Fire

Breakthrough Technologies

Fire?y Luciferase Complementation Imaging Assay

for Protein-Protein Interactions in Plants1[C][W][OA]

Huamin Chen,Yan Zou,Yulei Shang,Huiqiong Lin,Yujing Wang,Run Cai,

Xiaoyan Tang,and Jian-Min Zhou*

School of Agriculture and Biology,Shanghai Jiaotong University,Shanghai20040,China(H.C.,R.C.);National Institute of Biological Sciences,Beijing102206,China(H.C.,Y.Z.,Y.S.,H.L.,Y.W.,J.-M.Z.);and Department of Plant Pathology,Kansas State University,Manhattan,Kansas66506(X.T.)

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions.The previously described techniques,?uorescence resonance energy transfer and bimolec-ular?uorescence complementation,which are used widely for protein-protein interaction studies in plants,require extensive instrumentation.To facilitate protein-protein interaction studies in plants,we adopted the luciferase complementation imaging assay.The amino-terminal and carboxyl-terminal halves of the?re?y luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins,and that can be visualized with a low-light imaging system.A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants.We tested nine pairs of proteins known to interact in plants,including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant,proteins of the SKP1-Cullin-F-box protein E3ligase complex,the HSP90chaperone complex,components of disease resistance protein complex,and transcription factors.In each case,strong luciferase complementation was observed for positive interactions.Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity.Thus,the assay is simple,reliable,and quantitative in detection of protein-protein interactions in plants.

Noncovalent interactions among proteins are vital for all aspects of cellular processes.Thus,the identi-?cation and characterization of interacting proteins are key to our understanding of protein functions.A plethora of techniques have been developed to detect protein-protein interactions in vitro and in vivo(Piehler, 2005).The most widely used among these techniques is the yeast two-hybrid assay,which is ideal for large-scale screening for interacting proteins and the con-struction of protein interactomes(Fields and Song, 1989;Li et al.,2004).However,the yeast two-hybrid assay detects protein-protein interactions under het-erologous conditions,and results must be validated by assays under physiological conditions.Examination of protein-protein interactions under physiological con-ditions is often technically demanding and requires tedious procedures.For example,the co-immunopre-cipitation assay requires speci?c antibodies;lengthy procedures that are in?uenced by parameters such as schemes for protein extraction,binding,and washing; and expertise of individuals performing the experiment. Thus,the results are often variable from laboratory to laboratory.Tandem af?nity puri?cation represents a more advanced technique primarily designed to iden-tify new proteins in a protein complex in a native state (Puig et al.,2001;Rohila et al.,2006).

The development of reporter-based in vivo protein-protein interaction assays,such as?uorescence reso-nance energy transfer(FRET;Ha et al.,1996;Heim and Tsien,1996;Mahajan et al.,1998),the related technol-ogy bioluminescence resonance energy transfer(BRET; Xu et al.,1999;Subramanian et al.,2006),and bimo-lecular?uorescence complementation(BiFC;Hu et al., 2002)assays,has signi?cantly advanced the measure-ment of protein-protein interactions in vivo.These assays are instrumental for a number of important dis-coveries in mammalian studies.The application of FRET and BRET in plant biology,however,has encoun-tered signi?cant dif?culties despite sporadic successes (Shen et al.,2007).Both assays require sophisticated microscopy and computation.BiFC is relatively simple compared to FRET and BRET and has been used in a number of plant protein-protein interaction studies (Bracha-Drori et al.,2004;Walter et al.,2004;Dong et al.,2006;Quan et al.,2007).FRET and BiFC are technically challenging when a large number of pro-tein pairs are to be tested.Furthermore,the application of FRET and BiFC assays in plants is complicated by the auto?uorescence generated by cell wall,chloro-plast,and other cell structures.Finally,photobleaching

1This work was supported by the Chinese Ministry of Science and Technology(grant no.2003–AA210080to J.-M.Z.).

*Corresponding author;e-mail zhoujianmin@https://www.wendangku.net/doc/7b2867507.html,.

The author responsible for distribution of materials integral to the ?ndings presented in this article in accordance with the policy described in the Instructions for Authors(https://www.wendangku.net/doc/7b2867507.html,)is: Jian-Min Zhou(zhoujianmin@https://www.wendangku.net/doc/7b2867507.html,).

[C]Some?gures in this article are displayed in color online but in black and white in print.

[W]The online version of this article contains Web-only data.

[OA]Open Access article can be viewed online without a sub-scription.

https://www.wendangku.net/doc/7b2867507.html,/cgi/doi/10.1104/pp.107.111740

and phototoxicity caused by the external light source for excitation of?uorescence also restrict the applica-tion of the reporter-based assays in plants(Dixit et al., 2006).

Alternative reporter-based methods for protein-protein interactions have been developed using protein fragment complementation coupled with enzymatic assays.For example,expression of b-galactosidase fragments fused to interacting proteins reconstitutes the enzymatic activity in Escherichia coli(Rossi et al., 1997).Similarly,1-b-lactamase has been used to de-tect protein-protein interactions in mammalian cells (Galarneau et al.,2002).Protein fragment complemen-tation based on the reconstitution of murine dihydro-folate reductase(Remy and Michnick,1999)was used to detect NPR1-TGA2interaction in plants(Subramaniam et al.,2001).These assays typically require the addition of?uorescence-generating substrates and thus also suffer from the pitfalls of FRET and BiFC.Recently,an improved?re?y luciferase complementation imaging (LCI)assay was developed for protein-protein inter-actions in animals(Luker et al.,2004).The?re?y luciferase(LUC)enzyme is divided into the N-and C-terminal halves that do not spontaneously reassem-ble and function.LUC activity occurs only when the two fused proteins interact,resulting in reconstituted LUC enzyme,which can be detected by luminometer or a low-light imaging device.The assay measures dynamic changes in protein-protein interactions and can be used for both cell culture and whole animals. Because the luminescence was measured in the dark and is not affected by auto?uorescence,LCI is partic-ularly attractive for plant studies.A very recent report successfully used Renilla reniforms LUC complemen-tation assay to detect interactions of two pairs of plant proteins in protoplasts(Fujikawa and Kato,2007).The utility of the?re?y LCI in plant protein-protein inter-action studies remains to be tested.

In this study,we developed a series of constructs and comprehensively tested the utility of?re?y LUC-based LCI in plants.Tests with nine pairs of proteins that are known to interact with different strength in the plant cell showed that the?re?y LUC-based LCI assay is suitable for detecting protein-protein interactions in both protoplasts and intact leaves.The assay is simple, quantitative,highly sensitive,and can be used for transient expression or stable transgenic expression of the interacting proteins.The system provides a new tool for plant protein-protein interaction studies.

RESULTS

Constructs for LCI Assays

The?re?y LUC fragments2-416(NLuc)and398-550 (CLuc)were successfully used for protein-protein interaction assays in the mammalian system(Luker et al.,2004).These two fragments roughly correspond to the independently folded N-terminal and C-terminal domains that are linked by a disordered?exible region (Conti et al.,1996).To test the utility of LCI in plants, the NLuc and CLuc fragments were inserted into an expression cassette between the cauli?ower mosaic virus35S promoter and Rubisco small subunit termi-nator to form35S T NLuc and35S T CLuc,respectively (Fig.1).A Gly/Ser linker between the LUC fragments and the multiple cloning sites(Luker et al.,2004)was retained in the constructs to allow molecular mobility at the junction of the fusion proteins.Two sets of con-structs were made for LCI assays.The?rst set was made in a pUC19-based plasmid designed for transfection of protoplasts or particle bombardment into plant tis-sues.The second set was produced in pCAMBIA-based plasmid for generation of stable transgenic plants or Agrobacterium-mediated transient expression.Multiple cloning sites were inserted N terminus to the NLuc frag-ment and C terminus to the CLuc fragment.We selected nine pairs of proteins that are known to interact with different strength and possess a range of biochemical functions in the plant cell.

