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First published online April 23, 2004; 10.1104/pp.104.040139 Plant Physiology 135:25-38 (2004) © 2004 American Society of Plant Biologists
High-Throughput Fluorescent Tagging of Full-Length Arabidopsis Gene Products in Planta1Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794–5215 (G.-W.T., B.P., C.D.K., J.L., V.C.); Watson School of Biological Sciences (C.D.K.) and Cold Spring Harbor Laboratory (A.M., D.J.), Cold Spring Harbor, New York 11724; Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521–0124 (N.C., G.D., N.V.R.); and The Arabidopsis Information Resource (S.L., S.Y.R.) and Carnegie Institution of Washington (S.L., D.E., S.Y.R), Stanford, California 94305
We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.
Plants function as a complex network of interacting cell types, tissues, and organs. In turn, each cell represents an equally complex network of morphologically and functionally diverse subcellular structures. A comprehensive, systems-based understanding of plant physiology, morphogenesis, and development is impossible without thorough knowledge of protein expression and localization patterns within these supracellular and subcellular domains. Thus, there is a need for a sensitive high-throughput assay to simultaneously determine native subcellular localization and expression patterns of plant proteins in vivo (Girke et al., 2003  ).
To visualize localization and expression within individual cells and tissues, proteins are usually labeled with either a reporter or an antigenic tag, or are detected by specific antibodies. Enzyme reporters, such as
In most cases, the use of GFP as a protein expression marker has not been fully optimized. For example, GFP is typically fused to the N or C terminus of a target protein (Rolls et al., 1999 Tagged proteins are often expressed from a strong constitutive rather than from the native promoter, producing the protein in cells or under conditions where it does not normally function. Furthermore, overexpression may disrupt multiprotein complexes or can mask subtle localization patterns when there is an overabundance of the tagged protein, e.g. during plasma membrane targeting. To circumvent these problems, we designed a high-throughput strategy, termed Fluorescent Tagging of Full-Length Proteins (FTFLP). This procedure tags proteins at a selected internal site and incorporates the native gene regulatory sequences. The tagged proteins are then stably expressed in transgenic Arabidopsis plants to avoid ambiguities in interpretation of data from transient expression. Here, we describe how this experimental approach could be used for characterization of the Arabidopsis proteome.
The FTFLP Technique Fluorescent tagging of proteins to study their expression and localization patterns should ideally conform to the following three criteria: (1) the fluorescent reporter should be stable over the range of physiological conditions found in different subcellular compartments, such as pH, (2) the insertion of the reporter should minimally disturb the conformation of the target protein and preserve native targeting signals and posttranslational modification sites, and (3) expression of the tagged protein should occur from its native regulatory sequences to faithfully detect its developmental and/or tissue-specific regulation. Our FTFLP approach fulfills these three requirements.
As a fluorescent tag, we used the citrine variant of yellow fluorescent protein (YFP) (Griesbeck et al., 2001
We flanked the YFP open reading frame with flexible linker peptides (Doyle and Botstein, 1996
To further avoid effects of the YFP tag on native subcellular localization, the location of the tag relative to the target sequence was determined for each individual protein, based on computer-assisted predictions of protein folding and functional domains. We reasoned that it should be possible to devise a default tag location for most target proteins. The tag should ideally reside within a stretch of hydrophilic residues, outside of any specific protein domain, and near the C terminus. Thus, our default strategy was to insert the YFP tag 30 bp (10 amino acids) upstream of the stop codon. This location minimizes disturbance of the contiguous protein sequence, and protects the activity of membrane anchoring signals typically found within a few C-terminal amino acid residues (Casey, 1995
To faithfully reproduce the expression level and pattern of each target gene, our constructs included the 5' UTR and promoter sequences, the coding region with introns, and the 3' UTR and downstream sequence. Most intergenic distances in the Arabidopsis genome are around 2 kb, suggesting that the promoter sequences are contained in a relatively small region (The Arabidopsis Genome Initiative, 2000
