Exploiting a precise design of universal syntheticmodular regulatory elements to unlock the microbialnatural products in StreptomycesChaoxian Baia,b,1, Yang Zhanga,b,1, Xuejin Zhaoa,1, Yiling Hua,c, Sihai Xianga, Jin Miaoa, Chunbo Loua,2, and Lixin Zhanga,2

aChinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and MetabolicEngineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; bUniversity of Chinese Academy of Science, Beijing 100149,China; and cSchool of Life Sciences, Anhui University, Hefei 230601, China

Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved August 26, 2015 (received for review June 5, 2015)

There is a great demand for precisely quantitating the expressionof genes of interest in synthetic and systems biotechnology asnew and fascinating insights into the genetics of streptomyceteshave come to light. Here, we developed, for the first time to ourknowledge, a quantitative method based on flow cytometry and asuperfolder green fluorescent protein (sfGFP) at single-cell resolu-tion in Streptomyces. Single cells of filamentous bacteria wereobtained by releasing the protoplasts from the mycelium, andthe dead cells could be distinguished from the viable ones bypropidium iodide (PI) staining. With this sophisticated quantitativemethod, some 200 native or synthetic promoters and 200 ribo-somal binding sites (RBSs) were characterized in a high-throughputformat. Furthermore, an insulator (RiboJ) was recruited to elim-inate the interference between promoters and RBSs and im-prove the modularity of regulatory elements. Seven syntheticpromoters with gradient strength were successfully applied in aproof-of-principle approach to activate and overproduce thecryptic lycopene in a predictable manner in Streptomyces aver-mitilis. Our work therefore presents a quantitative strategy anduniversal synthetic modular regulatory elements, which will fa-cilitate the functional optimization of gene clusters and thedrug discovery process in Streptomyces.

synthetic biology | natural product | flow cytometry |single-cell resolution | modular regulatory elements

Streptomycetes are well known as the most abundant source ofbioactive secondary metabolites (1), including medically im-

portant antimicrobial agents [e.g., chloramphenicol from Strep-tomyces venezuelae (2)], agricultural chemicals [e.g., avermectinfrom Streptomyces avermitilis (3)], and anticancer agents and im-munosuppressants [e.g., rapamycin from Streptomyces hygroscopicus(4)]. However, the increasing difficulty of discovering novel drugsvia traditional high-throughput screening and the “one strainmany compounds” approach is frustrating pharmaceutical pro-ductivity (5, 6). Deciphering the genome sequences of Streptomycessurprisingly established the presence of a plethora of gene clustersencoding for yet-unobserved molecules, even in intensively in-vestigated Streptomyces coelicolor A3 (2), revealing a much higherpotential of novel bioactive agent production than originallyanticipated (7, 8). Therefore, the enormous number of naturalproducts that have been obtained likely represent only a tinyportion of the repertoire of bioactive compounds that can pos-sibly be produced. This has brought about extensive research intoapplied genomics aimed at investigating these new gene clusters,generally referred to as “cryptic,” “silent,” or “orphan” (9–11). Withdata on more than 12,000 in-house draft bacterial genomes, the po-tential for the discovery of a number of novel chemicals encrypted insilent biosynthetic gene clusters has been detected by genome mining.Many new strategies have been documented for “awakening”

poorly expressed and/or silent gene clusters in Streptomyces,enabling the discovery of new bioactive agents. Two main sub-stantially overlapped ways are used: physiological triggers (12, 13)

and synthetic or genetic manipulations (14–16). Even though thegenome size of Streptomyces is significantly smaller than that ofSaccharomyces cerevisiae, it has many more ORFs than itseukaryotic counterparts, a substantial part of which regulate thetranscriptional and translational machineries of gene clustersresponsible for the biosynthesis of secondary metabolites (7, 17,18). Thus, most activation approaches aim to stimulate the tran-scription of gene clusters. Nevertheless, a convenient and preciseapproach for characterization of the relevant transcriptional andtranslational elements in Streptomyces has become a bottleneck inthe effort to activate the cryptic gene clusters. Although antibioticresistance genes [e.g., the neomycin/kanamycin resistance gene (19)and chloramphenicol resistance gene (20)] were broadly used asqualitative reporters in Streptomyces, they are unable to do quanti-tation. Luciferase assay [e.g., luxBA operon (21) from Vibrio harveyiand luxCDABE operon (22) of the bioluminescent bacterium Pho-torhabdus luminescens], as well as chromogenic assay [e.g., xylE gene(23, 24) from Pseudomonas putida and gusA gene (25, 26) fromEscherichia coli], have wide application in quantitation. However,both of these assays are based on enzymatic reaction, and furtheractivity normalization for dry cell weight is required, which de-creases accuracy and is time-consuming. A GFP-based reportersystem is another approved strategy to qualitatively monitorspatial and temporal trafficking of proteins and other protein-related physiological processes (27–30). Although differentfrom the unicellular bacterium E. coli, Streptomyces grow byhyphal extension and exponential branching and ultimately formmulticellular network-structured pellets with diameters of up to