Interaction between Bacterial Effectors and Host Proteins Bacterial pathogens inject effector proteins into the host cells to regulate host susceptibility/resistance to the bacterium(Chisholm et al.,2006;Nomura et al., 2006).We previously showed that the Pseudomonas syringae effector protein AvrB targets Arabidopsis (Arabidopsis thaliana)protein RAR1to promote viru-lence(Shang et al.,2006).To test if such an interaction can be detected with the LCI assay,CLuc-AvrB was co-expressed with RAR1-NLuc in protoplasts.For

negative Figure 1.Constructs for LCI assays in plants.A,

Schematic diagrams of35S T NLuc and35S T CLuc

constructs.L,Gly/Ser linker between LUC fragments

and multiple cloning sites(MCS).rbs,Transcription

terminator derived from the Rubisco small subunit

gene.B,Diagram for LUC complementation resulting

from NLuc-and CLuc-fusion proteins.[See online

article for color version of this?gure.]

Luciferase Complementation for Protein-Protein Interactions

controls,we included SCaBP8that functions in salinity tolerance (Quan et al.,2007).As shown in Figure 2A,CLuc-AvrB and RAR1-NLuc coexpression led to strong LUC activity in the protoplasts that can be readily detected with a low-light imaging system after the addition of luciferin,the substrate for ?re?y LUC.In contrast,RAR1-NLuc coexpressed with SCaBP8con-struct showed only background level LUC activity.The rar1-29mutant carries a single amino acid substitution that speci?cally disrupts its interaction with SGT1b (Shang et al.,2006).This mutant showed normal inter-action with AvrB (Fig.2A).To determine if the ob-served LUC activity was caused by different levels of proteins expressed in the protoplasts,we examined respective NLuc and CLuc fusion proteins by western blot.The CLuc-SCaBP8protein was expressed at a level similar to CLuc-AvrB,and RAR1-NLuc protein was expressed at a similar level in all samples.The results indicate that the strong LUC activity was not caused by higher levels of CLuc-AvrB and RAR1-NLuc proteins expressed in the cell but resulted from a speci?c interaction between RAR1and AvrB.

The P.syringae effector AvrPto interacts with the tomato (Solanum lycopersicum )Ser/Thr protein kinase Pto (Tang et al.,1996);the latter subsequently triggers resistance through the association with the N terminus of the resistance protein Prf (NPrf)but not the C terminus of Prf (CPrf;Mucyn et al.,2006).The inter-action between AvrPto and Pto,however,has never,to our knowledge,been demonstrated in vivo.We tested if LCI can be used to detect such an interaction.Figure 2B shows that coexpression of Pto-NLuc with CLuc-NPrf,but not CLuc-SCaBP8or CLuc-CPrf,resulted in strong LUC activity.CLuc-NPrf accumulated to a level approximately 8-fold higher than CLuc-CPrf.How-ever,the reconstituted LUC activity of Pto-NPrf com-bination was approximately 60-fold greater than the Pto-CPrf combination.The results indicate that the Pto-NPrf interaction resulted in a signi?cant increase in LUC activity,con?rming previous co-immuno-precipitation results (Mucyn et al.,2006).Similarly,coexpression of CLuc-AvrPto with Pto-NLuc in Arabi-dopsis protoplasts resulted in strong complementa-tion of LUC activity.We recently showed that AvrPto Y89makes direct contact with Pto,and the AvrPto Y89D mutation abolishes the interaction in vitro (Xing et al.,2007).Coexpression of CLuc-AvrPto Y89D with Pto-NLuc failed to show LUC complementation,although the mutant and wild-type CLuc-AvrPto pro-teins accumulated to the same level in the plant cell (Fig.2C),indicating that the interaction detected by LCI is highly

speci?c.

Figure 2.Interactions of P.syringae effectors with host proteins in protoplasts.A,Interaction between AvrB and RAR1.B,Interaction between Pto and the N terminus of Prf.C,Interaction between AvrPto and Pto.The top panels show quanti?cation of LUC activity.Different letters above the bars indicate statistic difference at P ,0.01(t test).The images in the middle show microtiter plates containing protoplasts expressing the indicated constructs.The pseudocolor bar below shows the range of luminescence intensity in each image.The bottom panels show western blot for proteins isolated from protoplasts.Anti-full-length ?re?y LUC antibodies or the indicated speci?c antibodies (anti-RAR1;Shang et al.,2006;anti-CLuc antibodies;Sigma)were used to detect the indicated fusion proteins.The amount of protein loaded in each lane is indicated by Ponceau S staining of Rubisco on a representative protein blot.The data shown are representative of three independent experiments.[See online article for color version of this ?gure.]

Chen et al.

Interaction of the HSP90Complex Components

The HSP90protein complex plays an important role in plant innate immunity.HSP90and its co-chaperones,SGT1and RAR1,interact with each other,and all three components are required for disease resistance.Arabidopsis contains two SGT1genes,SGT1a and SGT1b ,both functions in stabilizing disease resistance proteins (Shirasu et al.,1999;Azevedo et al.,2002;Takahashi et al.,2003).The RAR1protein con-tains a CHORD I (Cys and His-rich)domain,a central Cys-rich domain,and a CHORD II domain.CHORD I is required for interaction with HSP90,whereas the CHORD II domain is required for interaction with SGT1.We showed previously that the rar1-29allele was compromised in the interaction with SGT1b in the yeast two-hybrid assay (Shang et al.,2006).Figure 3A shows that the LCI assay in protoplasts detected a speci?c interaction of RAR1with SGT1b,but not ScaBP8.The rar1-29mutant protein accumulated to a similar level as the wild-type RAR1but displayed much weaker interaction with SGT1b.Similarly,we tested the interaction of RAR1with SGT1a and HSP90.As shown in Figure 3,B and C,the full-length RAR1was capable of interacting with both SGT1a and HSP90.The CLuc-CHORD I and CLuc-CHORD II domain fusion proteins accumulated to a similar level as the full-length CLuc-RAR1protein,but showed only a background level LUC activity when coex-pressed with SGT1a-NLuc and HSP90-NLuc,respec-tively.These results are consistent with the respective

roles of CHORD I and CHORD II domains in the HSP90complex.

Protein-Protein Interactions between WRKY Proteins

Transcription factors WRKY18,WRKY40,and WRKY60play an important role in regulating plant immunity.Interestingly,these transcription factors are able to form homo-or heterodimers,and this interac-tion is the basis for a complex regulation of down-stream gene expression (Xu et al.,2006).We tested the utility of LCI for the interaction between WRKY40and WRKY18.As shown in Figure 4,coexpression of CLuc-WRKY18and WRKY40-NLuc in protoplasts strongly complemented the LUC activity compared to the neg-ative control protoplasts coexpressing CLuc-SCaBP8and WRKY40-NLuc.The Leu zipper motif of these WRKYs is required for the dimerization.Deletion of this motif (CLuc-WRKY18D)signi?cantly reduced the LUC complementation with WRKY40-NLuc,even though 2-to 3-fold more CLuc-WRKY18D protein was expressed in these protoplasts.These results indicate that the LCI assay is also useful for studying interac-tions among transcription factors.

Interactions between SKP1-Cullin-F-Box Protein E3Ubiquitin Ligase Complex Components

The SCF (SKP1-Cullin-F-box protein)complex is an E3ubiquitin ligase regulating 26S

proteasome-dependent

Figure 3.Interactions among the HSP90chaperone complex components in protoplasts.A,Interaction between SGT1b and RAR1.B,Interaction between SGT1a and RAR1.C,Interaction between RAR1and HSP90.CH I,CHORD I domain;CH II,CHORD II domain;HSP ,HSP90.The data shown are representative of three (A and B)or ?ve (C)independent experiments.[See online article for color version of this ?gure.]