We designed an efficient protocol for generation and in planta expression of tagged genes (Fig. 1). We first amplified the selected gene in two fragments, one from the predicted start of the promoter to the YFP insertion site (i.e. between primers P1 and P2 in Fig. 1), and the second comprising the rest of the coding sequence and 3'sequences (i.e. between primers P3 and P4 in Fig. 1). In addition to gene-specific sequences, primers P1 and P4 were tailed with sequences complementary to the Gateway primers (see below), and P2 and P3 were tailed with sequences complementary to the YFP linkers. We also amplified the YFP coding sequence with flanking linkers (Fig. 1). In a second round of PCR, all three amplified fragments acted as overlapping templates for long flanking homology (LFH) PCR (Wach, 1996
For stable expression in transgenic plants, each YFP-tagged gene was cloned and transferred into an Agrobacterium binary vector. For high throughput, we used the Gateway system (Invitrogen, Carlsbad, CA), which is based on bacteriophage  site-specific recombination (Landy, 1989). The Gateway cloning first produced a donor construct, containing the amplified TT-PCR product in the donor vector, pDONR207 (Invitrogen; Landy, 1989; see also www.invitrogen.com). The tagged gene was next recombined in vitro into the destination vector (Landy, 1989; see also www.invitrogen.com). A binary destination vector was constructed by subcloning the Gateway conversion cassette C.1 (Invitrogen) into the promoter-less T-DNA region of pBIN19, resulting in the pBIN-GW vector. To augment native expression of genes with relatively weak promoters, we also constructed a binary destination vector with tetramerized CaMV 35S enhancers in the T-DNA region of pMN20 (Weigel et al., 2000), resulting in pMN-GW. In our experiments, the efficiency of the first recombination reaction into the donor plasmid was 80% to 90%, whereas recombination into the binary destination vector was 100% efficient (data not shown). Following recombination into the donor vector, we completely sequenced all tagged gene clones to estimate the rate of sequence errors introduced by PCR. Each amplified gene carried between zero and three amino acid substitutions, corresponding to average amino acid substitution rate of 0 to 0.3/1 kb of amplified sequence. Additional details and step-by-step experimental protocols for FTFLP can be found on our web site, http://aztec.stanford.edu/gfp/. As proof of concept for the FTFLP approach, we fluorescently tagged a number of Arabidopsis genes whose protein products have diverse, known subcellular locations, such as the peroxisome, vacuole, plasma membrane, ER, Golgi, nucleus, and cytosol. Transgenic plants expressing these genes were produced by Agrobacterium-mediated transformation and examined for expression of the tagged transgenes in various plant organs, such as roots, hypocotyls, and cotyledons. On average, 20 to 40 primary transgenic plants were screened per construct, using a fluorescence dissecting microscope, followed by detailed analysis by confocal laser scanning microscopy. In most cases, 80% to 90% of plants exhibited fluorescent protein expression. In the following sections, we describe selected examples of patterns of subcellular localization and expression of known proteins (reference genes) as well as several proteins with unknown function and unassigned subcellular localization.
Peroxisomal Targeting
To demonstrate that the fidelity of subcellular targeting was not limited to protein tagging with YFP, we also tagged MFP2 and Pex5 with CFP. Figure 2 illustrates that the CFP-tagged MFP2 (sections D–F) and Pex5 (sections G–I) exhibited peroxisomal targeting identical to that of YFP-tagged MFP2 and Pex5, in different plant tissues, such as cotyledons, hypocotyls, leaves, and roots. The CFP-tagged genes also showed higher levels of expression but the same subcellular targeting specificities in the presence of the 35S enhancers (data not shown).
Tonoplast Membrane Targeting
Plasma Membrane Targeting Plasma membrane intrinsic protein 2A (PIP2A) of Arabidopsis, a channel protein (Cutler et al., 2000 ), was used to visualize protein targeting to the plasma membrane. YFP-tagged PIP2 expressed from its native promoter in the absence (not shown) or in the presence of 35S enhancers localized exclusively at the extreme cell periphery in all plant tissues, including hypocotyls (Fig. 4A), cotyledons (data not shown), and roots (Fig. 4B). This localization pattern could be interpreted as either plasma membrane or cytosolic because the cytoplasm is typically displaced to the cell periphery by the large central vacuole. However, the cytosolic compartment is characterized by transvacuolar strands, variations in cytosol thickness at the cell cortex, and rapid remodeling over time due to cytosolic streaming (Cutler et al., 2000). The absence of these patterns, together with the uniform and sharp pattern of peripheral labeling by YFP-PIP2A, indicates that the localization was in fact at the plasma membrane (also compare with cytosolic patterns in Fig. 7). We also distinguished plasma membrane targeting from cell wall localization by plasmolysis experiments, where the YFP-PIP2A fluorescent signal associated with the displaced membrane in plasmolysed cells (Fig. 4C).