Significance

To meet the increasing demands of drug discovery and bio-synthetic studies, we established a precise quantitative methodbased on flow cytometry at single-cell (protoplast) resolutionin Streptomyces for the identification of regulatory elements.A series of native or synthetic promoters and ribosomal bind-ing sites has been characterized. Moreover, an insulator wasdemonstrated to eliminate element–element interference. As aproof of concept, a native silent gene cluster was activated bythe synthetic modular regulatory elements in a predictablemanner. The universality of these elements is of high value tothe synthetic biology of Streptomyces.

Author contributions: C.L. and L.Z. designed research; C.B., Y.Z., X.Z., and Y.H. performedresearch; C.B., Y.Z., X.Z., Y.H., S.X., and J.M. analyzed data; and C.B., C.L., and L.Z. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1C.B., Y.Z., and X.Z. contributed equally to this work.2To whom correspondence may be addressed. Email: zhanglixin@im.ac.cn or louchunbo@gmail.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1511027112/-/DCSupplemental.

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2,000 μm (31). The network structure of the pellets appears as amixture of multiple layers of cells on a microscope slide, with thesizes of the pellets being too large to pass through the nozzle of acommercial FACS machine (32). This led to no description ofthe GFP-based quantitative measurement in Streptomyces.The work explained here was performed to overcome these

problems. We developed a quantitative method for gene expres-sion based on flow cytometry of protoplasts in Streptomyces. Thequantitative accuracy was improved by introducing propidiumiodide (PI) as another reporter to differentiate between viable anddead cells, and was further validated by quantitative PCR. Wedemonstrated the convenience and compatibility of this approachby characterizing a large number of native or synthetic promotersand RBSs. We also used the method to characterize the combi-nation of seven promoters and nine RBSs and to quantify thepredictability after introducing the RiboJ insulator (33). Thus, auniversal toolbox of synthetic modular regulatory elements hasbeen developed to systematically replace the indigenous promoterand RBS sequences to activate the expression of the cryptic geneclusters at various levels. The feasibility and efficiency of theuniversal cassettes were confirmed by the overproduction oflycopene in S. avermitilis.

Results and DiscussionA Flow Cytometry-Based Quantitative Method for Streptomyces. Forthe multicellular Streptomyces, we first developed a quantitativemethod at single-cell resolution (Fig. 1A). After introducing astrong promoter (kasOp*) with the superfolder green fluorescentprotein (sfGFP) gene into the S. venezuelae genome via site-specific recombination, the hyphae were treated with lysozyme torelease protoplasts into P10 buffer containing 10% (wt/vol) sucrose,which is very sticky. Although the sticky sucrose was important in

stabilizing the osmotic pressure of the protoplasts, it might damagethe fluidic system of the FACS machine. To conquer this problem,we sought an alternative buffer that is not only less sticky but alsocapable of maintaining the viability of the protoplasts. In addition,the membrane-impermeable dye, PI, was introduced to recognizethe cell death in the dead hyphae, of which the protoplasts can bestained and analyzed by confocal laser-scanning fluorescence mi-croscopy (Fig. 2 A and B). As a negative control, PBS buffer orwater did not maintain the osmotic pressure of the protoplasts;these lysed protoplasts can be stained by PI (Fig. 1B). Appropriateconcentration (0.4∼0.8 M) of NaCl or KCl in the buffer success-fully maintained the osmotic pressure of the protoplasts, resultingin ∼20% viable cells being obtained. Further optimization showedthat the use of the PBS buffer (pH 7.4) supplemented with 0.5 MNaCl can achieve more than 50% viability, which is almost twicethat achieved with the original P10 buffer (Fig. 1C). We thus notonly solved the problem accompanied by the morphogenesis ofStreptomyces for FACS processing but also developed a quanti-tative strategy at single-cell resolution for Streptomyces.Eight promoters were selected, based on extensive literatures

