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Targeting specificity of nuclear-encoded organelle proteins witha self-assembling split-fluorescent protein toolkitMayank Sharma1,*, Carola Kretschmer2, Christina Lampe1, Johannes Stuttmann2 and Ralf Bernd Klosgen1

ABSTRACTA large number of nuclear-encoded proteins are targeted to theorganelles of endosymbiotic origin, namely mitochondria and plastids.To determine the targeting specificity of these proteins, fluorescentprotein tagging is a popular approach. However, ectopic expression offluorescent protein fusions commonly results in considerablebackground signals and often suffers from the large size and robustfolding of the reporter protein, which may perturb membrane transport.Among the alternative approaches that have been developed in recentyears, the self-assembling split-fluorescent protein (sasplit-FP)technology appears particularly promising to analyze protein targetingspecificity in vivo. Here, we improved the sensitivity of this technologyandsystematicallyevaluated its utilization to determine protein targetingto plastids and mitochondria. Furthermore, to facilitate high-throughputscreening of candidate proteins we developed a Golden Gate-basedvector toolkit (PlaMinGo). As a result of these improvements, dualtargeting could be detected for a number of proteins that had earlierbeen characterized as being targeted to a single organelle only. Theseresults were independently confirmed with a plant phenotypecomplementation approach based on the immutansmutant.

This article has an associated First Person interviewwith the first authorof the paper.

KEY WORDS: Self-assembling split-GFP, Protein transport,Mitochondria, Chloroplast, Dual targeting, Golden Gate cloning

INTRODUCTIONMany nuclear-encoded proteins are targeted across cellularmembranes to reach their final destination in the cell, i.e. a sub-cellular compartment or cell organelle. To determine the specificityof such targeting, fluorescent protein tagging – the fusion of thecandidate protein with a fluorescent reporter (e.g. green fluorescentprotein, GFP) and subsequent in vivo imaging by fluorescencemicroscopy – is a popular approach. However, this widely utilizedmethod carries some inherent pitfalls (reviewed by Moore andMurphy, 2009). Firstly, the large size and intrinsic folding propertiesof the reporter protein might perturb membrane transport of thecandidate protein (Marques et al., 2004). Secondly, considerablebackground fluorescence signals occur as a result of proteinoverexpression, and hence saturation of the organelle transportmachinery is sometimes observed (Sharma et al., 2018a). And

finally, proteins targeted to an organelle in low amounts are difficultto visualize using this approach owing to weak fluorescence signalsoriginating from a limited number of protein molecules.

These constraints become particularly prominent when it comes tothe analysis of proteins targeted to mitochondria and/or plastids (Tanzet al., 2013). These organelle-targeted proteins usually carry an N-terminal transport signal (or transit peptide) to facilitate transport ofthe passenger protein across the membranes via organelle-specifictranslocation machineries. Amongst a repertoire of proteins encodedin the plant cell nucleus, more than 3000 are transported intomitochondria and/or plastids (Van Wijk and Baginsky, 2011; Raoet al., 2017).Whilemost of these proteins are targeted to only one typeof organelle, a group of proteins exists with dual-targeting specificity,i.e., they carry an ‘ambiguous’ transit peptide capable of translocatingthe passenger protein into both mitochondria and plastids (reviewedin Sharma et al., 2018b). Despite having dual-targeting properties,some of these proteins are still preferentially targeted to one of the twoorganelles. In this case, determining targeting specificity using aconventional fluorescent protein-tagging approach is particularlytedious and error-prone, as highly intensive fluorescence signalscoming from one organelle can mask signals of low-intensity comingfrom the other (Duchêne et al., 2005; Sharma et al., 2018a).

One possibility to circumvent these problems is to detect thefluorescence signals coming from each organelle in separate cells.This has become technically feasible with the invention of self-assembling split green fluorescent protein (sasplit-GFP) technology(Cabantous et al., 2005). Spontaneous self-assembly of split-GFPrelies on two highly engineered fragments derived from the‘superfolder’ GFP variant (sfGFP). The large fragment, GFP1-10OPT (hereafter referred to as GFP1-10), comprises ten N-terminalantiparallel β-sheets of GFP. The smaller fragment, GFP11 M3(hereafter referred to as GFP11), comprises only 16 amino acids andrepresents the C-terminal eleventh β-sheet of GFP. When broughtinto close proximity, the two fragments can assemble spontaneouslyto reconstitute the functional fluorophore without the need of anadditional interacting partner (Fig. 1A) (Cabantous et al., 2005). Forsubcellular protein localization studies, GFP1-10 is fused to atransport signal of known organelle specificity and analyzedtogether with a chimeric protein comprising the candidate proteinfused to GFP11. Fluorescence complementation is achievedspecifically and exclusively if the transport signal of the candidatemediates transport of GFP11 into the organelle housing GFP1-10.The fluorescence signal remains limited to the compartmentcontaining GFP1-10, irrespective of whether the GFP11-fusedcandidate protein is targeted to further subcellular compartments ornot (Fig. 1B). Thus, sasplit-GFP technology allows selective in vivoimaging of a protein of interest in a respective compartment withenhanced signal-to-noise ratio. Furthermore, the small-sized GFP11tag could be hypothesized to have a lower propensity for interferingwith membrane transport in comparison to the full-lengthfluorescent proteins used for direct fluorescent protein tagging.Received 12 February 2019; Accepted 1 May 2019

1Institute of Biology–Plant Physiology, Martin Luther University Halle-Wittenberg,Weinbergweg 10, 06120 Halle (Saale), Germany. 2Institute of Biology–Genetics,Martin Luther University Halle-Wittenberg, Weinbergweg 10, 06120 Halle (Saale),Germany.

