New Gateways to Discovery

This is the authors' abstract. We don't add key points for this paper.

Dr. Pangloss was right, at least for live-cell imaging in plants, in his contention that this is “the best of all possible worlds” (Voltaire, 1759). We are now able to look inside cells in detailed ways that the fathers of microscopy, Antoni van Leeuwenhoek and Robert Hook, could not have possibly imagined (Gest, 2004). Current state-of-the-art instrumentation has made routine an array of imaging techniques that were inaccessible to most researchers just a few years ago. Additionally, the number of autofluorescent proteins (AFPs) with which we can color intercellular structures increases almost daily, thus providing ever greater flexibility in the types of experiments that can be performed. Moreover, the completion of large-scale genome sequencing projects has sparked many derivative microscopy-based projects, which are essential to put the function into functional genomics and to realize the emergent field of systems biology (Murphy, 2005; Li et al., 2006; Pepperkok and Ellenberg, 2006). For indeed, if a picture is still worth a thousand words (Orenstein, 2000), consider all that will be learned when, by microscopy en masse, we visualize the thousands of proteins predicted to be encoded in the genomes of eukaryotes in general (Brasch et al., 2004; Rual et al., 2004; Matsuyama et al., 2006) and plants in particular (Sterck et al., 2007). This review is designed primarily to introduce researchers to the myriad of factors, beyond vector selection, that should be considered prior to embarking on protein localization experiments (Table I Key points for live-cell imaging in plants Key points for live-cell imaging in plants “In living color” is somewhat of a holy grail for cell biologists. Consider what it has meant to be able to paint cellular loci in living cells at will. First there was the green fluorescent protein (GFP; Chalfie et al., 1994) from which blue fluorescent protein, cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) forms were subsequently derived (Zhang et al., 2002). After the discovery of red fluorescent protein (RFP; Matz et al., 1999), colors such as banana, orange, cherry, tomato, and plum were produced (Shaner et al., 2004). This salad of mFruits (Shaner et al., 2004) has been followed by a palette with an ever-increasing number of colors (Brandizzi et al., 2004; Shaner et al., 2005; Giepmans et al., 2006; Stewart, 2006). To be more specific, the demonstration that the GFP isolated from the jellyfish Aqueora victoriae could be linked to proteins of interest to allow in vivo examination of protein localization and dynamics in real time has transformed cell biology in a manner similar to the effect of the PCR on molecular biology. Subsequent mutagenesis of GFP led to new spectral variants, which opened doors to powerful techniques to study protein dynamics and interactions in vivo, such as fluorescence recovery after photobleaching (Ward and Brandizzi, 2004; Bates et al., 2006), fluorescence resonance energy transfer (FRET; Hink et al., 2002; Bhat et al., 2006), and bimolecular fluorescence complementation (Bhat et al., 2006; Citovsky et al., 2006; Dixit et al., 2006). The impact of GFP and its many spectral variants ushered in the search for similar proteins, DsRed from coral being the first of major significance (Matz et al., 1999). Derivatives of DsRed, which is a tetramer in its native state, led to the isolation of monomeric forms displaying different colors, collectively called mFruits (Shaner et al., 2004). Most recently, a novel monomeric RFP, TagRFP, has been described (Merzlyak et al., 2007). This protein is brighter and more resistant to photobleaching than mRFP and can be used in combination with GFP in FRET experiments. This is a particularly exciting result as GFP, despite its overwhelming popularity as fusion partner for localization studies, has been of relatively little utility in FRET-based experiments, which are typically performed with the spectral variants CFP and YFP (Jares-Erijman and Jovin, 2006). To date, more than 35 AFPs that span an emission range from blue to red of the visible spectrum (460–595 nm) have been described (Stewart, 2006). Among these, GFP, DsRed, and most of their derivatives share a common property, namely that their fluorescence cannot be regulated easily. However, new AFPs have now been discovered and developed that are photoactivatible (PA-AFPs), photoconvertible, or photoswitchable. Briefly, the fluorescence from PA-AFPs, such as PA-GFP (Patterson and Lippincott-Schwartz, 2004; Runions et al., 2006) or DRONPA (Habuchi et al., 2005) is very low, but the AFP can be activated by a brief but intense pulse of excitation at a particular wavelength. Similarly, the fluorescence of photoconvertibles (Gurskaya et al., 2006) and photoswitchables (Chudakov et al., 2006) can be changed from one color to another, typically green to red or cyan to green, respectively, by a brief pulse of light of