(SI Appendix, Table S3), to measure their GFP fluorescence byflow cytometry. To validate the accuracy of the new method,their cognate mRNA level was also detected by real-timequantitative PCR. The results showed that the fluorescence ofthe protoplasts is consistent with the mRNA level of their hy-phae (R2 = 0.90), indicating that the flow cytometry-based GFPreporter correctly represented the activities of the promoters inStreptomyces (Fig. 3). Moreover, flow cytometry can process tensof thousands of individual cells within a few seconds and cansimultaneously monitor up to 20 different parameters [e.g., sidescatter (SSC), forward scatter (FSC), and up to 18-color fluores-cence]. These advantages of flow cytometry can facilitate further

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analysis of heterogeneity in a population of the multicellularStreptomyces. Meanwhile, PI was enrolled to exclude the inter-ference of dead cells, as a single parameter (GFP) cannot dis-tinguish the low GFP-expressing viable cells from the dead ones(Fig. 2 C and D), which therefore greatly improved the reliabilityof our approach and the accuracy of the results.In contrast, the most widely used quantitative methods in

Streptomyces, such as xylE, gusA, and luxCDABE, are based onenzymatic reactions that require further normalization of theiractivities to dry cell weight, thereby compromising the accuracyof the measurement. However, dead cells are inevitably presentwithin dry cells of Streptomyces mycelium and can further reducethe accuracy, as programmed cell death is occurring during thedevelopmental process (34, 35). Therefore, these traditionalmethods are far from satisfying the sophisticated elucidationof the regulatory elements for the biosynthesis of secondarymetabolites.

Characterization of Native or Synthetic Promoters and RBSs inStreptomyces. Apart from the advantage of single-cell resolu-tion, the flow cytometry-based method has the potential tocombine with high-throughput manipulation for Streptomyces. Forhigh-throughput application, 24-well plates were adopted for cul-turing and 96-well plates were used for treating the hyphae withlysozyme and harvesting protoplasts. Considering it has fastergrowth than other Streptomyces, S. venezuelae was preferentialas host for the verification of the procedure. In fact, we also con-firmed the application of the strategy in other Streptomyces such asS. avermitilis and S. coelicolor (SI Appendix, Fig. S2).To demonstrate the convenience of this method, we identified

195 native or synthetic promoters and 192 RBSs. In total, 15native or engineered promoters were chosen and insertedupstream of the sfGFP gene. As shown in Fig. 4A, the kasOp*promoter, originally obtained by Wang and coworkers (36),exhibited the strongest activity among all the promoters. Its activityis 20-fold higher than that of the widely used ermEp* promoter. Forthe purpose of acquiring stronger and versatile promoters, thekasOp* promoter was used as a template to construct two randomlymutated libraries. The first library was to randomize the nucle-otides downstream of the −10 sequences of kasOp*, and the

other library was made by mutating the spacer sequence betweenthe −10 and −35 regions of kasOp* to increase the diversity of thepromoters (SI Appendix, Fig. S3A). In the two libraries, six of180 synthetic promoters displayed stronger activity than that ofkasOp*, with five of them from the first library. In comparisonwith the original kasOp* promoter, the activities of the syntheticpromoters varied from 0.95 to 187.5%. Then we sequenced 44 ofthe promoters with gradually increased strength to facilitate fur-ther application (Fig. 4A and SI Appendix, Fig. S3B).Similarly, 15 native and 174 synthetic RBSs were characterized

in this study at the same time. Among the 15 native RBSs, theRBS of capsid protein from phage ϕC31 (37) showed the highestactivity (Fig. 4B and SI Appendix, Table S3). Thus, four librariesoriginated from the RBS of capsid protein were established toelicit a variety of RBSs. To obtain stronger RBSs, we firstlyrandomized the sequence either up- or downstream of the Shine-Dalgarno sequence (SI Appendix, Fig. S3A). As a result, 66 of130 RBSs were stronger than the template RBS, thereby re-vealing the potential of optimizing the local structure of mRNAto improve translational activity. Second, we partially mutatedthe core Shine-Dalgarno sequence to degenerate the strength ofthe RBS to obtain weaker RBSs. On the whole, we acquired 177synthetic RBSs with activity covering a 200-fold range (SI Ap-pendix, Fig. S3C) and sequenced 41 RBSs with varying strengthamong them (Fig. 4B).Moreover, the result of the time-course experiment (SI Ap-