*Author for correspondence (mayank796@gmail.com)

M.S., 0000-0002-6476-6262

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© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs230839. doi:10.1242/jcs.230839

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The sasplit-GFP system has already been applied to a wide rangeof organisms and adapted to elucidate a variety of cellular functionsor processes including, for example, in vivo protein solubility, sub-cellular localization of pathogen effectors, endogenous proteinlabeling for in vivo imaging, protein–protein interaction studies andmembrane protein topology determination (Cabantous and Waldo,2006; Van Engelenburg and Palmer, 2010; Machettira et al., 2011;Cabantous et al., 2013; Kamiyama et al., 2016; Henry et al., 2017).The principal suitability of the sasplit-GFP system for in vivoimaging of protein targeting in plant cells has also beendemonstrated recently (Park et al., 2017). In this case, transgenicArabidopsis thaliana lines expressing the organelle-targeted GFP1-10 receptor were transiently transformed with constructs expressinga candidate protein fused to the GFP11 tag. However, therequirement of transgenic ‘receptor’ plant lines and low transienttransformation efficiency hampers the utilization of this approachfor high throughput protein targeting studies. Along with a limitedyield of transformed cells, the relatively low brightness of sasplit-GFP prevented the visualization of proteins targeted in low amountsinto the organelles.Therefore, we systematically evaluated and optimized the sasplit-

GFP system for analysis of protein targeting specificity in plant cellsand assessed the effect of multimerization of the GFP11 tag on theintensity of the fluorescence signals inside mitochondria andplastids. A Golden Gate-based vector toolkit named PlaMiNGo(analysis of plastid and/or mitochondrial targeted proteinsN-terminally fused to GFP11 tags via Golden Gate cloning) wasdeveloped to facilitate high-throughput analyses of candidate

proteins. With this approach, dual targeting to mitochondria andplastids could be detected for several proteins that were previouslycharacterized as being targeted to a single organelle only.Importantly, plastid targeting of two of these proteins wasindependently confirmed using a phenotype complementation-based approach in stable transgenic Arabidopsis plants, whichproves the in vivo relevance of results obtained with the PlaMiNGosystem.

RESULTSThe sasplit-GFP system to determine protein targetingspecificityIn order to study protein targeting into plastids, the transitpeptide (FNR1-55) of a chloroplast protein, ferredoxin-NADP+

oxidoreductase (PETH) of spinach (Zhang et al., 2001), was fusedto the GFP1-10 receptor (the large fragment of the sasplit-GFPsystem) to facilitate its localization into plastids. For mitochondriallocalization of GFP1-10, the N-terminal 100 amino acid residues(mtRi1-100) comprising the pre-sequence of the mitochondrialRieske iron-sulfur protein (FES1) of potato (Emmermann et al.,1994) were used. Both transport signals had previously beencharacterized for their targeting specificity to a single organelle within vivo and in vitro approaches (Rödiger et al., 2011). In the initialexperiments, evaluating the suitability of the sasplit-GFP system forour purposes, each transport signal was likewise combined with aGFP11 tag (the small fragment of sasplit-GFP system). A sevenfoldrepeat of this GFP11 tag (GFP11×7; separated via a five-amino-acidlinker) was used, as such multiple GFP11 tags had been reported to

Fig. 1. Establishment of the sasplit-GFP system for in vivo organelle imaging. (A,B) Schematic drawings illustrating the principle of the sasplit-GFPsystem. (A) Two non-fluorescing fragments, GFP1-10 and GFP11, can self-assemble to generate a fluorescing GFP molecule. (B) Transport of bothGFP chimeras into the same organelle is essential to achieve fluorescence signals. tp, transit peptide. (C,D) The coding sequences of FNR1-55/GFP1-10 andFNR1-55/GFP11×7 (C), and mtRi1-100/GFP1-10 and mtRi1-100/GFP11×7 (D) were transiently co-expressed after Agrobacterium co-infiltration into the lowerepidermis of Nicotiana benthamiana leaves and analyzed by confocal laser scanning microscopy (CLSM). Image acquisition of transformed cells was donewith 20× objective in several z-stacks, which were subsequently stacked for maximum intensity projection. Representative cells (left panels) are presented asoverlay images of the chlorophyll channel (red) and the GFP channel (green). The strong chlorophyll signals in the background are derived from the largerchloroplasts of untransformed mesophyll cells underneath the epidermal cell layers. The squares highlight areas of the transformed cells that are shown inhigher magnification separately for the GFP channel and the chlorophyll channel, as indicated. Scale bars: 20 μm.

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intensify the fluorescence signals in mammalian cells (Kamiyamaet al., 2016). When these FNR1-55/GFP11×7 and mtRi1-100/GFP11×7constructs were co-expressed with the FNR1-55/GFP1-10 andmtRi1-100/GFP1-10 gene constructs, fluorescence signals wereexclusively obtained in those instances in which the sametransport signal was present in both chimeras, i.e. in plastids afterco-expression of FNR1-55/GFP1-10 and FNR1-55/GFP11×7 andin mitochondria after co-expression of mtRi1-100/GFP1-10 andmtRi1-100/GFP11×7 (Fig. 1C,D). When infiltrated alone, neither ofthese constructs generated any detectable fluorescence signal. Thechimeric GFP1-10 accumulated in the organelles and could bedetected in protein extracts from the infiltrated leaf tissue (Figs S1,S2). This demonstrates the suitability and specificity of the sasplit-GFP system for the analysis of protein targeting.Next, we wanted to evaluate the performance of this system