Luciferase Complementation for Protein-Protein Interactions

degradation of a variety of proteins and is central to plant development and responses to the environment (Callis and Vierstra,2000).SKP1directly interacts with the F-box proteins;the latter serve to recruit speci?c substrate proteins for degradation.F-box proteins form a super family with members such as COI1that regulates jasmonate signaling and EBF1and EBF2that regulate ethylene signaling (Xu et al.,2002;Guo and Ecker,2003;Potuschak et al.,2003).In Arabidopsis,SKP1is encoded by ASK1and ASK2.Both ASK1and ASK2interact with COI1to regulate jasmonate signal-ing (Xu et al.,2002).We tested if LCI could be used to detect the ASK1-COI1interaction.Coexpression of CLuc-COI1and ASK1-NLuc in protoplasts resulted in strong LUC activity (Fig.5).Cotransfection of an empty CLuc plasmid with ASK1-NLuc showed a much weaker activity that was approximately 14%of protoplasts coexpressing CLuc-COI1and ASK1-NLuc,suggesting a speci?c interaction between ASK1and COI1in plant cells.

EBF1and EBF2directly interact with their substrate protein EIN3(Guo and Ecker,2003;Potuschak et al.,

2003),a transcription factor,to regulate gene expres-sion.Because this interaction leads to the degradation of EIN3,such an interaction in vivo remains to be demonstrated.To test if LCI was capable of detecting EBF1-EIN3interaction in the plant cell,we used a truncated EBF1containing the Leu-rich repeat domain required for substrate binding but lacking the F-box domain.Coexpression of the CLuc-EBF1with EIN3-NLuc resulted in strong LUC activity in protoplasts,whereas the coexpression of CLuc-EBF1with SCaBP-NLuc showed only background level activity,indicat-ing that the interaction of an F-box protein with its substrate can be successfully detected by LCI (Sup-plemental Fig.S1).Although the EBF1-NLuc fusion protein was not detected by western blot,preventing a quantitative assessment of the protein-protein interac-tion,the results are nevertheless consistent with pre-vious yeast two-hybrid data (Guo and Ecker,2003;Potuschak et al.,2003).

Comparison of Reconstituted and Full-Length Fire?y LUC Activity

We compared protoplasts coexpressing SGT1a-NLuc and CLuc-RAR1with those transfected with a 35S T LUC (full-length)construct (Supplemental Fig.

S2).

Figure 4.Interaction between WRKY40and WRKY18.The WRKY18D construct lacks the Leu zipper motif.The data shown are representative of three independent experiments.[See online article for color version of this

?gure.]

Figure 5.Interactions between ASK1and COI1.The data shown are representative of four independent experiments.[See online article for color version of this ?gure.]

Chen et al.

The latter showed approximately30-fold stronger luminescence.The simple calculation based on cell number would be such that the reconstituted LUC possesses approximately3%of the native LUC activ-ity.However,the SGT1a-NLuc accumulated to only approximately10%of the full-length LUC protein, whereas the CLuc-RAR1protein was accumulated to a level similar to the full-length LUC.Because the ex-pression of CLuc-RAR1alone never resulted in signif-icant luminescence in numerous tests(less than?ve counts),the vast majority of CLuc-RAR1is unlikely to function in the absence of SGT1a-NLuc(Luker et al., 2004).Therefore,our adjusted estimate of the recon-stituted LUC activity is approximately30%of that of the full-length protein.

Agrobacterium-Mediated Transient Expression

for LCI Assays

Agrobacterium-mediated transient expression in Ni-cotiana benthamiana provides a convenient system for the rapid analysis of protein functions in plants.We therefore tested if Agrobacterium-mediated transient expression could be adopted for the LCI assay.Agro-bacterium strains carrying CLuc and NLuc constructs were simply mixed,in?ltrated into leaves of N.ben-thamiana,and the in?ltrated leaves were covered with plastic for2d to maintain humidity.Leaves coexpress-ing different constructs were then examined for LUC activity.CLuc-RAR1was tested for interactions with SGT1a-NLuc.Figure6shows that the expression of SGT1a-NLuc and the empty35S T CLuc vector or CLuc-RAR1construct and the empty35S T NLuc vec-tor did not show LUC complementation,whereas co-in?ltration of Agrobacteria containing CLuc-RAR1plus SGT1a-NLuc resulted in strong LUC complementa-tion.The LUC activity was approximately10-fold greater than the empty vector controls and7-fold greater than the negative control expressing SGT1-NLuc and CLuc-CHORD I,indicating a speci?c interaction.Notably, the Agrobacterium-based LCI assay was more sensitive and had very low background.We also determined the time course for LUC complementation following the coexpression of SGT1a-NLuc and CLuc-RAR1.Maxi-mum LUC activity was detected4to6d after in?ltra-tion of Agrobacterium containing SGT1a-NLuc and CLuc-RAR1,whereas leaves expressing SGT1a-NLuc and the negative control construct CLuc-CHORD I had only negligible LUC activity(Fig.7,A and B).

Western blot showed that maximum protein accumu-lation occurred between4to6d postin?ltration(Fig. 7C),indicating that the LUC activity is correlated with the SGT1a-NLuc and CLuc-RAR1protein level in the leaves.The CLuc-CHORD I and CLuc-RAR1proteins were expressed at a comparable level,indicating that the difference in LUC activity was not caused by dif-ferent amounts of proteins accumulated in the leaves. Similarly,Agrobacterium-based LCI assay detected spe-ci?c interaction between SGT1b-NLuc and CLuc-RAR1 (Supplemental Fig.S3).Although the accumulation of SGT1b-NLuc in leaves was too low to be detected by western blot,all three negative controls showed only a background level of luminescence that was at least 15-fold less than leaf panels expressing SGT1b-NLuc and CLuc-RAR1.

DISCUSSION

In this study,we explored the utility of LCI for protein-protein interaction studies in plants.

Using Figure6.Interactions between SGT1a and RAR1in N.benthamiana leaves.A,LUC image of N.benthamiana leaves co-in?ltrated with the agrobacterial strains containing SGT1a-NLuc and CLuc-RAR1.Arrows indicate leaf panels that were in?ltrated with Agrobacterium containing the indicated constructs.B,Quanti?cation of LUC activity in leaves expressing SGT1a-NLuc and CLuc-RAR1.The western blot below shows the expression levels of CLuc-and NLuc-fusion proteins.RAR1 derivatives were detected by anti-?re?y LUC antibodies,whereas SGT1a-NLuc was detected by anti-SGT1antibodies.Ponceau S staining shows equal loading of protein in lanes.Data were collected4d after in?ltration.The data shown are representative of three independent experiments.

Luciferase Complementation for Protein-Protein Interactions

protoplast-and Agrobacterium -based transient expres-sion,we tested the interactions for nine protein pairs in plants,including components of the SCF E3ubiquitin ligase complex,HSP90chaperon complex,bacterial effector-plant resistance protein complex,and tran-scription factors.The tested proteins possess a variety of biochemical functions,and the strength of interac-tions varies considerably from protein to protein.We observed expected LUC complementation for all pro-teins tested.Importantly,we included strict negative controls for protein-protein interactions,including unrelated proteins and/or mutant proteins that are speci?cally compromised in protein-protein interac-tions.Whenever possible,the protein level was deter-mined except for one protein pair.These allowed critical assessment of the detected LUC complemen-tation,indicating that LCI is well suited for plant protein-protein interaction studies.In previous studies in animal systems,the interacting proteins have been successfully positioned to both the N-terminal and C-terminal ends of the fusion construct to achieve com-plementation (Luker et al.,2004;Paulmurugan and Gambhir,2005),suggesting that the LUC reporter is suf?ciently ?exible for different construction strategy.Nonspeci?c interactions are an inherent problem associated with all protein-protein interaction assays.In our protoplast-based LCI assays,several negative controls showed a certain level of background signal.It is possible that the two halves of ?re?y LUC are capable of association when present at a high concen-tration.Nevertheless,the nonspeci?c LUC activity,as

determined by using mutant or truncated proteins that are known to interfere with protein-protein interac-tions,was signi?cantly lower than the positive inter-actions,indicating that nonspeci?c interaction does not impede the proper determination of true inter-actions.The speci?city of interactions was further enhanced when Agrobacterium -mediated transient ex-pression was used for LCI.LUC activity resulting from speci?c RAR1-SGT1interactions was 7to 15times greater than the negative control (SGT1-NLuc and CHORD I-CLuc)in the Agrobacterium -based LCI assay,indicating that the Agrobacterium -based transient ex-pression is particularly suited for protein-protein in-teraction studies in plants.