pendix, Fig. S4) reconfirmed that the engineered short and strongpromoter was less likely to interfere with the global regulatoryfactors (36), in comparison with the previously reported regula-tory elements, thereby providing a great opportunity to rationallydesign the pathway-specific regulatory system for a gene cluster.Therefore, the characterized synthetic promoter and RBS li-braries are of high application value for activation and optimi-zation of cryptic secondary metabolic pathways.

The RiboJ Insulator Enables Predictable Combination of Promotersand RBSs in S. venezuelae. As regulatory elements, promotersand RBSs are responsible for the transcriptional and translationalactivities of their downstream genes, respectively. However, the twofunctional elements might interfere with each other by the pro-moter escaping process (38) or by the formation of some structuresinaccessible to ribosomes on the messenger RNA. Different in-sulators have been reported to eliminate the unanticipated regulatory

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Fig. 2. Distinguishing viable individuals from dead ones by PI staining.Images of the PI-stained mycelium (A) and protoplasts (B) of S. venezuelaeharboring kasOp*-driven sfGFP were taken by a confocal laser-scanningmicroscope. (C and D) The dead individuals were separated from the viableones by PI staining. A weak promoter (ermEp*) (C) and a strong promoter(kasOp*) (D) were both demonstrated to show the advantage of PI staining.

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Fig. 3. Correlation of GFP expression with mRNA abundance, measured byquantitative RT-PCR. Open circle, gapdhp (SG); solid circle, rpsLp (SA); opensquare, rpsLp (RE); solid square, ermEp*; solid triangle, gapdhp (KR); solidupside-down triangle, rpsLp (TP); open triangle, rpsLp (CF); open upside-down triangle, kasOp*. Error bars, data are presented as mean ± SDobtained from at least three experiments performed on different days.

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element interaction in E. coli (33, 39). Here, we introduced thewell-elucidated RiboJ insulator between promoter and RBS toimprove the predictability of the combination. To test the mod-ularity and predictability of the insulated promoter and RBS, the

75-nt RiboJ was inserted between the pairwise combined sevenpromoters and nine RBSs, resulting in 63 promoter–RiboJ–RBSregulatory cassettes upstream of sfGFP (Fig. 5B). As for control,the RiboJ sequence was removed from these 63 combinatorialsequences (Fig. 5A). Moreover, a full factorial ANOVA modelwas applied to quantify the contribution of the interfered pro-moter::RBS (interaction term), the promoter, and the RBS to theGFP expression (40). ANOVA revealed that the contribution ofpromoter::RBS interaction without RiboJ (31%) (Fig. 5C) is muchlarger than that observed with RiboJ (3%) (Fig. 5D), indicatingthat the RiboJ insulator can also effectively eliminate the un-predictable interaction between the promoter and RBS. For in-stance, antagonistic interaction was observed in the combinationof a strong promoter (P6) and a strong RBS (R9) without RiboJ(Fig. 5E), whereas the GFP expression exhibited a modest cor-relation between promoters and RBSs with the presence of RiboJ(Fig. 5F). The contributions of other elements were comparable inthe two circumstances (promoter: 58% and 47% with and withoutRiboJ, respectively; RBS: 38% and 21% with and without RiboJ,respectively) (Fig. 5 C and D). Because the remaining experimentalerror contributes to only 1% in both cases, it was taken as a neg-ligible factor. These findings demonstrated that the RiboJ insulatorcould further improve the designability of the activation and opti-mization of secondary metabolic pathways in Streptomyces.