by determining the protein-targeting behavior of several dual-targeted proteins. For this purpose, we selected three previouslycharacterized nuclear-encoded organelle proteins from Arabidopsisthaliana with proven dual-targeting characteristics, namely TyrRS(tyrosyl-tRNA synthetase; At3g02660), GrpE (co-chaperoneGrpE1; At5g55200), and PDF (peptide deformylase 1B;At5g14660) (Berglund et al., 2009; Baudisch et al., 2014). Theexperiments employing eYFP (enhanced yellow fluorescentprotein) fusions indicated that all three proteins are targeted toboth endosymbiotic organelles as seen previously (Sharma et al.,2018a), either in comparable amounts (TyrRS1-91/eYFP) orpreferentially to either mitochondria (GrpE1-100/eYFP) orchloroplasts (PDF1-100/eYFP) (Fig. 2). The respective N-terminalamino acid sequences carrying the organelle transport signals of thecandidate proteins were fused to GFP11×7 tags and analyzed in oursystem. All three candidates (TyrRS1-91/GFP11×7, GrpE1-100/GFP11×7 and PDF1-100/GFP11×7) showed targeting to bothplastids and mitochondria when co-transformed with therespective organelle-targeted receptors, FNR1-55/GFP1-10 andmtRi1-100/GFP1-10 (Fig. 2). Even plastid targeting of GrpE1-100/GFP11×7 was clearly visible, which is remarkable considering thevague fluorescence signals obtained in this organelle with theclassical fluorescent reporter fusion (Fig. 2B). Thus, separation ofthe fluorescence signals for the two organelles into different cellsproved to be advantageous to determine the low plastid-targetingproperties of GrpE.

Multimerization of the GFP11 tag leads to fluorescencesignal enhancement in plastids but not in mitochondriaIn the initial experiments described above, we used GFP11×7, thesevenfold repeat of the GFP11 tag. However, the requirement orbenefits of such multiple GFP11 tags to enhance fluorescencesignals in mitochondria and plastids of plant cells were notsystematically assessed. Hence, we compared the fluorescencesignal intensity obtained in the two organelles with either seven(GFP11×7), three (GFP11×3) or a single (GFP11×1) repeat of theGFP11 tag when fused to the dual-targeted protein transport signalof TyrRS. It turned out that the use of a single GFP11 tag yieldsonly very faint signals in plastids while the GFP11×3 and GFP11×7tags significantly enhance the fluorescence signals (Fig. 3A). Incontrast, for mitochondria, no correlation between the number ofGFP11 tag repeats and fluorescence signal intensity was found.Instead, the signal intensities were largely similar for all threeconstructs (Fig. 3B). However, since the fluorescence signalsobtained with the single GFP11 tag in mitochondria were brighterthan in plastids, they are usually sufficient for proper visualizationof this organelle.

Construction of the PlaMiNGo toolkitTo facilitate easy combination of sasplit-GFP technology andfluorescence signal enhancement for high-throughput screening ofprotein targeting specificity, we developed a set of Golden Gate-based vectors. Designed to analyze the targeting specificity ofcandidate proteins to plastids and mitochondria, these vectorsenable rapid cloning of the candidate genes upstream of single ormultiple GFP11 tags. In this PlaMiNGo toolkit, we used highlyefficient gene regulatory elements, namely a ‘long’ 35S promoter(Engler et al., 2014) and ocs or rbcs E9 transcriptional terminators(De Greve et al., 1982; Coruzzi et al., 1984), to control expression ofthe chimeric genes (Fig. 4A; Fig. S3). Moreover, to avoid therequirement for performing co-transformation using two plasmids,we used a single transfer (T)-DNA expression system (Grefen andBlatt, 2012; Hecker et al., 2015) comprising two gene expressioncassettes. This has the advantage that each transformed cellexpresses both chimeras simultaneously. Consequently, the finalT-DNA vectors contain two gene expression cassettes between leftand right border elements. One of the expression cassettes encodesGFP1-10 gene chimeras to be targeted to either plastid ormitochondria by fusion with mtRi1-100 or FNR1-55, respectively,while the other expression cassette encodes either one, three orseven repeats of GFP11 tags. The latter expression cassetteadditionally carries a ccdB cassette upstream of the respectiveGFP11 tag, which can be replaced by the candidate gene in a singleGolden Gate reaction step, allowing for background-free selectionof positive clones. As a result, six vectors, three destined to analyzepotential plastid-targeting and three for mitochondria-targetinganalysis, were generated (Fig. 4A; Fig. S3).

To evaluate the functionality of these vectors, the dual-targetingtransport signal of TyrRS (N terminal 1–91 amino acids) was clonedupstream of the GFP11 tags of all six vectors and analyzed viaAgrobacterium-mediated transient transformation of Nicotianabenthamiana leaves. In comparison to the previous experimentsinvolving co-infiltration of two vectors, the signal intensities weresignificantly improved ∼ninefold in plastids and ∼threefold inmitochondria with the use of GFP11×7 and GFP11×1 tags,respectively (Fig. 4B; Fig. S4). Now, even in plastids a singlecopy of the GFP11 tag was sufficient for reliable detection of thereconstituted GFP fluorescence, although considerable signalenhancement could still be observed with GFP11×3 and GFP11×7tags. This suggests that the GFP11×7 tag, when co-expressed withthe plastid-targeted GFP1-10 receptor, should allow for detectioneven of minute amounts of plastid-localized proteins. Likewise,upon imaging of mitochondria the fluorescence signals obtainedwith the single GFP11 tag were stronger than the signals obtainedin the previous experiments using separate vectors. Still, asobserved earlier, no further improvement of signal intensitiesby multimerization of the GFP11 tag could be obtained inmitochondria (Fig. S4). Instead, artificial protein aggregates wereobserved in some cells expressing constructs with multiple GFP11tags (GFP11×3 and GFP11×7) (Movie 1).