a particular wavelength. These novel AFPs, therefore, facilitate tracking experiments in which the movement of a tagged protein from one subcellular locus to another can be monitored following activation. A further innovation is that of chromophore assisted laser inactivation (Vitriol et al., 2007), which is a light-mediated technique that can be used for AFP-tagged protein inactivation. Incorporation of these new AFPs into the vector systems described below is under way, which should be of great benefit to plant biologists seeking precise spatiotemporal control of their AFP fusion. As the number of AFPs increase, there is something of a revolution afoot with respect to microscopes by which these proteins can be detected. In the past 5 years all the major manufacturers of laser scanning confocal microscopes have commercialized instruments that permit high resolution images to be acquired at greater speed (e.g. Zeiss Live5), spectral resolution (e.g. Leica SP5), or laser synchronization (e.g. Olympus FV1000). Additionally, nonconfocal systems such as total internal fluorescence microscopes or specialized multiphoton lasers can be purchased as add-on units that are integrated into laser scanning confocal microscopes, thereby dramatically expanding their functionality. Furthermore, despite the high image quality of confocal laser scanning microscopy, the imaging frame rate is slow (typically <3 frames/s); therefore, addition of a spinning-disc unit on the confocal microscope can facilitate video-rate imaging of protein dynamics (Nakano, 2002; Wang et al., 2005). Prior to embarking on a study involving the use of AFP fusions, researchers should address two fundamental questions: (1) Where to attach the AFP to a protein of interest?, and (2) What promoter should be used to drive expression? With respect to the first question, the usual approach taken is that AFPs are fused to the amino (N) or carboxy (C) terminus. However, some proteins may not tolerate AFPs fused to one or either termini. So, it may be necessary to insert the AFP into an internal site (Tian et al., 2004; Li et al., 2006). Importantly, the use of AFPs should by no means be considered a replacement for alternative, and at times more appropriate, methods such as immunolocalization (Ruzin, 1999; Sauer et al., 2006). Additionally, proteins that contain N-terminal signal peptides may mislocalize if expressed as fusions to the C termini of AFPs (Simpson et al., 2001; Li et al., 2006). Moreover, if one is interested in studying a single protein and there is a requirement for the AFP fusion to function as closely as possible to the native protein with respect to localization, tissue specificity, timing, and level of expression, then it may be necessary to express the fusion in transgenic plants under control of the native promoter. At the other extreme, if there is a need to localize thousands of fusions in a relatively short period of time, then transient expression may be the most cost effective. It is currently impossible to use computational methods to accurately predict the effect of an AFP on a particular fusion (Li et al., 2006). Therefore, many of the current expression systems have been developed so that N- or C-terminal fusions can be tested (Tzfira et al., 2005; Earley et al., 2006; Chakrabarty et al., 2007). We consider it good general practice to test both types of fusions in preliminary studies prior to embarking on more intensive research. For example, our attempts to express the sonchus yellow net virus (SYNV) glycoprotein by fusing RFP to its C terminus were unsuccessful. Despite the fact that SYNV glycoprotein contains a predicted N-terminal signal peptide (Goldberg et al., 1991), the correctly targeted fusion was that in which RFP was placed in frame ahead of the signal peptide (Goodin et al., 2007). In a more detailed study, Simpson et al. (2001) found that approximately 50% of human proteins, including those targeted to the ER or mitochondria, localized to the same subcellular locus when expressed as N-terminal fusions to CFP or C-terminal fusions to YFP (Pepperkok and Ellenberg, 2006). While we prefer the simplicity of constructing N- or C-terminal fusions, Tian et al. (2004) have reported a highly successful breakthrough technology called fluorescent tagging of full-length proteins. Best suited to protein localization studies for plants with sequenced genomes, the fluorescent tagging of full-length proteins technique requires a triple-PCR procedure to incorporate an AFP into an internal locus near the C terminus of proteins of interest. To avoid effects of the AFP tag on native subcellular localization, the location of the tag relative to the target sequence has to be determined for each individual protein, based on computer-assisted predictions of protein folding and functional domains (Li et al., 2006). According to the authors the AFP tag should be placed within a stretch of hydrophilic residues, outside of any specific protein domain, and near the C terminus. Use of