Application of the Synthetic Modular Regulatory Elements forLycopene Overproduction in S. avermitilis. Lycopene synthase pro-teins are highly conserved but rarely expressed in most Strepto-myces. We therefore put effort into activating the silent lycopenebiosynthetic gene cluster in S avermitilis to explore the potentialof the universal synthetic modular regulatory elements for acti-vating cryptic gene clusters. Considering that there is enoughsupply of the universal acyclic precursors GPP, FPP, and GGPPin S. avermitilis, a set of synthetic promoters with RiboJ were insertedupstream of the lycopene biosynthetic cluster in S. avermitilis. Asexpected, we observed a correlation between lycopene production

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Fig. 5. Performance of the RiboJ insulator in Streptomyces to eliminate interferences between the promoter and RBS. (A and B) A scheme of the combinedseven promoters and nine RBSs to form a full combinatorial library of expression control elements without RiboJ (A) or with RiboJ (B). Heat maps show GFPfluorescence for all combinations of promoter (P, columns) and RBS (R, row) elements driving the expression of GFP reporter gene for the with-RiboJ (F) andwithout-RiboJ (E) cases. Each value was obtained by flow cytometry with three experimental duplications. Analysis of variances by full factorial ANOVA wasperformed for the without-RiboJ (C) and with-RiboJ library data (D). Promoter: GSV, gapdhp (SV); GSA, gapdhp (SA); GSG, gapdhp (SG); RTP, rpsLp (TP); GCF,rpsLp (CF); REL, gapdhp (EL); KSC, kasOp*. RBS: TER, terminase (ϕC31); TAP, tape measure protein (ϕC31); KSC, kasO (SC); TAI, tail protein (ϕC31); NUK,nucleotide kinase (ϕC31); HEL, helicase (ϕC31); CAP, capsid protein (ϕC31); GSG, GAPDH (SG); RCF, 30s ribosomal protein S12 (CF).

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Fig. 4. Relative strength of native and synthetic promoters and RBSs eval-uated in S. venezuelae ISP5230. (A) Relative strength of 15 native or engi-neered promoters (gray) and 44 sequenced synthetic promoters (white) withkasOp* (*) as the reference. The widely used promoter ermEp* is shown inred. (B) The relative strength of 15 native RBSs (gray) and 41 sequencedsynthetic RBSs (white) with the RBS of capsid protein from phage ϕC31 (*) asthe reference. Error bars, data are presented as mean ± SD obtained from atleast three experiments performed on different days.

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and promoter strength, and the highest production reached82 mg/g dry cell weight (Fig. 6 and SI Appendix, Table S7). Theextent of overproduction validated the strength of our syntheticpromoters, and the correlation between lycopene titer and pro-moter strength highlighted the predictive superiority endowed bythe addition of the insulator.Indeed, these synthetic promoters and insulator circumvent

the stringent endogenous regulatory system and not only activatethe gene cluster of lycopene but also facilitate overproduction.Insulation by RiboJ enables rational combination of promoters andRBSs, which will make the precise control of protein expressionlevel and the production of desirable metabolites possible.

ConclusionQuantitative characterization of regulatory elements in Strepto-myces is very important to secondary metabolic pathway re-constitution. The flow cytometry-based method has improved theresolution of the quantification of gene expression in the fila-mentous bacteria; that is, from the mycelium level to the single-cell level. The quantitative PCR experiments indicated that themRNA abundance was consistent with the cognate GFP ex-pression level. For further exploitation, we developed a versatiletoolbox of regulatory elements in Streptomyces by elucidationof 195 native or synthetic promoters and 192 RBSs. Moreover,an insulator was introduced to further improve the modularityand predictability of the regulatory elements. Indisputably, thequantitative method developed here and the modular promoterand RBS libraries have tremendous potential on the activation ofmore complex cryptic gene clusters enabling the discovery ofnew bioactive natural products and promote the rational designof heterologous pathways in Streptomyces.Siegl and colleagues developed a synthetic promoter library

for actinomycetes based solely on the −10 and −35 consensussequences of the widely used ermEp* promoter (26). Through aquantitative method, we can improve the modularity and greatlyincrease the dynamic range of these regulatory elements by up to1,000-fold. As a whole, synthetic promoters are advantageousover native promoters because they are less likely to underlieintracellular regulation. Moreover, Siegl’s work demonstrated

the consistent expression of the same promoters in not onlyStreptomyces lividans TK24 and Streptomyces albus J1074 but alsorare actinomycetes such as Salinispora tropica CNB-440 andSaccharothrix espanaensis DSM 44229. Therefore, the success ofactivating lycopene overproduction in S. avermitilis indicates thatour regulatory elements could be applied as well in other acti-nomycetes. In conclusion, the universal synthetic modular regu-latory elements we constructed will be beneficial to the syntheticbiology community and will facilitate the drug discovery process instreptomycetes as well as actinomycetes in general.