Among the methods measuring protein-protein in-teractions,the yeast two-hybrid method is most widely used because of the ease of the assay and suit-ability for large-scale screening.However,the protein-protein interactions are studied in a heterologous system that is prone to false positives.It is not uncom-mon that the interaction of two proteins occurs in the presence of additional proteins or cellular factors.The lack of theses factors in yeast also contributes to false-negatives in yeast two-hybrid assays.Like FRET and BiFC,LCI detects protein-protein in the native phys-iological environment and is thus relevant to biolog-ical problems under investigation.Unlike FRET and BiFC,the current LCI technology does not provide information concerning the subcellular location of the interaction (Fujikawa and Kato,2007),a caveat that can be addressed by protein colocalization

analysis.

Figure 7.Time course of SGT1a-RAR1interaction in N.benthamiana.A,LUC image of N.benthamiana leaves co-in?ltrated with the agrobacterial strains containing SGT1a-NLuc and CLuc-RAR1.B,Quanti-?cation of LUC activity in leaves expressing SGT1a-NLuc and CLuc-RAR1.C,Accumulation of fusion proteins in N.benthamiana leaves.Data were col-lected at the indicated days post in?ltration of Agro-bacterium .Mock,Leaves in?ltrated with water.Arrows indicate in?ltrated regions.The data shown are representative of ?ve independent experiments.

Chen et al.

The LCI assays described in this study have several advantages over FRET and BiFC assays.First,LCI assays are highly quantitative,which allows linear measurement of luminescence signals over the range of several orders of magnitude.Second,compared to FRET and BiFC,LCI samples the entire tissue or cell population,avoiding bias derived from individual cells.Third,FRET and BiFC assays are complicated by auto?uorescence generated by chlorophyll and cell wall.In contrast,LCI measures luminescence at dark and is not affected by the chlorophyll-and cell wall-generated auto?uorescence.In addition,LCI can be used to study protein-protein interactions at the or-ganismal level(Luker et al.,2004),and the technique is very useful for studying tissue-speci?c protein-protein interactions.Finally,LCI does not require the use of a microscope,and data can be collected within2min by using a low-light imaging system.This is particularly attractive for studying the dynamics of protein-protein interactions.The assay can also be done with a lu-minometer,so that a large number of protein pairs can be tested simultaneously.The Agrobacterium-mediated LCI assay requires minimum sample handling and laboratory training.This system enables simultaneous testing of multiple protein pairs with little effort.The ability to simultaneously examine a large number of interacting proteins is a prerequisite of protein inter-actome construction.Currently,this is done primarily by using the yeast two-hybrid assays and informatic tools(Piehler,2005).The availability of LCI as a simple tool for plant protein-protein interaction studies will facilitate the validation of protein interactome data collected from the yeast two-hybrid assays.

MATERIALS AND METHODS

Plants

Arabidopsis(Arabidopsis thaliana)ecotype Columbia plants and Nicotiana benthamiana plants were grown in a controlled growth room at24°C/20°C day/night with12h/d light and70%humidity.Six-week-old Arabidopsis plants were used for protoplast isolation.Seven-week-old N.benthamiana plants were used for Agrobacterium-mediated transient expression.

NLuc and CLuc Constructs

A plant gene expression cassette containing the cauli?ower mosaic virus 35S promoter and rbs terminator was excised from p35S-FAST(Yiji Xia, Danforth Plant Science Center)and ligated to pUC19at Eco RI and Hin dIII sites, resulting in35S-pUC19.CLuc and NLuc were PCR ampli?ed from CLuc-FKBP and FRB-NLuc(Luker et al.,2004)and ligated into35S-pUC19,resulting in 35S T NLuc and35S T CLuc plasmids.Derivative NLuc-and CLuc-fusion constructs were made by PCR amplifying the open reading frames of respec-tive genes with primers listed in Supplemental Table S1,digested with Kpn I and Sal I,or Kpn I and Pst I,and inserted into35S T Nluc or35S T Cluc plasmids.

For Agrobacterium-mediated transient expression in N.benthamiana,the expression cassette was excised from the35S T NLuc and35S T CLuc fusion constructs with Eco RI and Hin dIII and cloned into pCAMBIA1300to form pCAMBIA-NLuc and pCAMBIA-CLuc.The constructs were mobilized into A. tumefaciens strain GV3101.

Protoplast Transfection

Protoplasts were isolated from6-week-old ecotype Columbia plants according to Sheen(https://www.wendangku.net/doc/7b2867507.html,/sheenweb/).23105protoplasts were transfected with indicated constructs and incubated over-night in a24-well microtiter plate before LUC activity was measured(Li et al., 2005).

Agrobacterium-Mediated Transient Expression Agrobacterium tumefaciens(strain GV3101)bacteria containing indicated constructs were grown in Luria-Bertani medium at28°C overnight,pelleted, and resuspended to0.3OD in induction medium according to Bundock et al. (1995).The culture was then grown in induction medium for8to12h.Bacteria were then washed once with Murashige and Skoog medium containing10m M MES,pH 5.6,and resuspended in Murashige and Skoog-MES medium containing150m M acetosyringone to a?nal concentration of OD60050.5. Bacterial suspensions were in?ltrated into young but fully expanded leaves of N.benthamiana plants using a needleless syringe.After in?ltration,plants were immediately covered with plastic bags and placed at23°C for48h before bag removal.Plants were then incubated at28°C with16h light/d before the LUC activity was measured.

CCD Imaging and LUC Activity Measurement One millimolar luciferin was added to protoplasts or sprayed onto leaves, and the materials were kept in dark for6min to quench the?uorescence.A low-light cooled CCD imaging apparatus(CHEMIPROHT1300B/LND,16 bits;Roper Scienti?c)was used to capture the LUC image.The camera was cooled to2110°C and relative LUC activity was measured as described(He et al.,2004).An exposure time of2min with333binning was used for all images taken.Relative LUC activity is equivalent to luminescence intensity/ 200protoplasts or luminescence intensity/0.2mm2leaf area.Each data point consisted of at least three replicates,and three to?ve independent experi-ments were performed for each assay.t test was performed to determine statistic signi?cance of differences at P,0.01.

Western Blot

Total protein was extracted from equal amounts of protoplasts or leaves, and approximately100m g protein was fractionated through SDS-PAGE. Unless indicated otherwise,protein blot was hybridized with the rabbit anti-full-length?re?y LUC antibodies(Sigma),which react with both the N-terminal and C-terminal?re?y LUC fragments.The protein blot was detected with the ECL kit from Amersham Biosciences.Anti-RAR1and anti-SGT1antibodies were raised in-house as described previously(Azevedo et al., 2002).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1.Interaction between EBF1and its substrate.

Supplemental Figure https://www.wendangku.net/doc/7b2867507.html,parison of reconstituted and full-length LUC activities.

Supplemental Figure S3.Agrobacterium-based LCI assay for SGT1b-RAR1 interaction.

Supplemental Table S1.Primers used for plasmid construction.

ACKNOWLEDGMENTS

We thank David Piwnica-Worms for providing the NLuc and CLuc plasmids and Yan Guo for the CLuc-ScaBP construct.

