© 2013 Nature America, Inc. All rights reserved. rmbre luoelayer aleurone barley from isolated protoplasts with studied been have hnesb ih,srs n hormone species and various stress ion in light, of responses modulation by the channels and and signaling regulation, calcium activity, photo- synthesis regeneration, during wall ferentiation cell dif- embryogenesis, as division, cellular cell such observe synthesis, activities to and used processes been had plasts a efudi review a in protoplasts found plant be about can information are general constructs. assay More TEAMP discussed. reporter the also useful of limitations troubleshooting on and advantages information provide Applications, detailed and and advice (TEAMP) in expression protoplast transient libraries, of system mesophyll insertion a for as plant protocol a model such here resources, the in genetic available rich com- when with analysis genomics bined functional in to powerful especially manipulated is transiently method The be responses. and regulation cell-autonomous can investigate machineries translation microinjection. and transcription as and well as pathways metabolic and transduction Signal fusion, methods, PEG–calcium various and using protoplasts cell-based RNA e.g., into versatile DNA, delivered be as a can proteins such offer Macromolecules walls system. cell experimental without protoplasts Plant INTRODUCTION in mechanisms regulatory complex obtained of be analysis comprehensive can further results development. for the guidelines and and reliable growth h, immunity, offers physiology, system 6–8 whole- take procedures The transfection h. DNA 2–24 as and explorations isolation in cost-effective protoplast and The facile plasmid tests. hypothesis-driven enable of capability as transfection high-throughput PEG–calcium well and isolation, responses protoplast Physiological and includes culture. tested protoplast method extensively and The been here. DNA has presented that is protocol experiments well-established numerous A machineries. in the cellular analyze applied and to pathways approaches signaling proteomic diverse and of genomic functions genetic, biochemical, cellular, molecular, using experiments cell-based conducting using system expression gene transient The 1 analysis Yoo expression Sang-Dong gene transient for system Arabidopsis eotdmr hn4 er ago years 40 than first more was reported protoplasts plant of isolation The Overview (http://genetics. website lab Sheen the at mgh.harvard.edu/sheenweb/protocols_reg.html). seen be can procedure ye oivsiaecl type–specific and on gibberellin actions cell acid abscisic example, investigate For responses. cell to special from isolated types also were plasts ur cells guard n lelgtidcdH light–induced blue and ulse nie2 ue20;cretdatrpit1 coe 03 doi:10.1038/nprot.2007.199 2013; October 11 print after corrected 2007; June 21 online Published ([email protected]). S.-D.Y. to addressed be should Correspondence USA. 02114, Massachusetts eateto oeua ilg,MsahstsGnrlHsia,Bso,Msahsts USA. Massachusetts, Boston, Hospital, General Massachusetts , Molecular of Department 4 respectively. , 1,2 on-e Cho Young-Hee , a ayaesynthesis -amylase 1 + 3 n iepromneo h TEAMP the of performance live a and APs activity -ATPase and eohl rtpat:avraiecell versatile a protoplasts: mesophyll rbdpi thaliana Arabidopsis ii faba Vicia 2 1 .Proto- .Proto- 1,2 Arabidopsis e Sheen Jen & eeconstruct. gene maize the ( to microscopy fused enhancer 35S constitutive the by hrceitc ne ih irsoy( microscopy light under characteristics response ethylene constitutive rtpat eantecaatrsiso h eei akrud rmwihte eeobtained; were they wild-type which from from isolated backgrounds protoplasts genetic ( the source. of protoplast characteristics mesophyll the a retain protoplasts as used be to seventh) and Arabidopsis iue1 Figure bcdab edescribe We . | Arabidopsis Arabidopsis ln utbefrpools slto.Atrssidct h pia relae fit,sixth (fifth, leaves true optimal the indicate Asterisks isolation. protoplast for suitable plant eohl rtpat a rvna motn n estl olfor tool versatile and important an proven has protoplasts mesophyll  0) oeta 0 fpoolsswr rnfce ucsflywt h F reporter GFP the with successfully transfected were protoplasts of 90% than More 400). 1,2 rtpatioainadtasetgn xrsin ( expression. gene transient and isolation protoplast sitc lns ept h nyai ramn step treatment enzymatic the despite , intact activitiesas cellular and responses physiological same the of many tain (see collected washed released, and are protoplasts macerozyme, and fungal (see soil on of growing leaves from isolated nuaintm.T lo osiuieepeso ftransfected of expression constitutive allow To time. incubation (see expression 1d gene allow Fig. to incubation h proto- 2–24 into by followed DNA plasts, deliver to used is transfection calcium-mediated cell- a as system tool genomics TEAMP functional based the several established and we have sequence, laboratories genome other whole its of availability the with Since tde sn rtpat eeae rmsvrlpatspecies plant several from generated been protoplasts have using interest of studied genes of functions cellular diverse micro- injection), and electroporation PEG, (e.g., methods transfection DNA h EM rtcli ae nmspylpoolssfreshly protoplasts mesophyll on based is protocol TEAMP The fe h eeomn n infiatipoeeto various of improvement significant and development the After ctr1-1 Arabidopsis Col-0 .Aseietcnqei o eesr wn oteshort the to owing necessary not is technique sterile A ). uat(otm hwdfeetclua opooyand morphology cellular different show (bottom) mutant  2 eateto eeis avr eia col Boston, School, Medical Harvard Genetics, of Department tp,ethylene-insensitive(top), 0) ( 600). d eaetecnrlmdlsse npatbiology plant in system model central the became rtpat xrsigtencertree F driven GFP nuclear-targeted the expressing Protoplasts ) i.1b Fig. C4PPDK i.1a Fig. AUEPROTOCOLS NATURE Arabidopsis , c b .Rmral,teepoolssmain- protoplasts these Remarkably, ). eohl rtpat ( protoplasts Mesophyll ) aa rmtraesonudrfluorescence under shown are promoter basal .Atrcl al r eoe using removed are walls cell After ). 