Cell Wall Targeting Proline-rich protein 2A (PRP2) resides in the cell wall (Fowler et al., 1999 ). This specific subcellular localization was faithfully reproduced using the PRP2 gene tagged with CFP and expressed with 35S enhancers in hypocotyls (Fig. 4D), cotyledons, and roots (data not shown). The cell wall localization of the fluorescent signal remained after plasmolysis (data not shown), and was also observed using the YFP-tagged PRP2 expressed with and without 35S enhancers (data not shown).
Targeting to Plasmodesmata
Targeting to Cytoskeletal Elements
Targeting to the Nuclear Membrane and Proplastids Arabidopsis MFP1 associated factor (MAF1) preferentially localizes to the nuclear envelope and to proplastids (Gindullis et al., 1999 ; Jeong et al., 2003). YFP-tagged MAF1 exactly reproduced this subcellular localization pattern in roots (Fig. 6A) and leaves (data not shown), accumulating in a distinct perinuclear ring as well as associating with proplastid-like structures. Again, expression from the construct with 35S enhancers did not alter the subcellular targeting of the protein (data not shown).
Nuclear Targeting and Cell-Specific Expression VirE2-interacting protein 2 (VIP2, GenBank accession no. AF295433) of Arabidopsis is homologous to yeast Not2p and Drosophila Rga proteins, which are thought to mediate intranuclear interactions between chromatin proteins and the transcriptional complex (Frolov et al., 1998 ). Indeed, GUS fusions to VIP2 accumulate in the nucleus of tobacco and Arabidopsis cells (V. Citovsky, unpublished data). Here, our analysis of transgenic Arabidopsis expressing YFP-tagged VIP2 confirmed the nuclear targeting of this protein (Fig. 6, B and C). Virtually no signal was detected in the cytoplasm, indicating efficient nuclear import. Indeed, because the predicted size of the citrine-VIP2 fusion protein (approximately 85 kD) is substantially larger than the size exclusion limit of the nuclear pore (approximately 60 kD; reviewed in Dingwall and Laskey, 1991; Garcia-Bustos et al., 1991), its accumulation within the nucleus must result from the active process of nuclear import. The YFP-tagged VIP2 gene had an intriguing cell-specific pattern of expression. Figure 6 shows that the tagged protein was detected only in megasporocytes (section B) and tapetum cells (sections C and D), but it was not detected in seedling roots, leaves, or hypocotyls (data not shown). This specificity of VIP2 expression was confirmed by reverse transcription (RT)-PCR, which detected VIP2 transcripts in the flower buds (lane 1) and mature flowers (lane 2), but not in roots, leaves, or stems (lanes 3–5, respectively) of wild-type Arabidopsis (Fig. 6E). In control experiments, analysis of actin-specific transcripts generated similar amounts of PCR products in all samples, indicating equal efficiencies of the RT-PCR reactions (Fig. 6F). Thus, transgenic expression of the YFP-tagged VIP2 revealed novel information about the native cell-specific expression pattern of this gene as well as subcellular localization of its protein product.
Another example of an Arabidopsis nuclear protein is the VirE2-interacting protein 1 (VIP1), a basic Leu zipper protein thought to function in transcriptional complexes and, during Agrobacterium infection, facilitate nuclear import of VirE2 (Tzfira et al., 2001
Cytosolic Localization
To examine whether this tagged gene showed its known transcriptional regulation, we germinated the seedlings in light or in the dark. The YFP-tagged GSR1 exhibited the expected light-induced expression (Peterman and Goodman, 1991
More than one-third of Arabidopsis genes have no predicted function (Wortman et al., 2003 Figure 8 shows that At2g16530, augmented with 35S enhancers, was expressed predominantly in root tissues (sections A–C), especially in lateral root primordia (section C). The tagged protein exhibited perinuclear localization (Fig. 8B, arrow), remarkably similar to that of MAF1 (see Fig. 6A). Some of the tagged protein also accumulated at the cell periphery in the cytoplasm (Fig. 8, A–C). Native expression of A1g27090 was observed mainly in guard cells, in the cytoplasm, and in unidentified subcellular structures (Fig. 8D, arrows). At2g15240 was expressed in the root and accumulated in the cytoplasm (Fig. 8E), but was not detected in other organs (data not shown). At1g12170 was expressed specifically in the root tip (Fig. 8F, compare to expression of At2g16530 throughout the root in Fig. 8A) and was cytoplasmic (Fig. 8F). Finally, At1g80940 was exclusively nuclear in the leaf (Fig. 8G) and in petioles (Fig. 8H).