Received October25,2007;accepted November30,2007;published December 7,2007.

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Supplemental Data

Table S1. Primers for plasmid construction.

Primer name Sequence Protein Note

CLuc forward CAGAGCTCATGTCCGGTTATGTAAACAATC CLuc LUC398-550

CLuc reverse CAGGTACCGCCCCGGGACGCGTACGAGATC

NLuc forward ACGAGTCGACCAGATCTCGTACGCGTC NLuc LUC1-416

NLuc reverse AACTGCAGTCATCCATCCTTGTCAATCAAG

ASK1 forward ATGGATCCATGTCTGCGAAGAAGATTGTG ASK1 Full-length

ASK1 reverse ACGCGTCGACTTCAAAAGCCCATTGGTTC

COI1 forward TAGGTACCGAGGATCCTGATATCAAGAGGTG COI1 Full-length

COI1 reverse ACATGTCGACTCATATTGGCTCCTTCAGGAC

AvrB forward ATAGGTACCTTCATGGGCTGCGTCTCGTC AvrB Full-length

AvrB reverse ATAGTCGACTCAACGCCGCATCGGCATCAC

SCaBP8 forward CAAGGTACCATGACAACTGGCCGAC ScaBP8 Full-length

SCaBP8 reverse CAAGTCGACTCAGTCTTCAACCTCAGTGTTG

SGT1a forward CTTGGTACCATGGCGAAGGAGCTTGCTG SGT1a Full-length

SGT1a reverse CTTCTCGAGGATCTCCCATTTCTTGAGCT

SGT1b forward AGAGGTACCATGGCCAAGGAATTAGCAGAG SGT1b Full-length

SGT1b reverse AATCTGCAGTCAATACTCCCACTTCTTG

Full-length HSP90 forward TAGGATCCGTTGCGATGGCGGATGTTCAG HSP90.1

HSP90 reverse TTAGTCGACTTCCTCCATCTTGCTCTC

RAR1 forward ACGGGTACCATGGAAGTAGGATCTGCAAC RAR1 Full-length

RAR1-NLuc reverse ATTGTCGACGACCGCCGGATCAGGGCTGCTG

CLuc-RAR1 reverse ATTGTCGACTTAGACCGCCGGATCAGGGCTGCTG

CHORD I reverse AGTGTCGACTTAAACTGGTTTCTCAGTTGTGTG CHORD I RAR11-77

CHORD II forward GCAACGAAGAAGCTTCAGACAACTGAGAAACCAGTT CHORD II RAR11-11+72-226

WRKY18 forward GCTGGTACCATGGACGGTTCTTCGTTTC WKRY18 Full-length

WRKY18 reverse CCAGTCGACTTATGTTCTAGATTGCTCCATT

WRKY18D forward GAAATGGTTGGAGCAAGACTTGGAGAAGCTTCAGAG WRKY18D WRKY181-43+81-310

WRKY40 forward ACTGGTACCATGGATCAGTACTCATCCTC WRKY40 Full-length

WRKY40 reverse TCTGTCGACTTTCTCGGTATGATTCT

Pto forward GTAGGTACCATGGGAAGCAAGTATTCTAAG Pto Full-length

Pto reverse GACGTCGACAATAACAGACTCTTGGAGAC

AvrPto forward CCAGGTACCATGGGAAATATATGTGTC AvrPto Full-length

AvrPto reverse GTAGTCGACTCATTGCCAGTTACGGTAC

NPrf forward CAAGGTACCATGGCCAAGGAGTGTCGCGA NPrf Prf1-546

NPrf reverse AGGCTCGAGTTAGACAAGAATATATTCATG

CPrf forward CAAGGTACCATGTTTCTTCTCCAAATCAATAG CPrf Prf1481-1824

CPrf reverse CAACTCGAGCTAAGAGTCAAGGGGCTGTTC

EIN3 forward CGTGGTACCATGATGTTTAATGAGATGGGAATGTGTG EIN3 Full-length

EIN3 reverse TGAGTCGACGAACCATATGGATACATCTTGCTG

EBF1 forward AGAGGTACCGAGATTGATGTTCCTTCCAAGAT EBF1 LRR EBF1106-523

EBF1 reverse AGACTCGAGGGAGAGGATGTCACATTTG

Figure S1. Interaction between EBF1 and its substrate EIN3. The EBF1 construct contains the LRR domain but lacks the F-box domain. The data shown are a representative of 3 independent

experiments.

Figure S2. Comparison of reconstituted and full-length luciferase activities. Protoplasts expressing SGT1a-NLuc and CLuc-RAR1 and that expressing the full-length firefly luciferase (35S-LUC) were compared for LUC activity. The amount of fluorescence in equal amount of luciferase-free protoplasts was negligible. The data shown are a representative of 3 independent experiments.

Figure S3. Agrobacterium-based LCI assay for SGT1b-RAR1 interaction. A, Luciferase image of N. benthamiana leaves co-infiltrated with the Agrobacterial strains containing SGT1b-NLuc and CLuc-RAR1. Arrows indicate leaf panels that were infiltrated with Agrobacteria containing the indicated constructs. B, Quantification of luciferase activity in leaves expressing SGT1b-NLuc and CLuc-RAR1. The data shown are a representative of 3 independent experiments.

Girl On Fire中英文歌词

Girl On Fire She’s just a girl and she’s on fire她只是一个女孩子，她却在燃烧着 Hotter than a fantasy, longer like a highway 比幻想更张狂，比公路还要长 She’s living in a world and it’s on fire她就生活在我们的世界，她在燃烧着 Fill with catastrophe, but she know she can fly away 她的生活满是艰辛，但她知道她可以飞 Oh, she got both feet on the ground 噢，她站在这土地上 And she’s burning it down 把大地都点燃 Oh, she got her head in the clouds 她将头伸入云中 And she’s not backing down 就不会再回来 This girl is on fire 这个女孩燃烧着 This girl is on fire 这个女孩燃烧着 She’s walking on fire 她在火中行走 This girl is on fire 这个女孩燃烧着 Looks like a girl but she’s a flame 看起来是个女孩，可她却是一团火 So bright she can burn your eyes 如此明亮燃烧了你的眼睛 Better look the other way 最好不要直视她 You can try but you’ll never forget her name 可以试着去看，但这样你永不会再忘掉她的名字 She’s on top of the world 她站在世界的顶端 Hottest of the hottest girls say 最火的辣妹们说 Oh, we got our feet on the ground 哦，我们站在土地上 And we’re burning it down 我们将把这土地点燃 Oh, got our head in the clouds 噢，视野放在云端 And we’re not coming down 便再不回来 This girl is on fire 这个女孩燃烧着 This girl is on fire 这个女孩燃烧着 She’s walking on fire 她在火中行走 This girl is on fire 这个女孩燃烧着 Everybody stands as she goes by 她走过的时候所有人都停下了脚步 Cause they can see the flame that’s in her eyes 他们在她眼中看到了火焰 Watch her when she’s lighting up the night 看她将夜色点亮 Nobody knows that she’s a lonely girl 没有人知道女孩子的孤独 And it’s a lonely world 世界是冷漠的 But she gonna let it burn baby burn baby 但宝贝她要将世界点燃 This girl is on fire 这个女孩燃烧着 This girl is on fire 这个女孩燃烧着 She’s walking on fire 她在火中行走 This girl is on fire 这个女孩燃烧着 Oh, oh, oh…哦…… She’s just a girl, and she’s on fire她只是一个女孩子，她却在燃烧着