1,5 . etr1-1 idtp rmtn plants mutant or wild-type uat(ide n the and (middle) mutant a | elh 4-week-old healthy A ) O. NO.7 VOL.2 PROTOCOL  0) ( 400). | 2007 c Isolated ) 1 .PEG– | 1565 1 . © 2013 Nature America, Inc. All rights reserved. n,rgltr ucin n epne fAXIAproteins AUX/IAA of responses and functions regulatory signal- ing, auxin In elucidated. been have pathways signaling hormone by explorations. expanded high-throughput further be will with and integration years recent signifi- and in been broadened has growth cantly system TEAMP immunity, of physiology, application The plant development. in involved pathways transduction signal intracellular elucidate underlying mechanisms and molecular regulators key identify to used widely been proto- have plasts in assays and responses cell-autonomous and Physiological Applications and stability. interaction trafficking, activity,localization, protein investigate to methods be cal analyti- various using can scored and observed responses cell-autonomous is and TEAMP Physiological assays. reporter to system. of limited not the application TEAMP to the the new However, laboratories up set in help assay to controls posi- tive as serve can reporters These (ABRC). yteSenlbt h ulcsokcenter, stock public the the to lab Sheen the by FK-U-O F946C399A2110Bceilfl2 epne16 response flg22 13 Bacterial 13 response Cytokinin 5 5,13 response stress response General 12 Heat 15 response flg22 Bacterial 5,13 response response Auxin At2g19190 Ethylene promoter synthetic response box acid Abscisic 8xGCC At5g62920 19 At2g47730 At5g59720 control Transfection At4g23550 At2g23710 At5g52310 CD3-919 CD3-918 derivative 35S CD3-917 EF090416 EF090415 CD3-915 CD3-916 EF090414 pFRK1-LUC-NOS pGCC1-LUC-NOS CD3-914 EF090412 EF090413 CD3-913 pARR6-LUC-NOS CD3-912 pHSP18.2-LUC-NOS locus GenBank EF090411 pGST6-LUC-NOS EF090410 pWRKY29-LUC-NOS EF090409 CD3-911 pAtGH3-LUC-NOS pRD29A-LUC-NOS EF090408 pHBT-sGFP(S65T)-NOS Name A GTPase RAC 1566 1 TABLE ydvrepooesta a epn to respond ( signals can specific that promoters diverse ( by 1 assays gene TEAMP Table of in measurement expression for quantitative reporters as the used routinely are (GUS) maize used (5 the region untranslated with 35S C4PPDK derivative mosaic promoter cauliflower the genes, h anrgltr vn novdi nuepce cross- unexpected an signaling ethylene in and nuclear involved glucose be key event between to a talk regulatory shown been EIN3, main has of signaling, the ethylene control in stability factor transcription protein The terized. pd(e datgsadlimitations). and Advantages (see oped inducible devel- been also chemically has system expression gene a expression, gene PROTOCOL sn EM sas oeua ehnssudryn plant underlying mechanisms molecular assays, TEAMP Using Luciferase Arabidopsis 6 | (see O. NO.7 VOL.2 .Asto U eotr controlled reporters LUC of set A ). | ito sflrpre osrcsfrtasetepeso in expression transient for constructs reporter useful of List aa rmtradte5 the and promoter basal i.2a Fig. 7 10 LC and (LUC) ilgclRsuc Center Resource Biological al 1 Table n ui epnefactors response auxin and | Arabidopsis .T oto h ee of level the control To ). 2007 ¢ )havebeendonated UR nacris enhancer -UTR) | b AUEPROTOCOLS NATURE -glucuronidase eoisadgntcrsucsfor resources genetic and genomics i.2b Fig. etrsoknme rmtro neet(O)UeReferences Use (POI) interest of Promoter number stock Center Arabidopsis ¢ - 8 , 11 12 iue2 Figure evsa nitra oto.LCepeso sidcduo E ramn n hw linear shows and treatment DEX upon induced is kinetics. expression expression LUC control. internal an as serves 10 of The presence triplicates. and absence the in activation reporter ( interest of promoter the by driven construct gene ( maize the to fused interest; enhancer 355 constitutive the of control ( system. (TEAMP) protoplast U eotrgn sicue nte8x the in included is gene reporter LUC ( ATA the treatment, DEX ( domain activation transcription ( domain viral DNA-binding a bacterial a with constructed the (ATA), illustrates activator transcription picture artificial top The constructs. (DEX)-inducible dexamethasone the of representation bto itr) h T otostetasrpino N osrc hti rvnb ih oisof copies eight by driven is that construct DNA nucleus the a bacterial In of the transcription arrow). the black controls ATA the the by picture), (represented transcription (bottom gene target induce to nucleus the maize the with enhancer 35S constitutive the under driven is ATA The aebe charac- been have b infiaty the Significantly, . a c ilgclResource Biological 5PD eAGR LexA 35SPPDK 5PD GOI 35SPPDK POI NOS | omnyue lsi osrcsi h rnin xrsinin expression transient the in constructs plasmid used Commonly x8 oaiesnhs emntr ( terminator. synthase nopaline , LexA LUC LexA OP b guuoiae(U)atvt xrse rmteco-transfected the from expressed activity (GUS) -glucuronidase prtr(8x operator NOS 9 , 35STATA VP16 LexA-VP16-GR NOS Arabidopsis signaling charac- in and identified terized been mitogen-activated have conserved cascades (MAPK) evolutionarily kinase example, the protein For system. of TEAMP functions the in the studied be also can signaling hsh-ea i the via phospho-relay hw ob oetsprsoso h AKdpnetplant MAPK-dependent the of suppressors immunity AvrPtoB innate potent and be AvrPto to effectors, shown bacterium III pathogenic the type from of functions virulence h lcs esrhxkns HK)adispartners its and glucose nuclear (HXK1) HXK1-dependent 1 for signaling demonstrated hexokinase been sensor have glucose the envrfidb ueosgntcadpeoyi nlssof analyses phenotypic and genetic numerous corresponding by verified been salse stemi yoii euaoycircuitry regulatory cytokinin main the as established a ceai ersnaino euaoygn osrc ne the under construct gene regulatory a of representation Schematic ) LexA-OP oercnl,poenpoeninteractions protein–protein recently, More LUC NOS srlae rmtectpamHP0cmlxadmvsinto moves and complex HSP90 cytoplasm the from released is ) n h iiu 5 rmtr nteeapepeetd the presented, example the In promoter. 35S minimum the and ) 17 5 LexA-OP eohl rtpatassays. protoplast mesophyll VP16 hog plcto fteyattohbi concept, two-hybrid yeast the of application Through . Arabidopsis NOS n naeimn responses immune innate and n amla rt lccriodrcpo ( receptor glucocorticoid (rat) mammalian a and ) b Arabidopsis die osrc.