this location minimizes disturbances 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; Zhang and Casey, 1996). After deciding the best manner in which to construct AFP fusions for a specific set of experiments, the next critical choice is to determine the promoter to drive expression. Some consider that expression under the control of native promoters will always be superior to that of employing constitutive promoters, most commonly the constitutive cauliflower mosaic virus 35S promoter. However, this opinion ignores the fact that simply using the native promoter, or more often 1 to 2 kb of upstream sequence, does not take into account that promoter/gene duplication may affect expression levels, as will genomic context since the AFP fusion is unlikely to be expressed from the same genetic locus as the native gene (Bhat et al., 2006). Another common misconception is that expression from 35S, or even double-35S promoters, necessarily results in accumulation of fusion proteins at levels higher than would be achieved were the native promoter used. However, all such results are highly dependent upon the protein under investigation. Fusion of an AFP to a protein may stabilize it or make it less so. In systems such as Arabidopsis (Arabidopsis thaliana), where it is straightforward to obtain T-DNA insertional knockout alleles for particular genes of interest (Alonso et al., 2003), single gene complementation by AFP-fusion proteins implies their correct functionality in planta. Gateway-compatible binary vectors have greatly improved the cloning efficiency of AFP-tagging projects (Curtis and Grossniklaus, 2003; Earley et al., 2006). Briefly, Gateway cloning utilizes the site-specific recombination system utilized by λ phage to transfer DNA fragments between plasmids containing compatible recombination sites (Walhout et al., 2000). What makes this strategy so attractive is that once DNA clones of interest are captured into an entry vector (pDONR), they can be mobilized into a plethora of vectors that permit expression in bacteria, insect cells, yeast (Saccharomyces cerevisiae), animal, or plant cells. This avoids the frustrating situation commonly encountered with ligase-mediated cloning, namely that there are often no compatible restriction sites to permit easy mobilization from one vector to the next. This results in the need to reclone and resequence DNA fragments of interest, which may be cost and time prohibitive when done on a large scale. Once pDONR constructs are generated and validated, all downstream expression systems can be utilized, which increases the efficiency of sharing clones for genomics research (Hilson et al., 2003, 2004). Schematic representations of pSITE vectors. A, All modified pSAT6 cassettes were cloned into the pRCS2-ocs-nptII binary vector at the PI-Psp1 site. The ability to select transgenic plant cells is conferred by the nptII gene, the expression of which is controlled by the ocs promoter (P-OCS) and terminator (T-OCS). B, C-series pSITE vectors for Gateway recombination-mediated construction of binary vectors for expression of proteins of interest fused to the carboxy termini of AFPs. C, N-series pSITE vectors for Gateway recombination-mediated construction of binary vectors for expression of proteins of interest fused to the amino termini of AFPs. D, 0-series pSITE vectors for Gateway recombination-mediated construction of binary vectors for expression of native proteins. Protein expression is controlled by a duplicated CaMV 35S promoter (2X35S) and a tobacco etch virus translational leader (TL). All vectors employ the CaMV35S transcriptional terminator (TER). Nco1*, This restriction site was deleted to create the pSITE-NB and pSITE-0B vectors, thereby allowing translation to initiate at the native start codon on the gene of interest. This figure is a revision of that appearing in Chakrabarty et al. (2007). [See online article for color version of this figure.] The current set of pSITE vectors can be used to express native proteins or enhanced CFP, GFP, YFP, or RFP fusions to either the C or N termini of proteins of interest (Fig. 1). These vectors were validated with respect to five criteria essential for high-throughput protein localization studies associated with the study of plant-pathogen interactions. Such experiments require facile vector systems that permit (1) high-throughput construction of recombinant expression vectors, (2) protein expression in either transient assays or transgenic plants without the need for subcloning into different vectors, (3) the ability to efficiently deliver proteins and their interacting targets or substrates to the same cell, (4) expression of proteins in pathogen-infected cells and, (5) the ability to monitor membrane or protein dynamics in a large number of cells so as to permit rigorous statistical analyses (Chakrabarty et al., 2007). With respect to the first criterion, the pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. Thus, following high-throughput protein