Multicolor imaging of dual-protein targeting to twoorganellesA further objective of the study was to establish simultaneousmulticolor imaging with the sasplit-FP system. For this purpose, wemodified the GFP1-10 receptor to generate a yellow-shifted variant(YFP1-10) using a single amino acid substitution (T203Y) asreported earlier (Kamiyama et al., 2016). To test if this variant canassemble with GFP11 to generate a functional fluorophore insidethe organelles, the plastid (FNR1-55) or mitochondrial (mtRi1-100)

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Fig. 2. Comparison of fluorescent protein tagging and sasplit-GFP approach. The coding sequences of (A) TyrRS1-91/GFP11×7, (B) GrpE1-100/GFP11×7and (C) PDF1-100/GFP11×7 were transiently expressed with either FNR1-55/GFP1-10 (middle panels) or mtRi1-100/GFP1-10 (right panels) via Agrobacteriumco-infiltration into the lower epidermis of Nicotiana benthamiana leaves and analyzed by CLSM. For comparison the respective eYFP fusions, namely (A)TyrRS1-91/eYFP, (B) GrpE1-100/eYFP, and (C) PDF1-100/eYFP, were also transiently expressed and analyzed by CLSM (left panels). Both GFP and eYFPfluorescence signals are shown in false green color. For further details see the legend of Fig. 1. Scale bars: 20 μm.

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transport signals were separately fused to the N-terminus of YFP1-10. These fusions were co-infiltrated with a gene construct encodingthe dual-targeting transport signal TyrRS1-91 fused to eitherGFP11×1 or GFP11×7. When imaged with an YFP-specific filterset, fluorescence signals were solely obtained in mitochondria (withmtRi1-100/YFP1-10 and TyrRS1-91/GFP11×1) or in plastids (withFNR1-55/YFP1-10 and TyrRS1-91/GFP11×7), demonstrating that theYFP1-10 fragment can indeed assemble with GFP11 in bothorganelles (Fig. S5A,B).Next, we tested if multicolor imaging, i.e. the simultaneous

labeling of plastids and mitochondria with YFP1-10 and GFP1-10,respectively, within the same cell is possible. For this purpose, theFNR1-55/YFP1-10 fusion was co-infiltrated with a pMiNGo11-1:TyrRS vector comprising mtRi1-100/GFP1-10 and TyrRS1-91/GFP11×1. This should result in transformed cells co-expressingthe dual-targeted GFP11 (via fusion with TyrRS1-91) and twodifferent receptors targeted to two different organelles, namelyYFP1-10 to plastids (FNR1-55/YFP1-10) and GFP1-10 tomitochondria (mtRi1-100/GFP1-10). Indeed, the resultingtransformed cells emitted fluorescence signals of different spectrain the two organelles owing to reassembly of the GFP11 tag withboth the GFP1-10 and YFP1-10 receptors within mitochondria andplastids, respectively (Fig. S5C). However, in all transformed cells acertain degree of bleed-through of signals, i.e., the appearance of

YFP fluorescence signals in GFP channel and vice versa, could bedetected. The adjustment of the filter sets to avoid such bleed-through inevitably led to significant reduction of the fluorescencesignal intensity. In summary, the YFP1-10 derivative of the sasplit-GFP system is not yet perfect for multicolor imaging but representsa promising basis for development of such tools.

Analysis of protein targeting specificity with PlaMiNGoFinally, we examined the suitability of the PlaMiNGo toolkit usingeight candidates with presumed targeting specificity either toplastids, mitochondria, or to both organelles (Table 1). Three ofthese proteins, namely Gtred (Monothiol glutaredoxin-S15, alsoknown as GRXS15), GCS (Glycine cleavage system H protein 1,also known as GDH1) and GAPDH (Glyceraldehyde-3-phosphatedehydrogenase B, also known as GAPB) had earlier been reportedto be dual-targeted (Baudisch et al., 2014). Two other candidates,namely FNR (also known as PETH) and RbcS (small subunit ofRubisco from pea), are well-characterized plastid proteins(Highfield and Ellis, 1978; Zhang et al., 2001), while the residualthree candidates, namely mtRi (also known as FES1), ATPS (ATPsynthase subunit beta-3; At5g08680) and CoxIV (Cytochrome coxidase subunit IV) of yeast, are known for their mitochondrialtargeting specificity (Maarse et al., 1984; Emmermann et al., 1994;Baudisch et al., 2014). The protein fragments comprising the

Fig. 3. Effect of multiple GFP11 tags on fluorescence signal intensity. The gene-coding sequence of the dual-targeting transit peptide TyrRS1-91 wasfused with different numbers of GFP11 tags and co-expressed with either (A) FNR1-55/GFP1-10 or (B) mtRi1-100/GFP1-10 in Nicotiana benthamiana lowerepidermal cells and analyzed via CLSM. Image acquisition of transformed cells was done with 20× objective in several z-stacks, which were subsequentlystacked for maximum intensity projection. In the histograms, each bar represents mean±s.d. of average pixel brightness from nine maximum intensityprojected images, obtained from two independent experiments. ‘Measure’ feature of Fiji was utilized for quantification of pixel brightness. Scale bars: 50 μm. A.U.,arbitrary units.