( construct. -driven ceai ersnaino uieae( luciferase a of representation Schematic ) 16 . POI d m Arabidopsis n emntdby terminated and ) oul E.Vle r en frtoin ratio of means are Values DEX. soluble M eohl rtpat o xdtv stress oxidative for protoplasts mesophyll Relative LUC/GUS activity (+DEX/–DEX) mutants –20 100 120 C4PPDK 20 40 60 80 0 0 200 100 0 d suooa syringae Pseudomonas iecus nlsso h LUC the of analysis course Time ) w-opnn ytmhsbeen has system two-component aa promoter. basal ) 14 C4PPDK ohaitcadboi stress biotic and abiotic Both . NOS Arabidopsis aa rmtr Upon promoter. basal .( Time (min) 15 c UBQ10-GUS-NOS Schematic ) usqety the Subsequently, . GOI nvivo in mesophyll LexA-DB eeof gene , LUC 13 300 ,havebeen hc has which , reporter ) GR ). between ), 400 © 2013 Nature America, Inc. All rights reserved. ml T idn rti h1(e.2)adatnfilaments actin and 22) (ref. Rha1 protein binding GTP small o ahDAtaseto experiment transfection DNA each for inlvl npatmspylpoolss o ntne the a instance, provide to protoplasts For mesophyll in used system expression protoplasts. been gene inducible ligand has mesophyll receptor nuclear plant steroid mammalian in expres- protein levels and originally gene switches, sion control molecular to organisms, utilize other to in us identified allow plants ani- yeasts, and , mals among mechanisms regulatory and domains nte datg fteTAPsse wn oishigh protocols, its (10 protoplast protoplasts to other more owing some times system In 10–1,000 sensitivity. TEAMP is and protoplasts the of efficiency of number analysis small advantage requirement. relatively that laboratory another expression a minimal culture) for same gene requirement cell the The with mammalian for handled and be system can yeast biological There bacterial, costs. other medium, and do (including time culture experiments no in complex saving the is tremendous and transfection, measured a techniques represents DNA and which sterile after observed require h be not quantitative the 2–10 can bacteria, of within responses most e.g., As physiological systems, cells. and mammalian cell and heterologous than insect plants yeast, using to functions. relevance obtained physiological cellular data and and functional information greater provides molecular of often and protoplasts their mesophyll types mesophyll using learn in Analysis cell examined leaves. other to and in expressed in protoplasts occur ectopically expressed be that normally can activities tissues and are processes that system to Proteins TEAMP limited the not of application leavesis Importantly, in plants. observed whole responses of from the to isolated similar manner protoplasts physiological mesophyll abundant mature and Uniform limitations and Advantages scntutdwt atra N-idn oan( domain DNA-binding bacterial a with constructed is in depicted As ute ple orva eoewd eltp–pcfi transcript in type–specific profiles cell genome-wide reveal to applied been further have technique sorting isolation cell fluorescence-assisted protoplast and The method trafficking. vacuolar in studied been have of factors transcription map bZIP heterodimerization of a groups specific establish to protoplasts mesophyll in examined been also has interaction protein–protein ( oaiaino aiu rtishsbe eemnduigteGFP transport the tag using determined protein been has proteins various and of subcellularlocalization instance, targeting For trafficking. vesicle protein in functions machinery in also functions protein have localizations, proteins protein domain functional examine to to utilized fused extensively variants, been its and GFP e.g., ebaepoen7hv enietfidfrclrpattarget- chloroplast for identified been ing have of 7 protein acids membrane amino 7 C-terminal h osiuie3Sehne ue otemaize the to fused promoter enhancer 35S constitutive ( the receptor glucocorticoid ( (rat) domain activation transcription viral n oe notencest nuetre eetranscription gene target induce to nucleus the into moves and LexA-VP16-GR nteohrhn,tecnevdfntoso molecular of functions conserved the hand, other the On igecl mgn fdvrefloecnemre proteins, marker fluorescence diverse of imaging Single-cell 11,12,17–19 20 neetn euaoyrlso yai-ieAL rf 21), (ref. ADL6 dynamin-like of roles regulatory Interesting . Arabidopsis 6 pndxmtaoe(E)tetet h ATA the treatment, (DEX) dexamethasone Upon . Arabidopsis oe oe ftetasebaedmi and domain transmembrane the of roles Novel . iue2c Figure srlae rmtectpamHP0complex HSP90 cytoplasm the from released is ) evscnrsodt ies inl na in signals diverse to respond can leaves roots natfiiltasrpinatvtr(ATA) activator transcription artificial an , 24 . Arabidopsis GR .TeAAi rvnunder driven is ATA The ). 25 5 VP16 –10 . 7 18 ml ue envelope outer small r recommended are ) n mammalian a and ) . C4PPDK LexA-DB Arabidopsis basal ,a ), 26 12 23 . . h motneo rcieadeprclotmzto feach of over- optimization emphasized. be cannot empirical purpose researcher’s individual and the for practice experiment of importance results. straightforward,the and positive simple be false to appears and protocol the pitfalls though Even avoid to and mean- consistency to times ensure multiple repeated of be acquisition to need experiments the All results. ingful to contribute experience accumulated L2fnto nfl2 signaling flg22 in function FLS2 oteAR ovsal oio rtpattransfection protoplast monitor visually to donated marker ABRC We vital efficiency system. GFP the improved TEAMP an the to of using use the obtain data recommend to reproducible important very and is reliable 50%) than (greater efficiency fection oss h ellrntr hti hrceitco h original inresponse the from ethylene-insensitive lost of isolated protoplasts characteristic the mesophyll often is example, For that are plants. nature mutant which cellular the protoplasts responses, mutant possess The and cells. culture suspension features undifferentiated leaf cell maintain most protoplasts of mesophyll step Wild-type time-consuming establishment. the culture without experiments for diately manipulated most be and wild-type can proteins and genes system. inducible desired this using of expres- level the that sion indicating manner, controlled dosage–dependent also ligand is a LUC in of level linear expression shows The and kinetics. treatment expression DEX upon induced 8x is expression the LUC in included bacterial is the gene DNA of copies 2c a Fig. eight of transcription by (8x the driven operator controls is ATA that the construct nucleus, the In h attodcdsa well as decades two over developed past the been have protocols required. regeneration be protoplast will Such media