. Derfor uttrykt transiently effektor i stoffer av plante celler ikke kan være funksjonelt eller kvantitativt identisk effektor som leveres av T3SS på patogen infeksjon18. Videre kan fusjon av store fluorescerende tags effektor proteiner forstyrre riktig effektor levering og visualisering18,19. Derfor kan disse tilnærminger til analysen funksjonen T3E ikke fullt gjenspeiler den opprinnelige lokaliseringen av T3SS-utskilles effektor.
En grønn fluorescerende protein (GFP) består av en 11-strandet β-fat omslutter en sentral strand som inkluderer en chromophore20. Waldo et al. rapportert en roman split-GFP system som består av en liten del (GFP β tråd 11; GFP11) og en stor supplerende fragment (GFP β strand 1-10; GFP1-10)21. Fragmenter fluoresce ikke av seg selv, men fluoresce på tilknytningen selv når begge fragmentene er i nærheten med hverandre. For optimalisering av protein folding effektiviteten, ble robust folding varianter av GFP, dvs., sfGFP og sfGFPOPT, senere utviklet for delt GFP systemet20,21,22. Nylig mutert enkelt aminosyre varianter av sfGFP1-10OPT- sfYFP1-10OPT og sfCFP1-10OPT- som kan gjeninnføre med sfGFP11 fragment og Vis gule og cyanfargede fluorescens henholdsvis, ble generert23 . Videre sfCherry, et derivat av mCherry, kan deles inn i sfCherry1-10 og sfCherry11 fragmenter på samme måte som sfGFP23.
Dette systemet har vært tilpasset etikett og spore T3SS effektor i HeLa celler under infeksjon med effektor fra Salmonella24. Men var det tidligere ikke optimalisert for plante-bakteriell patogen vertssystemet. Nylig optimalisert vi delt GFP systemet basert på forbedret sfGFP1-10OPT å overvåke den subcellular lokaliseringen av T3Es levert fra P. syringae i anlegget celler25. For å lette lokalisering studier av T3Es til forskjellige subcellular rom i anlegget celler, et sett av transgene Arabidopsis thaliana ble planter generert for å uttrykke sfGFP1-10OPT i de ulike subcellular avdelinger 25. videre plasmider bærer en rekke organelle målrettet sfGFP1-10OPT for Agrobacterium-mediert forbigående overuttrykte og sfGFP11-merket vektorer for levering av T3SS-baserte effektor også genereres. Frøene av ulike transgene Arabidopsis linjer og plasmider å uttrykke T3Es rundt kan fås fra kilder som er nevnt i Tabellen for materiale26,27.
I den følgende protokollen beskriver vi en optimalisert system for å overvåke dynamikken i effektor av bakterier i verten cellene med delt sfGFP systemet. Infeksjon av planter uttrykke sfGFP1-10OPT med transgene Pseudomonas bærer rekombinant sfGFP11 plasmider resulterer i en levering av sfGFP11-merket effektor fra Pseudomonas inn i vert-cellen. Derfor disse proteinene er rekonstituert og translocate til bestemte effektor målet compartment(s). Pseudomonas syringae pv. tomat CUCPB5500 belastningen som slettes 18 effektor, ble brukt fordi denne belastningen viste lav eller ingen celledød i både A. thaliana og N. benthamianas28. Imidlertid kan alle materialer og trinnene som beskrives her erstattet eller endret for å tilpasse delt sfGFP systemet for undersøkelse av andre biologiske spørsmål eller optimalisering i de gitte laboratorieforhold.