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transport signals of these proteins were cloned as fusions withGFP11 tags into the PlaMiNGo vectors, additionally comprising theorganelle-targeted receptor fusions FNR1-55/GFP1-10 or mtRi1-100/GFP1-10.Five of the eight candidate proteins showed the same targeting

behavior in our assay system as reported in the literature (Table 1):mtRi and FNR showed exclusive transport into either mitochondriaor plastids, respectively (Fig. 5A,B) (Rodiger et al., 2011), and thedual-targeting candidates GCS, Gtred and GAPDH showedtransport into both organelles (Baudisch et al., 2014) (Fig. 5C–E).It should be noted though that in the case of GAPDH, mitochondrialtargeting was rather weak in our assays and could be observed only

in a few transformed cells. However, it was rather unexpected thatthe remaining three monospecific candidates, namely ATPS1-100,RbcS1-79 (SSU1-79) and CoxIV1-29, showed dual targeting in ourexperiments (Fig. 6). In the literature, mitochondrial targeting ofRbcS has already been described once (Rudhe et al., 2002) but theseresults were solely based on in vitro assays. In addition, dualtargeting of the yeast mitochondrial pre-sequence, CoxIV1-29, couldbe assumed considering the high degree of freedom of non-plantmitochondrial transport signals (Staiger et al., 2009). However, thisdual targeting was entirely unexpected for the plant mitochondrialprotein ATPS1-100, which has never previously been shown to targetto plastids when analyzed with in vivo fluorescent protein tagging

Fig. 4. PlaMiNGo toolkit. (A) Schematic representation of the vectors generated (see Fig. S3 for details of cloning process). (B) The dual-targeted TyrRS1-19

transit peptide was cloned in all six PlaMiNGo vectors to replace the ccdB cassette at the N-terminus of GFP11 tags (as indicated), and used to transformNicotiana benthamiana lower epidermis cells viaAgrobacterium infiltration. Image acquisition of transformed cells was donewith 20× objective in several z-stacks,which were subsequently stacked for maximum intensity projection. In all instances, fluorescence signals were observed in the respective organelles. Thequantification of signals is presented in Fig. S4. Scale bars: 50 μm.

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and in vitro protein transport experiments (Baudisch et al., 2014)(Fig. S6A).

Phenotype complementation confirms the plastid targetingproperties of ATPSThese unexpected results demanded independent confirmation.Thus, to re-evaluate the plastid-targeting properties of the transportsignal of mitochondrial ATPS, we applied a phenotypecomplementation approach. In this approach, we made use of theimmutans mutant of Arabidopsis thaliana that shows a white-greensectored (variegated) leaf phenotype and stunted growth whengrown under daylight conditions (Fig. 7A,B). The mutantphenotype is caused by the absence of a functional nuclear-encoded plastid protein, namely plastid terminal oxidase (PTOX,also known as AOX4) (Carol et al., 1999; Aluru et al., 2001).Complementation of the variegated phenotype of immutans requiresa functional PTOX protein in plastids (Fu et al., 2005) (Fig. 7). Toadapt PTOX for our analysis, we replaced the authentic transitpeptide of PTOX with the N-terminal 100 amino acid residues oftwo candidate proteins, namely ATPS and GCS (a validated dual-targeted protein), generating ATPS1-100/mPTOX and GCS1-100/mPTOX, respectively. Two further constructs encoding either theauthentic PTOX precursor or the transit peptide-free mature PTOXprotein (mPTOX) were generated for comparison. These genechimeras were expressed under the control of the CaMV35Spromoter in the immutans mutant background. As expected, boththe authentic PTOX precursor and GCS1-100/mPTOX were able tocomplement the variegated phenotype (Fig. 7C,E). In contrast, theexpression of mPTOX cannot complement the mutant phenotype(Fig. 7D). These results confirm that targeting of PTOX into plastidsis essential for complementation of the immutans variegatedphenotype. Remarkably, the expression of ATPS1-100/mPTOXalso resulted in mutant phenotype complementation (Fig. 7F).However, such complementation was observed in only four out ofsix transgenic lines analyzed, suggesting that plastid targeting ofATPS1-100/mPTOX is, in principle, possible but apparently lessefficient than with typical plastid-targeted transit peptides. Theseresults clearly underline the basic plastid-targeting property ofmitochondrial protein ATPS and thus re-confirm the resultsobtained with the sasplit-GFP technology established here.

DISCUSSIONThe goal of this study was to assess the suitability of the signal-enhanced sasplit-GFP system to determine the targeting specificityof nuclear-encoded organelle proteins and to develop tools for rapidcloning and subsequent analysis of their targeting behavior. The

characterization of the targeting specificity of organelle proteins iscrucial (i) to elucidate the functional properties of organelletransport machineries, (ii) to study evolutionary aspects of proteintargeting, and (iii) for biotechnological applications employingorganelles. The application of the sasplit-GFP system, asdemonstrated in this study, provides a novel toolbox to quicklydetermine the targeting properties of candidate proteins with highsensitivity.

Selective imaging and fluorescence signal enhancementwith sasplit-GFP technologySelective imagingThe selective imaging of organelles is one of the major advantagesof the sasplit-GFP system in comparison with classical fluorescentprotein tagging approaches. The requirement for the presence of thenon-fluorescing GFP1-10 receptor in a specific subcellular locationis the key for selective imaging (Kaddoum et al., 2010). In thisstudy, two transport signals, namely FNR1-55 and mtRi1-100, wereselected for localization of the receptor specifically within two sub-cellular locations, the plastid stroma and the mitochondrial matrix,respectively. As a result, fluorescence signals will appear only if theGFP11-tagged protein is completely imported into the same sub-cellular location and not if a protein is merely binding to theorganelle surface. This was otherwise difficult to distinguish withfluorescent protein-based approaches, specifically for mitochondriabecause of their small size.

Fluorescence signal enhancementThe self-assembling split-GFP molecules have been reported toproduce fluorescence signals of lower intensity than classicalfluorescent proteins (Köker et al., 2018). This problem can becircumvented with the use of multiple GFP11 tags. However, thetwo organelles respond differently to this modification. Whilesignal enhancement with multiple GFP11 tags works well inplastids, fluorescence signal enhancement could not be observed inmitochondria (Fig. 3). One possible reason might be the sizedifference between these organelles. Plastids are comparativelylarger in size and the proteins in the plastid stroma are moredispersed. Consequently, the chances for self-assembly of sasplit-GFP fragments in this organelle are lower than in mitochondria andthus fluorescence signal enhancement could be observed byincreasing number of GFP11 tags in plastids. Furthermore,differences in the physicochemical properties of the twoorganelles, e.g. pH, might likewise contribute to this phenomenon.