The complete sequence of the Arabidopsis genome has laid the foundation for functional characterization of its proteome. Elucidation of protein function often begins with homology-based predictions. However, in the absence of significant homology, determination of expression pattern and subcellular localization provide important clues to the potential function(s) of a gene. Thus, it is important to design a simple, reliable, and efficient procedure to directly assay native expression and subcellular targeting specificities of proteins in planta. To this end, we developed the FTFLP protocol, which is distinguished by three major features: (1) the fluorescent tag is inserted into the protein internally, minimizing interference with targeting signals at the N and C termini, (2) the tagged gene includes native 5', intron, and 3' regulatory sequences, allowing detection of the native promoter activity, and (3) the tagging procedure is simple, involving only two sequential PCR reactions followed by efficient recombination-based cloning into Agrobacterium binary vectors. These aspects make FTFLP the ideal methodology for high-throughput characterization of unknown Arabidopsis gene products. As proof of concept, we utilized FTFLP to tag Arabidopsis gene products with known subcellular targeting specificities. Each of the selected genes was tagged with the citrine variant of YFP and the cyan variant of GFP (CFP) using TT-PCR. We could efficiently amplify gene fusions up to 8 kb, corresponding to a median length of nontranscribed region of 1.5 kb, a transcribed region of 4.0 kb, and 5' and 3' regulatory sequences of 3.0 kb and 1.0 kb, respectively. Our analysis of the Arabidopsis genome revealed that approximately 40% of all its genes fall within these values, indicating that the FTFLP approach is suitable for tagging of a substantial proportion of the Arabidopsis proteome. Although the PCR resulted in some amplification errors, and an amino acid substitution rate of 0 to 0.3/kb, these changes had no effect on the known subcellular localization patterns of the reference proteins we tested. Indeed, most subcellular targeting sequences are short and partially redundant, so single amino acid changes would not be expected to change protein targeting.
To stably express the tagged proteins, we made two Gateway destination binary vectors. For native expression, pBIN-GW has a Gateway cassette in its T-DNA region and no additional regulatory sequences. The second vector, pMN-GW, carries a tetramerized CaMV 35S enhancer that functions to elevate gene expression without altering tissue-specific expression patterns (this work and Weigel et al., 2000
As any other technique, the current version of our FTFLP protocol has several limitations. First, notwithstanding our careful analysis of the predicted structure of each tagged protein for optimal tag placement, the internal YFP or CFP tag may still interfere with native targeting signals of some proteins. Furthermore, even if several tagged proteins retain their native localization patterns, e.g. peroxisomal or nuclear localization, other proteins with similar targeting specificities may be perturbed by the tag or by enhanced levels of expression from 35 enhancer-containing vectors. These considerations are especially important when tagging proteins with unknown function (see below), for which so little is known about the location of their functional domains, even using the Interpro database (Mulder et al., 2003
A second application for FTFLP is a high-throughput characterization of the Arabidopsis proteome. From close to 30,000 genes in the Arabidopsis genome, almost 30% could not be assigned to a functional category (Wortman et al., 2003
Conversion of Agrobacterium Binary Plasmids into Gateway Destination Vectors The pBIN19 plasmid was digested with EcoRI and HindIII, and the ends were filled-in with Klenow. Into these blunted ends, we ligated a blunt-ended Gateway conversion cassette (reading frame C.1; Invitrogen catalog no. 11797016). The resulting Gateway destination vector, designated pBIN-GW, has the following structure in its T-DNA region: T-DNA right border-NOS terminator<-NPTII<-35S promoter-attR1->CAT->ccdB->attR2-T-DNA left border. This vector has no regulatory sequences for expression of cloned genes and, thus, is useful for producing native levels and patterns of gene expression.