culture specialized sterile and responses, conditions growth and plant plant division long-term cell other study or to this protoplasts dedifferentiation use in To described regulation. of cycle buffer study the cell incubation for suitable not simple is protocol TEAMP the this Thus, protocol. in divide not genomics do functional in powerful the makes more protoplasts research. even mesophyll diverse system genetically TEAMP of use protoplast different the the for optimize conditions transfection to dopsis DNA empirically In and pathways. necessary performing signaling isolation is commonly specific to of it are analysis similar general, which genetic is the screens, TEAMP in regulators enhancer in applied new or plants suppressor the for mutant Conceptually, mutant screen from 27). to (ref. generated assays protoplasts B1 fumonisin of toxin use induced fungal death the cell programmed by in pathways signaling hormone eedn lcs n tyeesignaling ethylene and glucose dependent eitc itntfo hs ntewl ye(see type wild the in those from distinct teristics r ayscesu xmlso sn h EM sa with assay TEAMP the using of examples successful many are aiu uatpat,icuigteaayi fAF functions ARF7 of signaling analysis auxin the in including plants, mutant various o ucsfleprmns ihlvlo rtpattrans- protoplast of level high a experiments, successful For eohl rtpat a eioae rmfehlae of leaves fresh from isolated be can protoplasts Mesophyll niepatadaia elcluelns eohl protoplasts mesophyll lines, culture cell animal and plant Unlike cesosadmtns necniin r optimized, are conditions Once mutants. and accessions .I h xml rsne i hc h U reporter LUC the which (in presented example the In ). 19 ctr1-1 (see LexA-OP uat hwclua opooyadcharac- and morphology cellular show mutants i.1d Fig. 11 h naoitcitrcinbtenHXK1- between interaction antagonistic the , Arabidopsis n h iiu 5 rmtr(see promoter 35S minimum the and ) AUEPROTOCOLS NATURE .Crflpatc,ke bevto and observation keen practice, Careful ). etr1-1 LexA-OP 28–30 . n h osiuieethylene constitutive the and uatpat n sdimme- used and plants mutant 15 die osrc;see construct; -driven n oe fvrosplant various of roles and | O. NO.7 VOL.2 12 h eetrkinase receptor the , PROTOCOL i.1c Fig. | 2007 i.2d Fig. .There ). | Arabi- LexA 1567 ), - © 2013 Nature America, Inc. All rights reserved...... EQUIPMENT ln growth Plant PROCEDURE REAGENTS MATERIALS 1568 eaieysotpooeid(01 ih t23 at light h (10–13 photoperiod short relatively a 1| rtpatisolation Protoplast a possible. maintain to as Try much perturbation). mechanical as constant environment and constant temperature extreme flooding, drought, (e.g., changes environmental of utn ie odpeaainyed prxmtl 10 approximately yields preparation good A site. cutting the in problem a 3| be pathways. also signaling can hormonal low status experienced and cellular often defense stress–induced stress, have High of leaves. we However, stressed study leaves. from healthy protoplasts from the those with efficiency and to transfection temperature similar extreme look flooding, drought, may (e.g., perturbation) conditions mechanical stress constant from recovered leaves from prepared Protoplasts experiments. m (see 2| m humidity. relative DCA;Sga a.n.D-1383) no. cat. Sigma, (DACTAA; svr rtclt civ ihtaseto efficiency. transfection high achieve to critical very is ec-o etiue(E etaC2 nentoa qimn Company) Equipment 0.45- International CL2; Centra (IEC centrifuge Bench-top Hoefer) (TK100; Fluorometer Pharmigen) (Monolite3010; Luminometer Leica) DM5000; (Leica microscope Fluorescence SETUP) REAGENT (see assay Ampicillin GUS for mix substrate MUG SETUP) REAGENT (see buffer Protoplast SETUP) REAGENT (see solution transfection PEG–calcium SETUP) REAGENT (see solution MMG SETUP) REAGENT (see SETUP) solution REAGENT W5 (see solution (WI) incubation and Washing SETUP) REAGENT (see solution Arabidopsis Canada) Ltd., Products (Jiffy Jiffy7 Inc.) Horticulture, Gro 69580) (Sun no. 360 Metro-Mix cat. Fluka, (MU; methylumbelliferone M 1 Na M 0.2 MUG-1G) no. BioTechnology, Inc., cat. Gold (MUG; glucuronide 4-methylumbelliferyl a M using 0.1 sterilize T8650), no. cat. Biological, 0.45- (US 8.0) (pH Tris–HCl M E1501) 1 no. cat. (Promega, system D-9779) assay no. LUC cat. (Sigma, DTT T-8787) M no. 1 cat. 0.45- (Sigma, a X-100 using Triton sterilize (vol/vol) 15892), 20% no. cat. (Fisher, glycerol (vol/vol) 50% 0.45- a mM using 100 sterilize 7.8), (pH Tris–phosphate M 1 81240) no. cat. (Fluka, PEG4000 Japan) m Ltd., Co., Ind. Pharmaceutical (Yakult Japan) R10 Macerozyme Ltd., 0.45- Co., a Ind. using Pharmaceutical (Yakult sterilize A-6793), R10 Cellulase no. cat. (Sigma, BSA (wt/vol) 10% b 0.45- a using MgCl sterilize M P3911), no. 2 cat. (Sigma, KCl M 2 0.45- a using sterilize no.M4125), 1MCaCl cat. (Sigma, mannitol M 0.45- 0.8 a using sterilize M8250), Sigma, no. 5.7; cat. pH (MES, acid 4-morpholineethanesulfonic M 0.2 PROTOCOL Mratehnl(im,ct o M6250) no. cat. (Sigma, -Mercaptoethanol RTCLSTEP CRITICAL RTCLSTEP CRITICAL CRITICAL m m Grow u .–-mla tisfo h idepr fala sn rs hr ao ld ihu isecuhn tthe at crushing tissue without blade razor sharp fresh a using leaf a of part middle the from strips leaf 0.5–1-mm Cut flowering before seven) to five numbers leaf true 1a (usually Fig. plants 3–4-week-old from leaves well-expanded Choose | yig trlzto le Wamn a.n.6870-2504) no. cat. (Whatman, filter sterilization syringe m mfilter O. NO.7 VOL.2 trans 2 2 CO 2 Sga a.n.C92,seiieuiga0.45- a using sterilize C7902), no. cat. (Sigma, cesos o- n L and Col-0 accessions: Sga a.n.M22,seiieuiga0.45- a using sterilize M9272), no. cat. (Sigma, Arabidopsis ehv bandtebs eut ihti nyeprovider. enzyme this with results best the obtained have We 3 ). -1,2-diaminocyclo-hexane- Sga a.n.S7795) no. cat. (Sigma,  | TIMING 2007 o- n L and Col-0 h eeto fhatylae ttepoe eeomna tg savr motn atri protoplast in factor important very a is stage developmental proper the at leaves healthy of selection The  lnso ihrMtoMx30o if7si nagenos ra niomn-otoldcabrwith chamber environment-controlled an or greenhouse a in soil Jiffy7 or 360 Metro-Mix either on plants | TIMING AUEPROTOCOLS NATURE – weeks 3–4 m er er CRITICAL (ABRC) m aebe xesvl etdi u a.I general, In lab. our in tested extensively been have mfilter – h 4–5 N,N,N efudta h E source PEG the that found We ¢ ,N ¢ ttactcacid -tetraacetic m