The use of more efficient gene regulatory elements, i.e. promoterand terminator, in the PlaMiNGo toolkit also led to significant

Table 1. Sub-cellular localization of candidate proteins as determined with different approaches

Candidate sasplit-GFP* In vitro import‡ FP localization Gene accession

FNR Chloro Chloro2 Chloro2 M86349.1mtRi Mito Mito2 Mito2 X79332.1GCS Mito and chloro Mito and chloro2 Mito and chloro2 At2g35370GAPDH Mito and chloro Mito and chloro2 Chloro2 At1g42970Gtred Mito and chloro Mito and chloro2 Mito and chloro2, chloro4, mito5 At3g15660CoxIV Mito and chloro NA Mito6 SGD:S000003155ATPS Mito and chloro Mito2 Mito2 At5g08680RbcS Mito and chloro Mito and chloro1 Chloro3 XM_016585367

*This study.‡Import studies were performed with the authentic precursor proteins. 1Rudhe et al., 2002; 2Rödiger et al., 2011 and Baudisch et al., 2014; 3Nelson et al., 2007;4Cheng, 2008; 5Moseler et al., 2015; 6Köhler et al., 2003.NA, not available. Mito, mitochondria. Chloro, chloroplasts or plastids. FP, fluorescent protein.

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enhancement in fluorescence signal intensity. However, incombination with multiple GFP11 tags, such increased geneexpression can lead to the formation of aggregations in transformedcells, particularly if these tags are combined with mitochondria-targeting transport signals. The intrinsic property of the sasplit-GFPfragments to form dimers and aggregates (Cabantous et al., 2005) andcomparatively less efficient unfoldase activity of the mitochondrial

protein translocation machinery (Agarraberes and Dice, 2001) couldbe one of the possible reasons for this phenomenon. Since proteinunfolding prior to translocation is apparently more efficient inplastids, the multiple GFP11 tags can be efficiently imported into thisorganelle. By contrast, high expression of plastid-targeted GFP1-10alone could result in the appearance of faint fluorescence signals,probably due to the formation of dimers. In most instances, these faint

Fig. 5. Analysis of candidate proteinswith the PLaMiNGo toolkit. Genefragments encoding the transportsignals of either (A) FNR, (B) mtRi,(C) GCS, (D) Gtred or (E) GAPDH, werecloned upstream of GFP11×1 orGFP11×7 tags, in the two PlaMiNGovectors comprising either mtRi1-100/GFP1-10 (left panels) or FNR1-55/GFP1-10 (right panels), respectively.The resulting constructs were usedto transform the lower epidermis ofNicotiana benthamiana leaves viaAgrobacterium infiltration. For furtherdetails, see the legend of Fig. 1. Scalebars: 20 μm.

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signals are clearly distinguishable from ‘actual’ fluorescence signalsobtained via self-assembly of split-GFP, but experimental controlsshould still take these signals into account to avoid themisinterpretation of results (Fig. S7).

High sensitivity of sasplit-GFP systemFluorescence signal enhancement in combination with selectiveimaging makes the sasplit-GFP system highly sensitive with respectto targeting specificity determination. Subsequently, dual targetingof several proteins was newly detected with the PlaMiNGo toolkitdeveloped here, which had previously been missed owing to theinherent limitations of classical fluorescent protein taggingapproaches. For example, GAPDH shows dual targeting with thesasplit-GFP system, in line with the results of in vitro importexperiments (Baudisch et al., 2014). In contrast, when analyzedwith the classical fluorescent protein tagging approach, GAPDHappeared to be solely transported into plastids (Baudisch et al.,2014). Similarly, the transit peptide of RbcS is able to translocatethe GFP11 tag into mitochondria, but this property remainedundetected with the fluorescent protein tagging approach.Remarkably, supporting the above observation, such dualtargeting of the RbcS transit peptide was also witnessed in arecent study employing sulfadiazine-resistant plants (Tabatabaeiet al., 2019). Conversely, the dual targeting of the yeast CoxIVtransport signal had never been reported previously. The plastidtargeting of the yeast mitochondrial transport signal might be aconsequence of the fact that yeast does not contain plastids and thusthe transport signal of yeast mitochondria has not evolved to be able

to distinguish between the two endosymbiotic organelles (Huanget al., 1990; Staiger et al., 2009). However, such dual targeting wasmost unexpected for the transport signal of ATPS because neitherin vitro nor in vivo approaches had previously given any hint ofits plastid-targeting characteristic (Baudisch et al., 2014). Eventransgenic plants expressing ATPS1-100/eYFP did not show anyplastid localization (Fig. S6). However, with the use of the sasplit-GFP system the dual-targeting specificity of ATPS1-100 was clearlydetectable and this result could be independently confirmed by aphenotype complementation approach using immutans mutants(Fig. 6A, Fig. 7F). These observations highlight the high sensitivityof the sasplit-GFP technology over classical fluorescent proteintagging approaches. Nonetheless, it cannot be completely excludedat this point that the observed high sensitivity of the sasplit-GFPsystem is due to the use of vector sets carrying highly efficientregulatory elements.