Using a similar strategy, the pMN20 activation tagging plasmid (Weigel et al., 2000
Both pBIN-GW and pMN-GW vectors were propagated in the DB3.1 strain of E. coli (Invitrogen) strain carrying the gyrA462 gene which confers resistance to the toxicity of the ccdB gene (its protein product, a natural analog of the quinolone antibiotics, binds to the DNA gyrase subunit A and turns it into a poison; Bahassi et al., 1999
YFP and CFP tags were amplified from the pRSETB-Citrine (Griesbeck et al., 2001
For each gene, two sets of primers, P1/P2 and P3/P4 (see Fig. 1), were designed by a Perl script that takes into account the annealing temperature, the position of each primer within the genomic and protein sequence, and primer length and secondary structure in an iterative fashion until a suitable pair is detected; it uses part of the Primer3 program (available at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). For all unknown genes, TIGR's genome release version 4.0 was used. We obtained Interpro (Mulder et al., 2003
For the second PCR reaction (TT-PCR, see Fig. 1), a pair of standard, gene-nonspecific Gateway primers were designed. The forward primer, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCTCGATCCACCTAGGCT-3', contained the attB1 sequence, and the reverse primer, 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCGTAGCGAGACCACAGGA-3', contained the attB2 sequence. These primers were combined with three templates, i.e. the fluorescent tag and two gene fragments, in 20 µL of a mixture containing 100-ng long fragment (P1-P2), 50-ng short fragment (P3-P4), 50-ng YFP or CFP fragment, 1x ExTaq reaction buffer, 0.2 mM dNTP, 0.2 µM of each Gateway primer, and 0.02 units/µL ExTaq. TT-PCR was performed under the following conditions: 1 cycle at 95°C for 3 min; 20 cycles at 93°C for 30 s, 54°C for 30 s, and 68°C for 1 min/1 kb of the longest amplified sequence; and 1 cycle at 68°C for 1 min/1 kb of the longest amplified sequence.
The TT-PCR product was gel-purified as described above and recombined into the Gateway donor vector pDONR207 (Invitrogen) in 10 µL BP Clonase (Invitrogen) reaction containing 300-ng TT-PCR fragment, 150-ng pDONR207, 2 µL BP Clonase, and 2 µL BP Clonase buffer. Following overnight incubation at 25°C, 1 µL Proteinase K (2 µg/µL) was added, and the incubation continued for 10 min at 37°C, after which 1 to 2 µL of the reaction mixture was transformed into the DH5
Finally, the tagged genes were transferred from their donor constructs to both of the binary destination vectors pBIN-GW and pMN-GW in 10 µL of a single LR Clonase reaction containing 150 to 200 ng donor construct, 200 ng pBIN-GW and pMN-GW (1:1 w/w mixture), 5 units topoisomerase (Invitrogen), 2 µL LR Clonase (Invitrogen), and 2 µL LR Clonase buffer. After overnight incubation at 25°C, 1 µL of Proteinase K (2 µg/µL) was added, and the incubation continued for 10 min at 37°C, following which 2 µL of the reaction mixture was transformed into the DH5
The binary constructs were introduced into the A. tumefaciens strain GV3101 and the resulting bacterial cultures were used to transform Arabidopsis ecotype Columbia by the standard flower dip method (Clough and Bent, 1998
Live seedlings and plant tissue samples were mounted in water between number 1 1/2 coverglasses, using silicon vacuum grease to create spacers between the glass surfaces. Images were collected with one of three laser scanning confocal microscope systems, a Leica TCS SP2/UV (Wetlzer, Germany), a Zeiss LSM 5 Pascal (Jena, Germany), or a Bio-Rad MRC 1024 (Hercules, CA). In all cases, a high numerical aperture (1.2–1.3) water immersion objective (60–63x) was employed. A 488-nm or a 514-nm laser line from an argon ion was used to excited YFP and a 442-nm line from a HeCd laser or a 457-nm line from an argon ion laser was used to excite CFP.
Total RNA was extracted from 2.0 g of roots, leaves, stems, flower buds, and mature flowers of wild-type Arabidopsis using TRI Reagent (Molecular Research Center, Cincinnati), and reverse-transcribed with M-MuLV reverse transcriptase using the dT23VN primer (BioLabs). The resulting first strand cDNAs were PCR-amplified as described (Kang et al., 1995 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AF295433.
We thank Dr. Tsien for his generous gift of the pRSETB-Citrine plasmid. This is Carnegie publication number 1673. Received February 3, 2004; returned for revision March 9, 2004; accepted March 12, 2004.
1 This work was supported by a grant from the 2010 Project of the National Science Foundation to V.C., D.E., D.J., N.R., and S.R. 
2 These authors contributed equally to the paper. www.plantphysiol.org/cgi/doi/10.1104/pp.104.040139. * Corresponding author; e-mail jacksond{at}cshl.org; fax 516–367–8369.
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ABSTRACT
RESULTS
-glucuronidase (GUS; Jefferson, 1987
or DH10B strain of E. coli, and the recombinants were selected on a gentamycin-containing medium. Clones with the correct inserts were identified by colony PCR using a pair of vector-specific attL primers, i.e. forward attL1 primer: 5'-TCGCGTTAACGCTAGCATGGATCTC-3', and reverse attL2 primer: 5'-GTAACATCAGAGATTTTGAGACAC-3'.