mfilter m mfilter m m mfilter mfilter m 1 mfilter /11 aka 20 at dark h C/11–14 m mfilter m mfilter 7 rtpat e rmfehwih apoiaey1010leaves 100–150 (approximately weight fresh gram per protoplasts ...... a esoe t–20 at stored be can 0m C.Tepeae Islto a esoe tro temperature room at stored be can solution WI prepared The KCl. mM 20 solution WI fresh. prepared (22–25 5m MgCl mM 15 solution MMG temperature. ddH solution transfection PEG–calcium nac nyeslblt.Co tt omtmeaue(25 temperature room to it Cool solubility. enzyme enhance 55 KCl. mM at 20 solution and the mannitol Warm M 0.4 R10, macerozyme (wt/vol) 0.4% R10, cellulase solution Enzyme SETUP REAGENT o 0m nyesolution). enzyme ml 10 for CaCl solution W5 otiig1m U n MMgCl mM 2 and MUG mM 1 assaycontaining GUS for fresh. mix Triton prepared substrate (vol/vol) be MUG 1% should and buffer glycerol lysis (vol/vol) The 10% DACTAA, X-100. mM 2 DTT, mM 1 buffer lysis Protoplast solution. PEG autoclave transfection not protoplast Do better efficiency. However, relatively d. gives 5 solution within PEG The used prepared and PEG. temperature freshly dissolve room completely at stored to transfection be before can solution h PEG 1 least at solution PEG h nlezm ouinsol ecerlgtbon itrtefia enzyme final the Filter brown. light 0.45- clear a through solution be 70 should at solution preheated enzyme is final solution The MES the added, is powder enzyme purpose. experimental the to according determined mM 1–5 of Addition CaCl a.n.25389-000) no. cat. -elclueds Flo,ct o 3046) no. cat. (Falcon, dish culture 6-well 1620-2700) no. Scientific, cat. USA (SealRite; 55.517) tube no. microcentrifuge natural cat. round-bottomed 2-ml (Sarstedt, tube round-bottomed 30-ml 1483) no. cat. Scientific, (Hausser hemacytometer Reichert 0.1-mm-deep Neubauer Improved 65-2222N) no. cat. (75 mesh Nylon (100 55411-055) no. dish cat. Scientific, Petri VWR blade; (single-edged blade razor Generic 2 2 2 otiig02Mmnio n 0 MCaCl mM 100 and mannitol M 0.2 containing O ,1–5mM n MKl h rprdW ouincnb trda room at stored be can solution W5 prepared The KCl. mM 5 and 1 C). 1 )udrlwlgt(50–75 light low under C) 2 h rprdMGslto a esoe tro temperature. room at stored be can solution MMG prepared The . rpr MMS(H57 otiig14m al 2 mM 125 NaCl, mM 154 containing 5.7) (pH MES mM 2 Prepare rpr MMS(H57 otiig05Mmnio and mannitol M 0.5 containing 5.7) (pH MES mM 4 Prepare  b rpr MMS(H57 otiig04Mmnio and mannitol M 0.4 containing 5.7) (pH MES mM 4 Prepare m mratehnl(pinl n .%BSA. 0.1% and (optional) -mercaptoethanol 5mm 25 ,lbrtr itr,Crln ilgclSupplies, Biological Carolina sifters, laboratory m, rpr 0m E p .)cnann .%(wt/vol) 1.5% containing 5.7) (pH MES mM 20 Prepare Arabidopsis 1 b C. mratehnli pinl n t s hudbe should use its and optional, is -mercaptoethanol m 1 rpr 5m rspopae(H78 containing 7.8) (pH Tris–phosphate mM 25 Prepare yig le eieit er ih(100 dish Petri a into device filter syringe m o 0mnt nciaeDAeadpoessand proteases and DNAse inactivate to min 10 for C 2 o 0m nyeslto;VRScientific, VWR solution; enzyme ml 10 for m CRITICAL lnsaevr estv oalkinds all to sensitive very are plants rpr 0m rsHl(H8) (pH Tris–HCl mM 10 Prepare rpr 04%(tvl E40 in PEG4000 (wt/vol) 20–40% Prepare h nyeslto hudbe should solution enzyme The 2 h rprdGSasysubstrate assay GUS prepared The . m Em –2 m s 2 CRITICAL . –1 m n 40–65% and ) 1 CRITICAL )adad1 mM 10 add and C) 1 m o – min. 3–5 for C eoethe Before CRITICAL  Prepare 5mm 25 2 © 2013 Nature America, Inc. All rights reserved. eayoee.Rs h rtpat ykeigo c o 0min. 30 for ice on m keeping by protoplasts the Rest hemacytometer. ietdi 06 lo nyeslto) o otn xeiet,1–0lae ietdi –0m nyeslto ilgive will solution enzyme ml 5–10 in digested leaves 10–20 experiments, routine For solution). enzyme 0.5–1 of ml 40–60 in digested transfection). 15| osbewtottuhn h rtpatple.R-upn rtpat t2 at protoplasts Re-suspend pellet. protoplast the touching without possible and transduction signal regulation, expression gene 12| of study the for used localization. be and should processing protoplasts trafficking, prepared protein freshly h, 24 least at N-E–acu transfection DNA-PEG–calcium re fe etesiln oin hc niae h ees fprotoplasts. of release the indicates which motion, m swirling gentle a after green 6| significantly. decreased is yield protoplast and penetrate cannot 5| solution enzyme the cutting, during paper the forceps.on flat-tip of pair a using m submerged) (completely strips the of 4| stain). green dark and (juicy tissue (8" m m 13| prxmtl 30–50 approximately other in recommended 7| processes regeneration and dedifferentiation the for important be protocols. each might protoplast responses physiological for stress eliminate optimized might the and However, be stressful cells. is protoplast to dark leaf the the has However, of in time Col-0. h) digesting (16–18 of leaves The case of genotypes. incubation Prolonged in genotype. and and strips ecotypes ecotype leaf different from for released significantly are differs protoplasts efficiency isolation most digestion, h 3 After empirically. optimized h rprto fpamdDAuigteeooia slgain spoie nteSenlbwbie(http://genetics.mgh. website lab Sheen 14| the on provided on information is Additional gradient efficiency. transfection CsCl effector economical high any support the express harvard.edu/sheenweb/protocols.html). using to not appropriately DNA does purified plasmid that plasmid be of control preparation to a the has with ratio compensated DNA plasmid is plasmid the amount The optimize reporter. plasmid DNA the plasmid to or Whenever recommended total amount. highly same the half the is only changed, it needs is However, reporter sensitive amount 5:4:1. highly a of example, ratio For sensitivity. reporter’s the each at on depending used are reporter control transfection a 11| swirling. ? gentle by pellet protoplast the re-suspend m and possible as supernatant much 10| 75- the wetting after protoplasts containing solution solution. enzyme W5 the with Filter filtration. protoplast before water excess 9| 8| tips. pipette and pipettes regular with them m TROUBLESHOOTING RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL  otnetedgsin ihu hkn,i h akfra es tro eprtr.Teezm ouinsol turn should solution enzyme The temperature. room at h 3 least at for dark the in desiccator. shaking, a without digestion, using dark the