大大世界英文版歌词

大大世界英文版歌词 篇一：Bigbigworld-Emilia大大世界歌词 Big big world-Emilia 大大世界Im a big big girl 我是个重要的女孩，In a big big world !在这个大千世界里。Its not a big big thing if you leave me.即 使你离开我，也没有什么大不了的But I do do feel.但我真的真的感觉到.That I too too will miss you much.我将会非常非常想念你！Miss you much ! 非常想念你！I can see the first leaf falling.我看见第一片落叶。Its all yellow and nice.是那样金黄而美好。Its so very cold outside.外面是那么的冷。Like the way Im feeling inside.如同我内心的感受。Im a big big girl ! 我是个重要的女孩，In a big big world ! 在这个大千世界里Its not a big big thing if you leave me.即使你离开我，也没有什么大不了的。But I do do feel.但我真的真的感到。That I too too will miss you much.我将会非常非常想念你Miss you much ! 非常想念你Outside its now raining.现在外面正在下雨。And tears are falling from my eyes.而眼泪正从我眼中滑落。Why did it have to happen ? 这一切为何要发生？Why did it all have to end ? 这一切又为何要结束？Im a big big girl ! 我是个重要的女孩！In a big big world ! 在这个大千世界里！Its not a big

Faded-Alan Walker(中英文歌词-左右对照)

Faded 歌手-Alan Walker 专辑Faded (Restrung) You were the shadow to my light Did you feel us Another start You fade away Afraid our aim is out of sight Wanna see us Alive Where are you now Where are you now Where are you now Was it all in my fantasy Where are you now Were you only imaginary Where are you now Atlantis Under the sea Under the sea Where are you now Another dream The monsters running wild inside of me I'm faded I'm faded So lost I'm faded These shallow waters never met What I needed I'm letting go A deeper dive Eternal silence of the sea I'm breathing Alive Where are you now Where are you now Under the bright But faded lights You've set my heart on fire Where are you now Where are you now Where are you now Where are you now Where are you now Atlantis Under the sea Under the sea Where are you now Another dream The monster running wild inside of me I'm faded I'm faded So lost I'm faded 你是我生命之光中的一道暗影你能感受彼此的存在吗 下一个开始 你的身影渐渐模糊 唯恐我们的目标迷失在视野只希望我们能 依然活着 你如今身在何方 你如今身在何方 你如今身在何方 难道这一切只是我的幻想 你如今身在何方 你只是我虚幻的存在 你如今身在何方 亚特兰蒂斯 没于汪洋 没于汪洋 你如今身在何方 又是一场梦 狂野的怪兽驰骋在我心深处我憔悴不堪 我憔悴不堪 迷失方向憔悴不堪 那些镜花水月从未目及 却如此渴望 只是顺其自然 深沉海底 无尽的沉默与海中 我呼吸着 依然活着 你如今身在何方 你如今身在何方 明亮的灯光 却依然黯然失色 你点燃了我的心火 你如今身在何方 你如今身在何方 你如今身在何方 你如今身在何方 你如今身在何方 亚特兰蒂斯 没于汪洋 没于汪洋 你如今身在何方 又是一场梦 狂野的怪兽驰骋在我心深处我怕憔悴不堪 我憔悴不堪 迷失方向憔悴不堪

lightyourfire夜空中最亮的星英文版歌词

Sometimes I may wonder why It’s hard to find The reason you cry The reason you always hide When the day is passing by It comes the night I look at the stars Hoping that they could guide Now I see it’s all about the perfect time That could make two hearts collide Sparkling, once or twice Only love can light your fire Maybe I can fly away a thousand miles But you’re always in my mind Oh every time I look into your eyes Oh my love can light your fire Sometimes I may lose my sight And wear a disguise I do wanna try I do wanna lead my life Meet me under shining lights Please don’t be shy I’ll be waiting right here For the rest of my life Now I see it’s all about the perfect time That could make two hearts collide Sparkling, once or twice Only love can light your fire Maybe I can fly away a thousand miles But you’re always in my mind Oh every time I look into your eyes Oh my love can light your fire Sometimes I may wonder why It’s hard to find The reason you cry The reason you always hide

fire歌词中英文对照

Oh if there's one thing to be taught 哦如果有件事是要记住的那会是 it's dreams are made to be caught 梦想是用来追逐的 and friends can never be bought 朋友千金难换 Doesn't matter how long it's been 要花多久时间并不重要 I know you'll always jump in 我知道你会投入其中 'Cause we don't know how to quit 因为我们不知道该如何放弃 Let's start a riot tonight 今晚就让我们放纵一下 a pack of lions tonight 今晚就让我们像狂野的狮子 In this world he who stops 在这个世界一旦他停止 won't get anything he wants 他就不会有任何收获 Play like the top one percent 百里挑一竭尽全力till nothing's left to be spent 全情投入 Take it all ours to take 接受这一切 Celebrate because 我们要开始庆祝因为 We are the champions 我们是神勇斗士 Setting it off again 再次出发 Oh we on fire 哦我们热血沸腾 We on fire 我们热血沸腾 Running our own campaign 奔向我们的冠军之路 Doing the whole shebang 努力奋斗 Oh we on fire 哦我们热血沸腾 We on fire 我们热血沸腾Get up起身Stand up站立

Set fire to the rain歌词 经典

Set fire to the rain I let it fall, my heart, 我使我的心下坠 And as it fell, you rose to claim it, 而当它下坠时，你出现认领它， It was dark and I was over, 这是黑暗的，我已经结束， Until you kissed my lips and you saved me, 直到你亲吻我的嘴唇，并且救了我， My hands, they were strong, but my knees were far too weak, 我的手，他们是强有力的，但我的膝盖太过薄弱， To stand in your arms without falling to your feet, 如果要不落后于你的步伐，站在你的怀里 But there's a side to you that I never knew, never knew, 但有你有我永远不知道的一面，永远不知道， All the things you'd say, they were never true, never true, 所有你说的事情，，它们从来不真实，从来不真实 And the games you'd play, you would always win, always win, 和你玩的游戏，你总是会赢，永远赢， But I set fire to the rain, 不过，我放火烧了雨， Watched it pour as I touched your face, 看着它在我触摸你的脸时倾盆而下， Well, it burned while I cried, 那么，当它燃烧时我痛哭， 'Cause I heard it screaming out your name, your name, 因为我听到它尖叫着你的名字，你的名字， When laying with you I could stay there, 当我能和你躺在这里， Close my eyes, feel you here forever, 闭上我的双眼，感觉你永远在这里， You and me together, nothing is better, 你和我在一起，没有什么更好， 'Cause there's a side to you that I never knew, never knew, 因为你有我永远不知道的一面，永远不知道， All the things you'd say, they were never true, never true, 所有你说的事情，，它们从来不真实，从来不真实 And the games you'd play, you would always win, always win, 和你玩的游戏，你总是会赢，永远赢，

【必克音乐会】I See Fire(双语歌词)

【必克音乐会】I See Fire（双语歌词） 【歌曲信息】 所属专辑： The Hobbit: The Desolation Of Smaug （Original Motion Picture Soundtrack） 演唱者： Ed Sheeran 作词： Ed Sheeran 作曲： Ed Sheeran 编曲： Pete Cobbin, Ed Sheeran 【歌词内容】 Oh, misty eye of the mountain below Keep careful watch of my brothers' souls And should the sky be filled with fire and smoke Keep watching over Durin's son If this is to end in fire Then we should all burn together Watch the flames climb high into the night Calling out for the rope, sent by and we will Watch the flames burn on and on the mountain side And if we should die tonight Then we should all die together Raise a glass of wine for the last time Calling out for the rope

Prepare as we will Watch the flames burn on and on the mountain side Desolation comes upon the sky Now I see fire, inside the mountain I see fire, burning the trees And I see fire, hollowing souls And I see fire, blood in the breeze And I hope that you'll remember me Oh, should my people fall Then surely I'll do the same Confined in mountain halls We got too close to the flame Calling out father hold fast and we will Watch the flames burn on and on the mountain side Desolation comes upon the sky Now I see fire, inside the mountain I see fire, burning the trees And I see fire, hollowing souls And I see fire, blood in the breeze And I hope that you'll remember me

play with fire歌词

play with fire歌词 Hilary Duff I can’t believe it’s really you Been so long, you look good I hear you’re doing really well Don’t ask me, let me tell you How I’ve been since when you left Since you let me for dead Finally every tear has dried I’ve wiped you from my life Do you remember all the times you said you’d call me Cause I remember all the reasons people warned me And now I hear you saying that you still adore me But if you think I’d ever get with you again Then you could just Love me, love me Feed the flame If you want me back again Burn to the sky Higher and higher Baby can you play with fire Love me, love me