Materials and MethodsStrains, Media, and Growth Conditions. E. coli strains were cultivated at 37 °Cin Luria-Bertani medium or on Luria-Bertani agar plates. DH5α and ET12567(pUZ8002) were used as E. coli hosts for plasmid construction and E. coli–Streptomyces conjugation, respectively. Mannitol soya flour medium wasused for conjugation, and malt extract–yeast extract–maltose medium wasused for liquid inoculation of the spores of S. venezuelae ISP5230. Themedia were supplemented with different antibiotics at appropriateconcentrations as follows: ampicillin at 100 μg/mL, apramycin at 50 μg/mL,nalidixic acid at 25 μg/mL, kanamycin at 25 μg/mL, and chloramphenicol at25 μg/mL Spores of S. avermitilis WT transformants were used to inoculate a250-mL flask containing 25 mL seed medium [glucose (5 g), soy flour (15 g),and yeast extract (5 g) per liter, at pH 7.2], and the culture was allowed togrow with shaking (220 rpm) at 30 °C for 2 d. A 1-mL aliquot of the culturewas used to inoculate a 250-mL flask containing 50 mL production medium[glucose (60 g), (NH4)2SO4 (2 g), MgSO4•7H2O (0.1 g), K2HPO4 (0.5 g), NaCl(2 g), FeSO4•7H2O (0.05 g), ZnSO4•7H2O (0.05 g), MnSO4•4H2O (0.05 g),CaCO3 (5 g), and yeast extract (2 g) per liter at pH 7.0] (41). The samples wereincubated with shaking (220 rpm) at 28 °C for 5 d.

Preparation of Protoplasts from Streptomyces. Broth culture (1 mL) containingthe mycelia was spun down and washed by 10% (wt/vol) sucrose once andresuspended in 1 mL of the P10 buffer supplemented with 5 mg/mL of ly-sozyme. After incubation at 37 °C for 1 h, protoplasts were filtered throughcotton wool to remove the mycelia. For high-throughput manipulation, 24-square deep well microtiter plates were used for the cultivation of Streptomyces,and 96-well filter plates with cottonwool were used for preparation of protoplasts.

PI Staining. Before the FACS test, dilute 100 μL protoplasts suspension with900 μL PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 atpH 7.4) supplemented with 0.5 M NaCl, 1 mg/mL kanamycin, and 5 μg/mL PI.The samples were kept in the dark at room temperature (25 °C) for 5 min.The PI-stained protoplasts were measured by flow cytometry with an exci-tation wavelength of 488 nm and an emission wavelength of 585 nm.

Confocal Laser-Scanning Fluorescence Microscopy Analysis of S. venezuelae.The PI-stained samples of liquid mycelium and protoplasts were observed un-der Leica SP8 confocal laser-scanningmicroscopeat excitationwavelengths of 488and 561 nm and emission wavelengths of 500∼550 nm (green) and 570∼650 nm(red), respectively. Images were merged using the Leica confocal software.

Quantitative Measurement of GFP Expression by Flow Cytometry. The pro-toplasts were analyzed by BD FACSCalibur Flow Cytometer with a 488-nmexcitation laser and the FL1 (530/30 nm band-pass filter) detector. Eachsample collected 50,000 events, and the data were further acquired using BDFACSuite software and analyzed by FlowJo 9.3.2 software (Tree Star, Inc.). Theparameters of the FACS setting were as follows: FSC-E00, SSC-650, FL1-400,FL2-400; threshold: FSC-50, SSC-400. The fluorescence of each sample was thegeometric mean of all of the measured cells and was normalized to thecorresponding FSC value, which indicates the size of the cells.

Statistical Analysis. A heat map was generated from FACS data reflecting GFPexpression by MATLAB R2011b (MathWorks, Inc.). ANOVA was performed bySPSS v. 22.

ACKNOWLEDGMENTS.We thank Keqian Yang andHuarong Tan for the helpfuldiscussions and comments on the manuscript, and thank Guoqing Niu, XiaolanZhang, Pei Huang, Jiaqian Cao, and Jingjing Xu for the technical help. This workwas supported by funding in part from theMinistry of Science and Technology ofChina (Grants 2013CB734000 and 2011CBA00805) and the National NaturalScience Foundation of China (Grant 31470818). L.Z. is an awardee of the NationalDistinguished Young Scholar Program in China.

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