Continue the in min 30 for strips leaf infiltrate Vacuum sides both dipping by ml) 5–10 in leaves (10–20 solution enzyme prepared the into gently and quickly strips leaf Transfer hc o h ees fpoolssi h ouinudrtemcocp;tesz of size the microscope; the under solution the in protoplasts of release the for Check iueteezm/rtpatslto iha qa oueo 5slto eoefitaint eoeudgse eftissues. leaf undigested remove to filtration before solution W5 of volume equal an with solution enzyme/protoplast the Dilute ahacen75- clean a Wash d 110 Add rtpat hudbgnt etea h otmo h ueb rvt fe 5mn eoeteW ouina uhas much as solution W5 the Remove min. 15 after gravity by tube the of bottom the at settle to begin should Protoplasts d 10 Add d 100 Add essedpoolssa 2 at protoplasts Re-suspend 100 at flow-through the Centrifuge 1)o o ftesldadcenlbrtr ec,wihpoie o odspotades npcino wounded/crushed of inspection easy and support good for provides which bench, laboratory clean and solid the of top on 11")  10 6 m rtpat,eog o oeta 510smls(1–2 samples 25–100 than more for enough protoplasts, m m N (10–20 DNA l fPGslto,adte i opeeyb etytpigtetb hnl i otnsmlsfreach for samples ten to six (handle tube the tapping gently by completely mix then and solution, PEG of l lprotoplasts(2 meit ipn n umrigo efsrp svr rtclfrpools il.We efsrp r re out dried are strips leaf When yield. protoplast for critical very is strips leaf of submerging and dipping Immediate fcl epnei eie,ke rtpat tro eprtr.Atog h rtpat a ekp niefor ice on kept be can protoplasts the Although temperature. room at protoplasts keep desired, is response cold If osuyterl facniaergltr he lsisepesn euaoyefco,aseicrpre and reporter specific a effector, regulatory a expressing plasmids three regulator, candidate a of role the study To ti o eesr orlaealtepoolssfo efsrp.B etewt rtpat,btyucnhandle can you but protoplasts, with gentle Be strips. leaf from protoplasts the all release to necessary not is It ihrsed(200 speed higher A ieto iedpnso h xeietlgas eial epne n aeil sd n tnest be to needs it and used, materials and responses desirable goals, experimental the on depends time Digestion hnetebaeatrctigfu ofielae.W otnl u evso ic fcenwiepaper white clean of piece a on leaves cut routinely We leaves. five to four cutting after blade the Change m m m. yo ehwt ae ormv tao tems snral eti 5 tao)te remove then ethanol) 95% in kept normally is mesh (the ethanol remove to water with mesh nylon m m fpamdDAo –0k nsz)t -lmcouetube. microfuge 2-ml a to size) in kb 5–10 of DNA plasmid of g    10 TIMING 10 4 rtpat)admxgently. mix and protoplasts) 5 g g ml fcnrfgto a ept nraepools recovery. protoplast increase to help may centrifugation of ) ople h rtpat na3-lrudbtoe uefr12mn eoeas Remove min. 1–2 for tube round-bottomed 30-ml a in protoplasts the pellet to –1 esta o 0samples 20 for h 1 than Less nW ouinatrcutn el ne h irsoe( microscope the under cells counting after solution W5 in  10  4 rtpat e sample). per protoplasts 10 5 ml –1 nMGslto eta omtemperature. room at kept solution MMG in AUEPROTOCOLS NATURE Arabidopsis eohl rtpat is protoplasts mesophyll  | 0)uiga using 100) O. NO.7 VOL.2 m PROTOCOL yo mesh nylon m | 2007 | 1569 © 2013 Nature America, Inc. All rights reserved. rti aeig h EM ytmi otsial o h td feryeet nsga rndcinptwy,gn regulation gene m pathways, transduction signal in events early m of study the death. for cell suitable most and is system TEAMP The labeling. protein rtpat r ufiin o eotrezm sas 10 assays, enzyme reporter for sufficient are protoplasts 18| process. transfection the stop to tube the inverting 17| m 16| 1570 ytei g2ppiedrvdfo atracnb de o 0mno o h nlsso rti iaeatvt rearly or activity kinase protein of analysis the for m h transfection. DNA 1 ( after or system min min expression 15–30 10 gene inducible for the added For be respectively. responses, can transcription bacteria from derived peptide flg22 synthetic C F assay GFP (C) rtpatclueadharvest and culture Protoplast to examined systematically be should number ? protoplast and DNA condition. plasmid optimal of ratio the the or find DNA plasmid of quality the 20%), than (less rtpat hudrmi nattruhu h slto,taseto,clueadhretn procedures. harvesting and culture transfection, to isolation, used m the throughout be intact can remain GFP should as protoplasts such ( reporters conditions transfection Visual purpose. DNA experimental optimal each determine for empirically determined be should concentration the of sticking prevent m protoplast to during stress s) hypoxia prevent (1–2 to time m mm short 0.1 approximately a is for solution WI serum the incubation. calf of sterile depth The (vol/vol) surface. plastic 5% the with to protoplasts coated normally is plate culture The m 19| etr ltaayi n 10 and analysis blot western A U assay LUC (A) 23| m 20| ’ 22| concern. a is growth 21| bacterial if transfection DNA after incubation protoplast the during added eotrassays Reporter B U assay GUS (B) PROTOCOL ii esr h ursec fM sn fluorometer. a using MU of fluorescence the Measure (iii) ii s 5–20 Use (iii) TROUBLESHOOTING i)Atr5mnicbto nie etiuea 1,000 at centrifuge ice, on incubation min 5 After (ii) i)Ad09m . Na M 0.2 ml 0.9 Add (ii) RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL RTCLSTEP CRITICAL STEP CRITICAL STEP CRITICAL RTCLSTEP CRITICAL i iwlv ellriaeo F rGPfso rtis(sn rtpat bandi tp2)udrfloecnemicro- fluorescence under 22) Step in obtained protoplasts (using proteins fusion GFP or GFP of image cellular live View (i) i d 100 Add (i) i d 10 Add (i) AS POINT PAUSE etiuea 100 at Centrifuge 400–440 with mixture sufficient). transfection is the min Dilute (5 min 15 to up for temperature room at mixture transfection the Incubate essedpoolssgnl ih1m Ii ahwl fa6wl iseclueplate. culture tissue 6-well a of well each in WI ml 1 with gently protoplasts Re-suspend eea sascnb are u sn pinA(U sa) GSasy rC(F assay). (GFP C or assay) (GUS B assay), (LUC A option using out carried be can assays Several nuaepoolssa omtmeaue(20–25 temperature room at protoplasts Incubate eoetespraatadfez ape ndyice. dry on samples freeze and supernatant the Remove 100 at centrifugation by protoplasts harvest and Re-suspend | m cp ( scopy plasts. t37 at O. NO.7 VOL.2 RTCLSTEP CRITICAL 1 C.  m m ftepools yaefo tp2Ai)it 100 into 23A(ii) Step from lysate protoplast the of l 0–0)wtotayadtoa apepeaainsteps. preparation sample