<table>
	<tbody>
		<tr>
			<td>Arabidopsis transgenic lines</td>
			<td></td>
			<td></td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>CYTO-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69831</td>
			<td></td>
		</tr>
		<tr>
			<td>NU-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69832</td>
			<td></td>
		</tr>
		<tr>
			<td>PT-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69833</td>
			<td></td>
		</tr>
		<tr>
			<td>MT-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69834</td>
			<td></td>
		</tr>
		<tr>
			<td>PX-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69835</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69836</td>
			<td></td>
		</tr>
		<tr>
			<td>GO-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69837</td>
			<td></td>
		</tr>
		<tr>
			<td>PM-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69838</td>
			<td></td>
		</tr>
		<tr>
			<td>Organelle-targeted sfGFP1-10OPT plasmid</td>
			<td></td>
			<td></td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>CYTO-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97387</td>
			<td></td>
		</tr>
		<tr>
			<td>NU-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97388</td>
			<td></td>
		</tr>
		<tr>
			<td>PT-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97389</td>
			<td></td>
		</tr>
		<tr>
			<td>MT-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97390</td>
			<td></td>
		</tr>
		<tr>
			<td>PX-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97391</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97392</td>
			<td></td>
		</tr>
		<tr>
			<td>GO-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97393</td>
			<td></td>
		</tr>
		<tr>
			<td>PM-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97394</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfCherry1-10</td>
			<td>Addgene</td>
			<td>97403</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfYFP1-10</td>
			<td>Addgene</td>
			<td>97404</td>
			<td></td>
		</tr>
		<tr>
			<td>CYTO-sfCFP1-10</td>
			<td>Addgene</td>
			<td>97405</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">sfGFP11-tagged Gateway compatible vector for T3SS-based effector delivery system</td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>pBK-GW-1-2</td>
			<td>Addgene</td>
			<td>98250</td>
			<td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-1-4</td>
			<td>Addgene</td>
			<td>98251</td>
			<td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-2-2</td>
			<td>Addgene</td>
			<td>98252</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-2-4</td>
			<td>Addgene</td>
			<td>98253</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-1-2</td>
			<td>Addgene</td>
			<td>98254</td>
			<td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-1-4</td>
			<td>Addgene</td>
			<td>98255</td>
			<td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-2-2</td>
			<td>Addgene</td>
			<td>98256</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-2-4</td>
			<td>Addgene</td>
			<td>98257</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens GV3101</td>
			<td></td>
			<td></td>
			<td>Csaba Koncz and Jeff Schell, The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet. 204,383-396 (1986); Resistant to gentamycin (50 ug/ml) and rifampicin (50 ug/ml)</td>
		</tr>
		<tr>
			<td>Pseudomonas syringae pv. Tomato CUCPB5500</td>
			<td></td>
			<td></td>
			<td>Kvitko, B. H. et al. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog. 5 (4) (2009).; Resistant to rifampicin (100 ug/ml)</td>
		</tr>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plant germination media</td>
			<td></td>
			<td></td>
			<td>Add 2.165g/L Murashige &#38; Skoog powder, 10 g/L sucrose to water. Adjust to pH 5.8 and add 2.2 g/L phytagel. Autocalve.</td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium including vitamins</td>
			<td>Duchefa Biochemie</td>
			<td>M0222</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Duchefa Biochemie</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma-Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media</td>
			<td></td>
			<td></td>
			<td>Add 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl to water. For solid media, add 15 g/L micro agar. Autoclave.&#160; Allow solution to cool to 55 &#176;C, and add antibiotic if needed.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>BD Bioscience</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bioscience</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duchefa Biochemie</td>
			<td>S0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro agar</td>
			<td>Duchefa Biochemie</td>
			<td>M1002</td>
			<td></td>
		</tr>
		<tr>
			<td>King&#39;s B media</td>
			<td></td>
			<td></td>
			<td>10 g/L protease peptone #2, 1.5 g/L anhydrous K2HPO4, 15 g/L of agar to water. Autoclave. Cool down to 55 &#176;C and add sterile 15 ml/L glycerol, 5 ml/L MgSO4 to the medium. Add antibiotics if needed.</td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>BD Bioscience</td>
			<td>212120</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>1551128 USP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Duchefa Biochemie</td>
			<td>G1345</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Bioscience</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol-Glutamate (MG) liquid media </td>
			<td></td>
			<td></td>
			<td>Add 10 g/L of mannitol, 2 g/L of L-glutamic acid, 0.5 g/L of KH2PO4, 0.2 g/L of NaCl, and 0.2 g/L of MgSO4 to water. Adjust to pH 7</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Duchefa Biochemie</td>
			<td>M0803</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamic acid</td>
			<td>Duchefa Biochemie</td>
			<td>G0707</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>NIST200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Infiltration buffer</td>
			<td></td>
			<td></td>
			<td>10 mM MES (2-(N-morpholino)-ethane sulfonic acid), 10 mM MgCl2, 150 &#181;M acetosyringone. pH 5.6; Prepare a fresh buffer before use.</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Duchefa Biochemie</td>
			<td>M1503</td>
			<td>Prepare 100 mM (pH 5.6) stock in water. Filter sterilize.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Prepare 100 mM stock in water. Autoclave.</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma-Aldrich</td>
			<td>D134406</td>
			<td>Prepare 150 mM stock in DMSO.</td>
		</tr>
		<tr>
			<td>Confocal microscope equipments/materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>710 laser scanning confocal system</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axio observer Z1 inverted microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>ThermoFisher</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
	</tbody>
</table>