localization, any construct of interest can be used to generate transgenic plants if necessary, without the need for subcloning into alternate vectors. Additionally, the ability to clone directly into a complete binary vector eliminates the two-step cloning procedure required for assembly of pSAT derivatives. Transient expression of AFPs from pSITE vectors. A to F, Expression of proteins whose subcellular localization cannot be determined in silico. Fluorescence from DAPI (A) and GFP (B) in the nucleus of a cell expressing a GFP:PYDV-P protein. The overlay of A and B is shown in C. Fluorescence from DAPI (D) and RFP (E) in the nucleus of a cell expressing a RFP:PYDV-N protein. The overlay of D and E is shown in F. Although both PYDV-N and -P proteins are entirely localized to the nucleus, analysis of their primary structure failed to identify karyophillic domains. G to J, Expression of AFP fusions in callus cells of N. benthamiana. G, GFP fluorescence of transgenic callus cell expressing mGFP5-ER. H, Overlay of G and I. I, RFP fluorescence following agromediated expression of RFP-SYNV-P from a pSITE vector. J, Differential interference contrast image of cell shown in G to I. K to V, Expression to study differential protein localization in pathogen-infected cells. Shown are confocal micrographs of RFP fusions of SYNV proteins expressed in SYNV-infected and mock-inoculated mGFP5-ER transgenic N. benthamiana plants. Fluorescence images for GFP, RFP, and the corresponding overlay are shown for each fusion expressed in SYNV-infected cells. Only the overlay is shown for fusions expressed in mock-inoculated leaves. Sections from top to bottom show localization of RFP:P (K–N), RFP:N (O–R), and RFP:M (S–V). Sections K to V are reprinted from Goodin et al. (2007). Like Vero cells, which to localization of the human projects in plants require a cell or a system (Simpson et al., Thus, cell have been used for localization of Arabidopsis proteins et al., 2005; et al., 2005). In a similar callus cells derived from N. benthamiana can be used (Fig. and However, an attractive is to use a highly facile means to express proteins in plants, which simply with of transformed with binary vectors of interest et al., Goodin et al., 2002; using vectors, such as that contain any constructs of interest in transient expression assays can be used to generate transgenic plants for further In addition to another great of in N. benthamiana is the by which such as to DNA (Chakrabarty et al., Goodin et al., or to (Goodin et al., can be into prior to examination of tissue by This approach to both the quality and of of thus the quality green on images that any frame of to of the and facile techniques are not in Arabidopsis or other plants. Therefore, N. benthamiana has been used in the context of it is being in a myriad of studies in plant particularly in where localization is being linked to interactions et al., 2007), or complementation of studies in Arabidopsis et al., 2007). micrographs localization of AFPs targeted to a of subcellular All are expressed from pSITE vectors in N. benthamiana. with transient expression expression in transgenic plants. expressing a fusion were not resistant to was from the to with sites where cell proteins used were the and protein of a plant cell of A to F, scanning confocal micrographs of N. benthamiana cells in the localization of expression of the from two loci in mock-inoculated cells to in SYNV-infected cells Transient expression of was in mGFP5-ER plants. G, of expression in mock-inoculated and SYNV-infected cells. Expression were into two with one light or two using are shown on the those with transgenic plants are on the The of are shown at the bottom of the Sections A to and in G are reprinted from Chakrabarty et al. (2007). The effects of on the localization of proteins are but few of a field of research that to determine the effect of on the localization of proteins et al., 2005; 2006; et al., 2007). The great that transient expression of proteins to that using transgenic plants. Therefore, in addition to the essential of the transient expression assays in plants will to the of proteins and membrane dynamics in pathogen-infected cells will in the between in protein localization and gene expression (Goodin et al., 2005). A, The of is that light that the of two media of at greater than the critical result in the being of the At the of an is produced in the of the which is in the of are in fluorescence with high signal to B, of an N. benthamiana expressing C and D, micrographs time of ER and fusion. that the of fusion at loci where at the termini (C) or within (D) micrographs ER and fusion the speed and resolution of F, that and the sites of ER fusion. While these are to a of they to to be and to new imaging techniques and even if this is not the best of all possible our the best possible the on the of this article and the of live-cell imaging in plants, we to whose was not in this We for the prior to This is with the of the of the as article number