If you want me Never know just what you got Til’ it’s gone, you freak out But I’m not falling for that game Boys like you, never change You made me feel I wasn’t enough Wasn’t enough for your love But it was insecurity that made you run, it wasn’t me So don’t you sit there trying to give me more excuses I don’t have time for this, I’m off to play in Houston And I’m too busy with the million things I’m doing You can’t make up for what you done But you still try to be the one Love me, love me Feed the flame If you want me back again Burn to the sky Higher and higher Baby can you play with fire Love me, love me If you want me Oh, by the way,

歌词

Rolling in the deep There's a fire starting in my heart 我怒火中烧 Reaching a fever pitch and it's bringing me out the dark熊熊烈焰带我走出黑暗 Finally, I can see you crystal clear 最终我将你看得一清二楚 Go ahead and sell me out and I'll lay your ship bare去吧出卖我我会让你一无所有 See how I'll leave with every piece of you看我怎么离你而去带走你的一切 Don't underestimate the things that I will do不要低估我将来的所作所为 There's a fire starting in my heart 我怒火中烧 Reaching a fever pitch and it's bring me out the dark 熊熊烈焰带我走出黑暗 The scars of your love remind me of us你的爱情伤痕让我想起了我们曾经的甜蜜 They keep me thinking that we almost had it all它们总在提醒我我们几乎拥有了一切 The scars of your love, they leave me breathless你的爱情伤痕让我窒息 I can't help feeling 我不禁心生感触 We could have had it all 我们本该拥有一切 Rolling in the deep 内心深处爱恨交织 You had my heart inside your hand 你俘虏了我的芳心 And you played it to the beat 但是你玩弄它伴着每一次心跳 Baby, I have no story to be told 宝贝我没有故事可讲 But I've heard one on you and I'm gonna make your head burn但是我听说了一件有关你的事情我会让你焦头烂额 Think of me in the depths of your despair在绝望的深渊中想起我 Making a home down there as mine sure won't be shared就在那里安家吧因为我的家园绝对没你的份 Could have had it all 本该拥有一切 Rolling in the deep 内心深处爱恨交织 You had my heart inside your hand 你俘虏了我的芳心 But you played it with a beating 但是你玩弄它还一顿打击 Throw your soul through every open door打开每一扇门将你的灵魂驱赶出去 Count your blessings to find what you look for细数你的祷告知道你在寻找什么 Turn my sorrow into treasured gold 把我的悲痛化作珍宝 You'll pay me back in kind and reap just what you've sown我要让你付出同样的代价你自作自受自食其果 We could have had it all 我们本该拥有一切 We could have had it all 我们本该拥有一切 It all, it all, it all 一切一切一切 (Tears are gonna fall, rolling in the deep)（眼泪快要掉下来，内心深处爱恨交织） You’ve got the words to change a nation but you’re biting your tongue You’ve spent a lifetime stuck in silence afraid you’ll say something wrong If no one ever hears it, how we gonna learn your song? So come on come on, come on come on You’ve got a heart as loud as lions so why let your voice be tamed? Maybe we’re a little different, there’s no need to be ashamed You’ve got the light to fight the shadows so stop hiding it away Come on, come on

Wake 歌词

《Wake》--Hellsong Young and Free 歌词翻译：冯石 At break of day, in hope we rise. 破晓时分，我们满怀希望醒来 We speak Your Name, we lift our eyes. 我们喊出你的名字，睁开我们的双眼Tune our hearts to Your beat. 让我们的心跟随你的节奏 Where we walk, there You'll be. 无论我们去哪里，你都在我们身边With fire in our eyes, our lives a light. 我们眼里闪烁着火苗，我们的生命就像一束光 Your love untamed, it's blazing out.

你的爱无法驯服，熊熊燃烧 The streets will glow forever bright. 街道永远发光明亮 Your glory's breaking through the night. 你的荣耀划过那夜空 You will never fade away, Your love is here to stay 你永远不会消逝，你的爱永存 By my side, in my life, shining through me everyday. 在我身边，在我生命里，每天闪耀You will never fade away, Your love is here to stay 你永远不会消逝，你的爱永恒 By my side, in my life, shining through me everyday. 在我身边，在我生命里，每天闪耀

Fire 歌词 中英文对照教学内容

F i r e歌词中英文 对照

《Fire》歌词 Oh if there's one thing to be taught 哦如果有件事是要记住的那会是it's dreams are made to be caught 梦想是用来追逐的 and friends can never be bought 朋友千金难换 Doesn't matter how long it's been 要花多久时间并不重要 I know you'll always jump in 我知道你会投入其中 'Cause we don't know how to quit 因为我们不知道该如何放弃 Let's start a riot tonight 今晚就让我们放纵一下 a pack of lions tonight 今晚就让我们像狂野的狮子 In this world he who stops 在这个世界一旦他停止 won't get anything he wants 他就不会有任何收获 Play like the top one percent 百里挑一竭尽全力 till nothing's left to be spent 全情投入 Take it all ours to take 接受这一切 Celebrate because 我们要开始庆祝因为 We are the champions 我们是神勇斗士 Setting it off again 再次出发Oh we on fire 哦我们热血沸腾 We on fire 我们热血沸腾 Running our own campaign 奔向我们的冠军之路 Doing the whole shebang 努力奋斗 Oh we on fire 哦我们热血沸腾 We on fire 我们热血沸腾Get up起身Stand up站立 Throw your hands up 甩动你们的双手 Welcome to the other land of 欢迎来到梦想之家 dreamers brothers sisters others 兄弟姐妹们 Yeah we on fire like that 是的我们如此的热血沸腾 Oh the bond is deeper than skin 这联系浓于骨肉关系 The kind of club that we're in 像是置身于激情似火的夜店 The kind of love that we give 像是给予情侣般浓烈的爱 Oh ever since the dawn of mankind 哦自人类发展的曙光起 we all belong to a tribe 我们都属于一个部落 It's good to know this one's mine 我很高兴知道这部落属于我的 Let's start a riot tonight

fire中英文歌词

shake me in to the night 轻摇着让我入睡 and im an easy lover 我只需要简单的爱 take me into the fight 让我投身于战斗 and im an easy brother 我只是普通的兄弟 im on fire 我置身于火焰 burn my sweet effigy 焚烧我那甜美的雕像 im a road runner 我只是在随波逐流 spill my guts on a wheel 让车轮碾压我的身体 i wanna taste uh-huh 我想品尝这味道 and im on fire 我置身于火焰 and im on fire 我置身于火焰 im on fire .......... 我置身于火焰...... wire me up to machines 把我捆在机械上 i'll be your prisoner 我会成为你的囚徒 find it hard to believe 如此难以置信 you are my murderer 你是我的凶手（我被你所杀）im on fire behind you 我置身于在你的身后火焰

heads a fallen sky 面对着坠落的苍穹 im on fire 。。。我置身于火焰 im on fire 。。。我置身于火焰 im on fire .......... 我置身于火焰...... move on you've got to move on 前进，你得继续前进you've got to get it to the level 你得达到全新标准and get your shake on 让全身摇摆起来 move on you've got to move on 前进，你得继续前进you've got to get it to the level 你得达到全新标准and get your shake on 让全身摇摆起来