Modularity of PlaMiNGO toolkitThe PlaMiNGO vector toolkit is based on the principle of modularcloning (Weber et al., 2011). Hence, the components of the toolkitcan easily be rearranged for in vivo imaging of proteins targeted tovarious other sub-cellular compartments. The vectors constructed inmodule I and II (Fig. S3) are binary vectors and can be utilized forplant cell transformation via Agrobacterium or via several othermethods, e.g. protoplast transformation or particle bombardment.The gene of interest can be cloned upstream of one of the GFP11tags of module I vectors with a single Golden Gate cloning reaction.Similarly, GFP1-10 can be targeted to the different sub-cellular or

Fig. 6. Dual localization of presumedmonospecific candidate proteins.Gene fragments encoding the transportsignals of either (A) ATPS, (B) CoxIV or(C) RbcS (SSU), were cloned upstreamof the GFP11×7 tag in the two PlaMiNGovectors comprising either mtRi1-100/GFP1-10 (left panels) or FNR1-55/GFP1-10 (right panels). The resultingconstructs were used to transformthe lower epidermis of Nicotianabenthamiana leaves via Agrobacteriuminfiltration. For further details, see thelegend of Fig. 1. Scale bars: 20 μm.

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even sub-organellar compartments via cloning of the specifictransport signal N-terminally in a Golden Gate ‘ready’ vectorpTEI177. These vectors carry a ccdB negative selection cassetteupstream of GFP1-10 or GFP11 tags for background-free cloning ofthe candidate gene. Consequently, the two vectors carrying GFP11and GFP1-10 gene chimeras should be co-expressed in a single cellin order to determine protein targeting specificity to the organelle ofinterest.

Significance of high dual targeting frequencyThe results obtained in this study strongly suggest that the number ofdual-targeted proteins is much higher than previously assumed.More than just increasing the list of such proteins, these findingsalso highlight the evolutionarily conserved nature of organelletranslocation machineries in plant cells. Even if some of theseproteins are mistargeted to the wrong organelle, for example due toprotein overexpression, the technique reveals the fundamentaltargeting properties of the respective transport signal. The reasons forsuch widespread dual targeting of nuclear-encoded proteins cannotbe clearly defined at this stage. On the one hand, it supports thehypothesis that dual targeting is an evolutionary remnant (Staigeret al., 2009). On the other hand, it highlights that the acquisition ofdual-targeting properties might still be an ongoing process, whichallows for the development of completely new biochemical pathwaysin an organelle (Martin, 2010; Xu et al., 2013).

MATERIALS AND METHODSMolecular cloningGeneration of vectors for co-infiltrationThe GFP1-10 and GFP11×7 fragments were amplified by PCR fromplasmids pcDNA3.1-GFP (1-10) and pACUH-GFP11×7-mCherry-β-tubulin(deposited by the Bo Huang lab; Addgene #70218 and #70219) and clonedinto pRT100mod-based vectors (Baudisch et al., 2014) either withdigestion–ligation or with restriction-free cloning (Bond and Naus, 2012).Primers for gene amplification are summarized in Table S1. The genesequence coding for the N-terminal 91 amino acids of TyrRS was amplifiedfrom a vector provided by Elzbieta Glaser (Stockholm University, Sweden)and cloned accordingly (Baudisch et al., 2014). The above constructs,comprising promoter and terminator regions (CaMV35S::Gene of interest:

GFP1-10/GFP11×7::t35S), were later sub-cloned into a Golden Gate-compatible pLSU4GG binary vector using a BsaI restriction–ligationreaction (Erickson et al., 2017). Golden Gate cloning was performed in a15 µl reaction with the following conditions: 2.5 units of T4 DNA ligase(Thermo Fisher Scientific), 5 units of BsaI (Thermo Fisher Scientific),1×BSA, 20 cycles of incubation at 37°C for 2 min and 16°C for 5 min, finaldeactivation and denaturation at 50°C for 10 min and 80°C for 10 min,respectively. The construction of respective eYFP chimeras is described inSharma et al. (2018a).

Construction of the PlaMiNGo toolkitThemodular cloning principle and DNA fragments of the Plant Parts I and IItoolkits were used for vector construction (Weber et al., 2011; Engler et al.,2014; Gantner et al., 2018). The modules utilized for cloning of GoldenGate-based vectors are illustrated in Fig. S3. Golden Gate reactions wereperformed with 20 fmol of each DNA module with the followingconditions: 2.5 units of T4 DNA ligase (Thermo Fisher Scientific),5 units of BsaI or BpiI (New England Biolabs), 30 cycles of incubation at37°C for 2 min and 16°C for 5 min, final denaturation at 80°C for 10 min.When required, the restriction–ligation reactions were subsequentlysupplemented with fresh ligation buffer and ligase for terminal ligation,and incubated for ≥3 h at 16°C. Ligation mixtures were transformed intoDh10b or ccdB survival II cells (Thermo Fisher Scientific) and grown onplates with appropriate selective medium. PCR primers for module 0cloning and gene sequences are summarized in Tables S2 and S3. TheYFP1-10 construct was generated by introducing a point mutation in GFP1-10. Two fragments were amplified via PCR from GFP1-10 template usingprimer set 1 (GFPf- tttgaagacataATGTCCAAAGGAGAAGAAC; YFPr-tttgaagacatGataTGAGAGGTAGTGATTATCAG) and primer set 2 (YFPf-tttgaagacattatCAAACAGTCCTGAGCAAAG; GFPr-tttgaagactaaagCTA-ACTTCCGCCGCCACCTG), and later ligated via BpiI Golden Gatereaction to generate YFP1-10.