additional any without 400–600) m fpools yi ufrt h rznpoolssadmxvgrul yvreigfr2st utr h proto- the rupture to s 2 for vortexing by vigorously mix and protoplasts frozen the to buffer lysis protoplast of l |  ftelst n 100 and lysate the of l ape a esoe t–80 at stored be can Samples 2007 ngnrl h rtpaticbto iei – o N nlssad21 o nyeatvt nlssand analysis activity enzyme for h 2–16 and analysis RNA for h 2–6 is time incubation protoplast the general, In eas s 2wl 05m I r2-el(.5m I ltsfrlre-cl xeiet 5–0 samples). (50–100 experiments larger-scale for plates WI) ml (0.25 24-well or WI) ml (0.5 12-well use also We tmlscnb ple uigteicbto codn oteeprmna upss o xml,the example, For purposes. experimental the to according incubation the during applied be can stimulus A ti o eesr opromeprmnsudrseiecniin ngnrl miiln(50 Ampicillin general. in conditions sterile under experiments perform to necessary not is It h rtpaticbto eprtr a ob piie codn oeprmna needs. experimental to according optimized be to has temperature incubation protoplast The rnfcintm hudb piie empirically. optimized be should time Transfection rtpattaseto a esae po ondpnigo h xeietlnes prxmtl 10 Approximately needs. experimental the on depending down or up scaled be can transfection Protoplast PEG optimal The concentration. final delivery. PEG DNA 10–20% of using type achieved this be for can unnecessary efficiency transfection is High DNA carrier of use The TIMING g | o i tro eprtr sn ec-o etiueadrmv supernatant. remove and centrifuge bench-top a using temperature room at min 2 for AUEPROTOCOLS NATURE ute iuinwt . Na M 0.2 with dilution Further 2 CO 6 – h 1–2 rtpat o N xrcinadmcora nlss ftepools rnfcinefiinyi low is efficiency transfection protoplast the If analysis. microarray and extraction RNA for protoplasts 3 oso h reaction. the stop to  TIMING m U i Poea omaueLCatvt ihaluminometer. a with activity LUC measure to (Promega) mix LUC l –4h 2–24 1 i.1d Fig. ni ute analysis. further until C m 5slto tro eprtr n i elb etyrcigor rocking gently by well mix and temperature room at solution W5 l 4–5 2 1 , CO g )frtedsrdpro ftime. of period desired the for C) al 1 Table rtpat r eurdfrpoenlbln n immunoprecipitation or and labeling protein for required are protoplasts o min. 2 for 3 o U sa hudb eemndempirically. determined be should assay GUS for .I rtpat r eie rmhatyla aeil,most materials, leaf healthy from derived are protoplasts If ). g o min. 2 for m fMGsbtaemxadicbt o 010min 30–180 for incubate and mix substrate MUG of l i.2c Fig. , d ,DXcnb de oteprotoplasts the to added be can DEX ), m gml –1 a be can ) 3–4 © 2013 Nature America, Inc. All rights reserved. rtpat.Teseicrpre edu au hudb omlzdwt hto otasetdcnrlrpre rvnb a by driven reporter control co-transfected a of that with normalized be e.g., promoter, should constitutive value readout reporter specific The protoplasts. rbe osberao Solution reason Possible yield protoplast Low Problem ucino nefco sn h edu faseicrpre ncl-uooosrsoss h feto euaoycandidates regulatory of regulatory effect the The determine responses. cell-autonomous ( to reporter in is specific reporter assay specific a TEAMP with a the transfected of of are readout use that the typical using A effector analysis. an reliable of a function ( for general efficiency transfection in protocol 50% this than higher with 60–90% approximately is efficiency transfection Protoplast RESULTSANTICIPATED in found be can advice Troubleshooting ? h 1–2 approximately 23: transfected) Step being samples h of 2–24 approximately numbers 20–22: on Steps (depending h 1 approximately 13–19: collected) on Steps (depending being leaves h of 3–4 approximately numbers 5–12: on (depending Steps h 1 approximately 2–4: Steps weeks 3–4 1: Step .Jcbe,JV ec,LR oto ftasrpinof transcription of Control L.R. Beach, & J.V. Jacobsen, vacuoles. 3. and protoplasts plant of isolation the for method A E.C. Cocking, 2. and maize in transduction Signal J. Sheen, 1. http://npg.nature.com/ at online available reprintsandpermissions is information permissions and Rights http://www.natureprotocols.com at online Published interests. STATEMENT INTERESTS COMPETING J.S. to Health GM060493) of (R01 Institutes National (IOB-0217191, has Foundation and work IOB-0618292) Science National This and DBI-0077692 the system. from TEAMP grants the improve by supported and been test to efforts their for laboratory ACKNOWLEDGMENTS 2 TABLE  fcec o-ult N eueteeooia slgain ehdfrpamdDAprfiain h detailed The purification. DNA plasmid for method gradient CsCl economical the use We solution PEG autoclave not Do storage. and sources on depends quality PEG DNA Low-quality problem solution PEG efficiency transfection Low TROUBLESHOOTING TIMING 7–7 (1985). acid. 275–277 abscisic and gibberellin by protoplasts aleurone barley in genes 187 Physiol. Plant 2–2 (1960). 927–929 , | rulsotn table. Troubleshooting 127 4617 (2001). 1466–1475 , etakfre n urn ebr fteSheen the of members current and former thank We tml plcto eawy etadotmz h epnetmn n nlz ra ocnrtosof concentrations broad analyze and timing response the optimize and test always We incubation and response resting, protoplast for time the and empirically conditions determined Optimize be should Ratio application Stimuli quality protoplast Poor ratio Protoplast/DNA ( morphology and development age, leaf Check treatment) heat and quality (enzyme solution enzyme of activity Check had have We soil. and conditions) storage and digestion (growth enzyme Inefficient seeds of quality the condition Check leaf Poor growth plant Poor Accession h uhr elr ocmeigfinancial competing no declare authors The UBQ10-GUS-NOS Arabidopsis al 2 Table 8 eohl protoplasts. mesophyll . a al 1 Table ayaeadrRNA and -amylase . Arabidopsis inldsg ocvrawd ag hnanwsga sapidfrtefis time first the for applied is signal new a when range wide a cover to dosage signal website lab Sheen the on available is protocol mutant and accession each for time digestion leaf Optimize nutrients and intensity, watering light photoperiod, humidity, temperature, conditions: growth from optimize vary and Check may soil of batch quality to The occasionally. batch soil of types different with problems isolation protoplast in better perform h) (10–13 L and Col used, are bolting Arabidopsis a eqatfidwti –2hatrtaseto fmesophyll of transfection after h 2–12 within quantified be can ) Nature Nature 316 ceso n mutants) and accession rwhcniini eedn nteacsinue.A evsbefore leaves As used. accession the on dependent is condition growth , 0 a,L,Cen,AY,Nbu .