To Build a Fire Summary

《生火》是著名美国作家，捷克伦敦的著名短篇故事之一。 描写的是一个人独自在寒冷中行走，最终抵御不住严寒而冻死的故事。《生火》是一篇经典的自然主义作品。故事中的人藐视自然，却被自然挫败。 叙述在大雪纷飞的寒冬之际，一只狗跟着一位旅人，徒步横越阿拉斯加。故事主要是想告诉读者这位旅人之所以能让狗乖乖地跟在他身边， 完全是因为他身上有可以用来生火的火柴，可以提供这只狗在冰天雪地所需要的温暖，并不是因为狗对主人的忠心。 半个月后的一个雪夜里，这只狗悄悄的离开了这位旅人，原因是旅人不小心将身上的火柴弄湿，不能再像之前一样生火，让它度过漫漫长夜。杰克伦敦在故事的最后写道：“这只狗在严冬的夜空下低吠，还是未见主人有生火的动静， 他觉的眼前的这位主人，似乎已是无法再满足它所要的温暖，只好夹着尾巴，低着头离开，在月光下继续寻找另一个可能给予它温暖火光的主人。” 英文版简介 To Build A Fire and Other Storiesis the most comprehensive and wide-ranging collection of Jack London's short stories 口口ailable in paperback. This superb volume brings together twenty-five of London's finest, including a dozen of his great Klondike stories, vivid tales of the Far North were rugged individuals, such as the Malemute Kid face the violence of man and nature during the Gold Rush Days. Also included are short masterpieces from his later writing, plus six stories un口口ailable in any other paperback edition. Here, along with London's famous wilderness

Faded中英字幕歌词打印版

Faded 歌手：Alan Walker 作曲: Jesper Borgen/Gunnar Greve Pettersen/Alan Walker/Anders Froen You were the shadow to my light 你是我生命之光中的一道暗影Did you feel us 你能理解我们吗Another Star 另一颗行星You fade away 你逐渐消失Afraid our aim is out of sight 恐惧我们的目标迷失在视野Wanna see us 希望我们互相理解Alive 活着Where are you now?Where are you now?Where are you now? 你身在何方Was it all in my fantasy 难道这一切都在我的幻想里Where are you now 你身在何方? Were you only imaginary 你只是虚幻的不存在吗？Where are you now 你身在何方? Atlantis 亚特兰蒂斯Under the sea Under the sea 在海底Where are you now 你身在何方? Another dream 另外的梦想The monster's running wild inside of me 狂野的怪兽驰聘在我心深处I'm faded我憔悴不堪I'm faded我憔悴不堪 So lost, I'm faded 所以迷失，憔悴不堪I'm faded ~~~ 我憔悴不堪So lost, I'm faded 所以迷失，憔悴不堪These shallow waters never met 那些从未见过的水中之影What i needed 我需要的I'm letting go 只是顺其自然A deeper dive 深沉海底Eternal silence of the sea 无尽的沉默于海中I'm breathing 我的呼吸声Alive. 活着Where are you now Where are you now 你身在何方? Under the bright but faded lights 明亮的灯光却已经黯然失色You set my heart on fire 你点燃了我的心火Where are you now Where are you now 你身在何方? ... ... Where are you now你身在何方? Atlantis亚特兰蒂斯 Under the sea Under the sea 在海底Where are you now 你身在何方? Another dream 另外的梦想The monster's running wild inside of me 狂野的怪兽驰聘在我心深处I'm faded I'm faded 我憔悴不堪So lost, I'm faded所以迷失，憔悴不堪 I'm faded ~~~ 我憔悴不堪So lost, I'm faded 所以迷失，憔悴不堪... ... 1

2NE1的Fire中文歌词和中文译音

I go by the name CL ofIt’s been a long time coming But we’re here now And we about to set the roof on fire baby(oh oh) Y ou better get yours Cause I’m getting mine {All}Eh eh eh eh eh eh ehh ,{Sandara}2ne1 Eh eh eh eh eh eh ehh {Sandara}Y ou’gotta ring the alarm Eh eh eh eh eh eh ehh . We 2ne1 EH eh eh eh eh eh ehh Hey hey hey hey {CL}Come in come in come in在另一个世界里 只把烦恼苦恼统统抛到身后 La la la la 用我毫不造作的哼唱 Ha ha ha ha 更为了让你无法嘲讽我 Now let’s {敏智}跳舞跳舞跳舞跳舞Wanna get down 我心中的梦想梦想梦想梦想比世人的都大 为达到那随心所欲的自由境界Tonight tonight {朴春}我的目光比星光还要闪耀 心中的热情比火光还要有热量 不要永远but 不能丢弃oh~ {All}我想疯疯疯疯疯疯疯疯掉 要更快快快快快快快快去奔跑 去那高高的建筑上去那蔚蓝的天空上 想大大大大大大大大声呼喊 {Sandara}Y ou got that fire 在我心中嘭嘭嘭的燃烧 Y ou gotta drop it like it's hot 现在别想停下来 Ooooh that fire 在你脑海中扑扑扑的燃烧 I gotta drop it like it's hot 别想停下来 {CL}Get up get up get up 哪怕摔了好几个跟头 就算曾经信任的世界又再次的背叛了你 {All}我我我我我绝对不会像傻瓜一样哭泣 哦哦哦哦哦不会像其他人那样耍伎俩 {敏智}我会一直走到尽头follow follow me 我的心如同呼吸般快速跳动 我为什么不讨厌这样有意思吧 不要害怕Ready go 奔向比以往更美好的明天 Let let let let’s go {朴春}我的目光比星光还要闪耀 心中的热情比火光还要有热量 不要永远but 不能丢弃oh~ {All}我想疯疯疯疯疯疯疯疯掉 要更快快快快快快快快去奔跑 去那高高的建筑上去那蔚蓝的天空上

英文歌曲 Fire fly

Fire fly （萤火虫） When I said go I never meant away （当我说离开时我从不打算离开） You ought to know the freaky games we play （你应该知道我们玩的任性游戏）Could you forgive and learn how to forget （你可以原谅而且学习该如何忘记吗）Hear me as I'm calling out your name （听到我呼唤你名字的时候） Firefly come back to me （萤火虫飞回我身边） make the night as bring as day （使夜晚像白天一样明亮） I'll be looking out for you （你轻轻的告诉我） Tell me that your lonely too （告诉我你也很孤单） Firefly come lead me on （萤火虫过来指引我） follow you into the sun（跟随你接触太阳） that's the way it ought to be（我们应当这样做） Firefly come back to me （萤火虫飞回我身边） You and me（你和我） we shared a mistery （我们分享朦胧的感觉） We were so close （我们是如此接近） like honey to the bee （像是一对亲密的蜜蜂） And if you tell me how to make you understand （而且如果你告诉我该如何使你了解）I'm minor in a major kind a way （在主要的前提下我使用次要的） Firefly come back to me （萤火虫飞回我身边 make the night as bring as day（使夜晚像白天一样明亮） I'll be looking out for you（你轻轻的告诉我） Tell me that your lonely too （告诉我你也很孤单） Firefly come lead me on （萤火虫过来指引我） follow you into the sun（跟随你接触太阳） that's the way it ought to be（我们应当这样做） Firefly come back to me（萤火虫飞回我身边） Fly firefly through the sky （萤火虫飞越天空） come and play with my desire （过来与我的渴望嬉戏） Don't be long,don't ask why（不要太久，不要问为什么） I can't wait another night （我不可以等到下一个夜晚） Wait another night （等到下一个夜晚） Don't be long （不要太久） Fire...fly（萤火...虫） Firefly come back （萤火虫飞回我身边） Firefly come back to me （萤火虫飞回我身边） make the night as bring as day （使夜晚像白天一样明亮） I'll be looking out for you （你轻轻的告诉我） Tell me that your lonely too （萤火虫过来指引我） Firefly come lead me on（萤火虫过来指引我） follow you into the sun （跟随你接触太阳） that's the way it ought to be （我们应当这样做） Firefly come back to me（萤火虫飞回我身边）