split green fluorescent protein system to visualize effectors delivered from bacteria during infection

Bakterier, en av de viktigste forårsaker agentene fra ulike anlegg sykdommer, skiller en rekke effektor proteiner til verten anlegget celle å styrte immunsystemet anlegget. Under infeksjon leveres cytoplasmatiske effektor til verten stoffer via en type III sekresjon system (T3SS). Etter levering i anlegget celle mål effector(s) den spesifikke compartment(s) for å modulere verten celleprosesser for overlevelse og replikering av patogen. Selv om det har vært noen undersøkelser på den subcellular lokaliseringen av effektor proteiner i verten cellene å forstå deres funksjon i virusets ved å bruke fluorescerende proteiner, undersøkelse av dynamikken i effektor direkte injiseres fra bakterier har vært utfordrende på grunn av inkompatibilitet mellom T3SS og fluorescerende proteiner.
Her beskriver vi vår siste metode for en optimalisert delt superfolder grønne fluorescerende protein system (sfGFPOPT) å visualisere lokalisering av effektor levert via bakteriell T3SS i vert cellen. SfGFP11 (11th β-strand av sfGFP)-merket effektor skilles ut gjennom T3SS kan settes sammen med en bestemt organelle målrettet sfGFP1-10OPT (1-10th β-strand i sfGFP) ledende til fluorescens utslipp på stedet. Denne protokollen gir en prosedyre for å visualisere rekonstituert sfGFP fluorescens signalet med en effektor protein fra Pseudomonas syringae i en bestemt organelle på Arabidopsis og Nicotiana benthamiana planter.

Β fat oppbygning GFP består av elleve β tråder og kan deles inn i to fragmenter, 1-10th strand (GFP1-10OPT) og den 11th (GFP11)-stranden. Selv om ingen av to fragmenter fluorescerende selv, kan selv montert sfGFP avgir fluorescensen når to fragmenter finnes i nærheten (figur 1A). I dette systemet, sfGFP1-10OPT-uttrykke Arabidopsis eller N. benthamiana planter er inokulert med Pseudomonas<...

Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Split grønne fluorescerende Protein System å visualisere effektor levert fra bakterier under infeksjon

Fluorescerende protein-baserte tilnærminger til overvåke effektor utskilles av bakterier i vertsceller er utfordrende. Dette skyldes inkompatibilitet mellom fluorescerende proteiner og type-III sekresjon systemet. Her, brukes en optimalisert delt superfolder GFP system for visualisering av effektor utskilles av bakterier i verten anlegget celle.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Research

JoVE Journal

Biology

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Overvåking av Ubiquitin-proteasome aktivitet i levende celler ved hjelp av en Degron (DGN)-destabilisert grønt fluorescerende protein (GFP)-baserte reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Visualisere Bakterier i Nematoder med fluorescerende Mikroskopi

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA Screening for å identifisere Ubiquitin og Ubiquitin-lignende system regulatorer av biologiske mekanismer i dyrkede pattedyrceller

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

En ny Avklaring metode for å visualisere Galle Degeneration Under Liver Metamorphosis i Sea Lamprey ( Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...