&W,H A Tae ntbcoand tobacco in GTPases RAC H. Wu, & C. Nibau, A.Y., Cheung, L., Tao, 10. C.K. Worley, 9. gene light-inducible for required is activity phosphatase Protein J. Sheen, 6. oxidative of analysis Functional J. Sheen, & G. Tena, W.L., Chiu, Y., Kovtun, 5. .Nri,SR,Myr ..&Cli,J h nrnof intron The J. Callis, & S.E. Meyer, S.R., Norris, 8. in analysis expression Transient V. Walbot, & J.R. Wet, de K.R., Luehrsen, 7. electrogenic activates light Blue J.I. Schroeder, & L. Simoncini, S.M., Assmann, 4. htcnanAXIAproteins. AUX/IAA contain that bodies protein nuclear active proteolytically of formation auxin-induced mediate signalling. (1993). quantitative expression. a gene chimeric is of and determinant location in conserved is genes polyubiquitin maize. in expression USA Sci. Acad. plants. in cascade kinase protein mitogen-activated stress-activated (1985). (1992). gene. reporter luciferase firefly using plants of protoplasts cell guard in pumping ion er lnsgonudrarltvl hre photoperiod shorter relatively a under grown plants ln J. Plant tal. et 97 9024 (2000). 2940–2945 , erdto fAxIApoen sesnilfrnra auxin normal for essential is proteins Aux/IAA of Degradation 21 MOJ. EMBO 5–6 (2000). 553–563 , AUEPROTOCOLS NATURE i.1d Fig. 12 i.1a Fig. ln Cell Plant 4730 (1993). 3497–3505 , .I sncsayt have to necessary is It ). ) ii faba Vicia ln o.Biol. Mol. Plant 17 ehd Enzymol. Methods 3928 (2005). 2369–2383 , | rbdpi thaliana Arabidopsis O. NO.7 VOL.2 . Nature 21 PROTOCOL 318 895–906 , 216 | ,285–287 2007 397–414 , Arabidopsis rc Natl. Proc. | 1571 © 2013 Nature America, Inc. All rights reserved. 4 ule,B he,J. Sheen, & B. Mueller, 14. 0 e,Y,Km . i,Y wn,I dnicto fasga htdistinguishes that signal a of Identification I. Hwang, & Y. Kim, D., Kim, Y., Lee, 20. W. Chiu, 19. A. hexokinase1 Ehlert, nuclear 18. of functions Regulatory J. Sheen, & S.-D. Yoo, Y.-H., Cho, 17. P. He, 16. T. Asai, 15. in circuitry Two-component J. Sheen, & I. Hwang, 13. restores factor7 response Auxin T.J. Guilfoyle, & G. Hagen, S.B., Tiwari, S., Wang, 11. 1572 2 aaiaa . o,S-.&Sen .Dfeeta euaino EIN3 of regulation Differential J. Sheen, & S.-D. Yoo, S., Yanagisawa, 12. PROTOCOL ewe h hools ue neoemmrn n h endomembrane the and system membrane envelope outer chloroplast the between (1996). S group and C factors. transcription group bZIP of map heterodimerization a of establishment protoplasts: signalling. glucose in complex in MAPKKK Nature (2007). http://stke.sciencemag.org/cgi/cm/stkmcm;CMP_10021 press), the in Pathways, Map (Connections transduction. (2003). mutant protoplasts. in genes auxin-responsive of expression the tblt yguoeadehln inligi plants. in signalling ethylene and glucose by stability | O. NO.7 VOL.2 tal. et 415 nvivo in tal. et tal. et tal. et 7–8 (2002). 977–983 , Arabidopsis pcfi atra upesr fMM inligusra of upstream signalling MAMP of suppressors bacterial Specific ln Cell Plant A iaesgaln acd in cascade signalling kinase MAP Nature . nierdGPa ia eotri plants. in reporter vital a as GFP Engineered ln Cell Plant w-yrdpoenpoenitrcinaayi in analysis interaction protein-protein Two-hybrid | 2007 413 17 Arabidopsis naeimmunity. innate 9919 (2005). 1979–1993 , 8–8 (2001). 383–389 , 13 | ln J. Plant 1529 (2001). 2175–2190 , AUEPROTOCOLS NATURE Cell 127 yoii inligpathway. signalling cytokinin 46 9–0 (2006). 890–900 , 7–8 (2006). 579–589 , Cell 125 Arabidopsis 6–7 (2006). 563–575 , Arabidopsis Arabidopsis Nature ur Biol. Curr. naeimmunity. innate 425 yoii signal cytokinin efmesophyll leaf c.STKE Sci. 521–525 , Arabidopsis 6 325–330 , 2007 4 inam K. in Birnbaum, role 24. critical a play filaments Actin I. Hwang, & S. Kim, M., Park, H., Kim, 23. E.J. Sohn, 22. 1 i,J.B Jin, 21. 8 ec,AR atn .Lresaepools slto n eeeainof regeneration and isolation protoplast Large-scale L. Marton, & A.R. Wenck, 28. sucrose nine of Identification C. Lauriere, & H. Barbier-Brygoo, M., Boudsocq, 25. 0 asn .&Pskwk,J utr epneof response Culture J. Paszkowski, & J. Masson, 30. 29.Damm,B.,Schmidt,R.&Willmitzer,L.Efficienttransformationof T. Asai, 27. inactivation regulable and movable A K.R. Yamamoto, & S.J. Salser, D., Picard, 26. aulrtafikn tteGlicmlxi ln cells. plant in (2005). complex Golgi the at trafficking vacuolar proteins. cargo soluble of trafficking vacuolar (2001). 1511–1526 rmtetasGlintokt h eta aul in vacuole central the to network trans-Golgi the from rbdpi thaliana Arabidopsis 12 pathways. signaling salicylate-dependent and ethylene-, jasmonate-, saline and in hyperosmotic stresses by activated 2 kinases protein 1-related nonfermenting (2003). 1956–1960 sdtrie ygot odtoso oo plants. donor of conditions growth by determined is thaliana receptor. glucocorticoid the of 54 domain binding steroid the within function 8313 (2000). 1823–1835 , 0318 (1988). 1073–1080 , tal. et sn ietgn rnfrt protoplasts. to transfer gene direct using tal. et tal. et rbdpi thaliana Arabidopsis uoii 1idcdcl et in death cell B1-induced Fumonisin tal. et e yai-iepoen D6 sivle ntrafficking in involved is ADL6, protein, dynamin-like new A h1 an Rha1, eeepeso a fthe of map expression gene A . Biotechniques Arabidopsis . .Bo.Chem. Biol. J. 18 a5hmlg ly rtclrl nthe in role critical a plays homolog, Rab5 4–4 (1995). 640–643 , ln Cell Plant o.Gn Genet. Gen. Mol. 279 rbdpi thaliana Arabidopsis Arabidopsis Arabidopsis 15–16 (2004). 41758–41766 , ln J. Plant Arabidopsis ln Cell Plant 15 0717 (2003). 1057–1070 , 2 rtpat requires protoplasts root. 2–3 (1992). 829–833 , 217 Arabidopsis . 17 –2(1989). 6–12 , ln Cell Plant Science 888–902 , protoplasts ln Cell Plant 302 Cell 13 , , CORRIGENDA Corrigendum: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis Sang-Dong Yoo, Young-Hee Cho & Jen Sheen Nat. Protoc. 2, 1565–1572 (2007); doi:10.1038/nprot.2007.199; published online 21 June 2007; corrected after print 11 October 2013 In the version of this article initially published, the concentration given for 2.5 mM Tris-phosphate (pH 7.8) when making up protoplast lysis buffer was incorrect. The correct concentration should be 25 mM. The error has been corrected in the HTML and PDF versions of the article.

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