Cloning of candidate proteins into PlaMiNGo vectorsThe candidate targeting signals (TyrRS1-91, FNR1-55, mtRi1-100, ATPS1-100,GCS1-100, GAPDH1-100, Gtred1-100, RbcS1-79 and CoxIV1-29) wereamplified from the corresponding cDNA templates (Nelson et al., 2007;Berglund et al., 2009; Baudisch et al., 2014) and cloned via standard GoldenGate reaction (see above) into PLaMiNGo vectors in exchange for a ccdBnegative selection cassette, upstream of GFP11 tags. Overhangs of thefragment to be cloned were AATG at the 5′ end and TTCG at the 3′ end. Two

Fig. 7. Complementation analysis of the immutansmutant of Arabidopsis thaliana. (A,B) Phenotypeof wild-type Col-0 (A) and immutans mutant (B) plants.(C–F) Gene constructs encoding either the full-lengthprecursor PTOX (PTOX) (C), the mature PTOX (mPTOX)(D) or mPTOX fused to the transit peptides of thecandidate proteins GCS (E) or ATPS (F), were used totransform immutans mutant plants. At least threeindependently transgenic lines were examined forcomplementation of the variegated immunats phenotype.The pictures are from representative 5-week-old T2generation plants.

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additional nucleotides were inserted into some of the fusions to maintain thereading frame, resulting in an additional codon for an alanine residue.

Cloning for complementation of the immutans phenotypeThe gene sequence coding for mature PTOX57-295 (mPTOX) was amplifiedfrom a cDNA clone provided by Steven Rodermel (Iowa State University,USA) and sub-cloned via restriction-free cloning into pRT100mod-basedvectors downstream of the gene sequences coding for the transport signals ofeither GCS1-100 or ATPS1-100. Additionally, the PTOX full-length gene andcoding sequence for mPTOX protein (lacking the transit peptide) werecloned into empty pRT100mod vectors, downstream of the CaMV35promoter, via restriction digestion and ligation. The candidate geneconstructs with promoter and terminator (CaMV35S::Candidate:mPTOX::t35S) were then cloned into a Golden Gate-compatible pLSU4GG binaryvector with BsaI restriction–ligation reaction.

Agrobacterium infiltration of Nicotiana benthamianaThe constructs were transformed into electro-competent cells ofAgrobacterium tumefaciens GV3101 (pMP90) (Koncz and Schell, 1986).Agrobacterium infiltrations of 6–8-week-old fully expanded leaves ofNicotiana benthamiana plants, grown in soil under long-day conditions(16 h:8 h light:dark cycle) in a greenhouse, at a day temperature of 21°C,night temperature of 19°C and 50–60% relative humidity, were performed asdescribed by Sharma et al. (2018a). For co-infiltration, each bacterial strainwas adjusted to OD600=0.8 and mixed in a 1:1 ratio prior to infiltration.

Microscopy and imagingConfocal laser scanning microscopy was carried out as described by Sharmaet al. (2018a). For GFP–YFP dual-channel imaging, the 493–518 nm (GFP)and 519–620 nm (YFP) filter ranges were used to collect the emittedfluorescence signals. When required, brightness and contrast of the imageswere equally adjusted for each image to avoid any discrepancy invisualization of signal intensities.

Signal quantificationFor quantification of signal intensities, infiltration of all relevant constructs wascarried out on different spots of the same leaf. At least three images from eachinfiltration spot were used for quantification. Quantification of the signals wasperformedwith raw images using the Fiji program (Schindelin et al., 2012). Forthe purpose of quantification, image acquisition was done with the 20×objective in 7–8 z-stacks covering the epidermal cell layer and later stacked toproject the maximum intensities. The mean gray values of stacked images werecalculated using the ‘Measure’ option of Fiji and further utilized forcomparison of the fluorescence signal strength in arbitrary units (A.U.).

Growth and transformation of immutans plantsFor the purpose of stable transformation, the Arabidopsis thaliana immutansseeds (provided by Steven Rodermel, Iowa State University, USA) weregerminated at 5 μmol m−2 s−1 light in a 8 h:16 h light:dark cycle. After threeweeks of germination, the seedlings were transferred to 50 μmol m−2 s−1

light. For induction of flowering, the plantlets were transferred to>150 μmol m−2 s−1 light and incubated in a 16 h:8 h light:dark cycle.Floral-dip transformation was performed as described by Davis et al. (2009).At least three independent T2 transgenic lines were selected in each case forthe analysis of phenotype. After germination at >150 μmol m−2 s−1 light in a8 h:16 h light:dark cycle, these plants were transferred to a 16 h:8 h light:dark cycle. Pictures presented in Fig. 7 are from 5-week-old plants grown in>150-μmol m−2 s−1 light.

AcknowledgementsWe would like to thank Theresa Ilse for assistance in cloning, Elzbieta Glaser(Stockholm University, Sweden), Martin Schattat (Martin Luther University Halle-Wittenberg, Germany) and Bo Huang (University of California, San Francisco, USA;via Addgene) for kindly providing constructs and Steven Rodermel (Iowa StateUniversity, USA) for providing immutans mutants and the PTOX clone.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: M.S., R.B.K.; Methodology: M.S., C.K., C.L., J.S.; Validation:C.K., C.L.; Formal analysis: M.S.; Investigation: M.S.; Resources: R.B.K.; Datacuration: M.S.; Writing - original draft: M.S.; Writing - review & editing: M.S.,J.S., R.B.K.; Visualization: M.S., J.S., R.B.K.; Supervision: M.S., R.B.K.; Projectadministration: M.S., R.B.K.; Funding acquisition: J.S., R.B.K.

FundingThis work is supported by Martin Luther University Halle-Wittenberg, Halle(Saale), Germany and M.S. was supported by a fellowship funded by theErasmus Mundus Action 2 program of the European Union (BRAVE project).

Data availabilityThe vectors of PlaMiNGo toolkit have been deposited to Addgene, https://www.addgene.org/browse/article/28203315/.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.230839.supplemental

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