Understanding how genes regulate plant biology is crucial to addressing the challenges faced by modern agriculture. Plant transformation is a powerful tool to investigate how plant genes regulate biology. When combined with fluorescence microscopy, transgenic plants allow for live imaging of proteins in their native cellular environment. Previously, we developed a series of Gateway-compatible tissue-specific plant transformation vectors [1]. Here we expand the toolkit of available Agrobacterium-mediated plant transformation vectors with additional fluorophores and extend the set to include a set of modular Gateway- and Gibson assembly-compatible vectors for protoplast transformation.
pLCS Protoplast Vectors
Protoplasts are a powerful system for transient expression of plant genes. Protoplasts are well suited to synthetic biology, allowing for a rapid cycle of design, testing, and iteration [2]. We created a collection of protoplast expression vectors known as the pLCS vector series. Each pLCS vector carries the UBIQUITIN10 (UBQ10) promoter, which typically drives the expression of UBQ10, a uniform and widely-expressed transcript in Arabidopsis [3]. In plants, the UBQ10 promoter displays a more uniform expression pattern than many commonly used viral promoters, such as the CaMV 35S promoter. In each pLCS vector, the UBQ10 promoter drives the expression of a fluorescent protein (FP), followed by a Gateway cassette (attR1, chloramphenicol resistance gene, ccdB gene, attR2), and the octopine synthase (OCS) terminator. The backbone of pLCS vectors contains the AmpR gene, which encodes beta-lactamase and confers resistance to ampicillin and carbenicillin in bacteria (Fig. 1A). Thus, a Gateway LR clonase reaction with an entry vector produces a vector that ultimately express the protein of interest with an N-terminal fluorescent protein tag.
We first created pLCS100 by subcloning the UBQ10 promoter, a gene encoding EYFP, the gateway cassette, and OCS terminator from pUBQ10-YFP-GW [1] into the pUC19 [4] backbone. As EYFP is a weak dimer, and dimerization can create artifacts that influence the behavior of the tagged protein, we then created pLCS101 by replacing EYFP with monomeric mVenus [5] (Fig. 1).
mNeonGreen is an exceptionally bright monomeric fluorescent protein with an excitation and emission spectra between that of GFP- and YFP-derived fluorescent proteins [6]. To take advantage of this exceptionally bright fluorophore, we created pLCS107, which contains mNeonGreen in place of mVenus (Fig. 3A). StayGold is a recently developed highly photostable and bright FP enabling long imaging times with minimal photobleaching [7]. To take advantage of this new FP, we created pLCS110. StayGold is a dimer and users should be aware of potential artifacts due to its dimerization. During the preparation of this manuscript, a monomeric version of StayGold, known as mStayGold, containing the mutation E138D, was described in a preprint [8]. Whereas the data shown herein were collected using the dimeric StayGold, we have deposited the updated version of pLCS110 containing the monomeric mStayGold with Addgene.
To enable multi-color imaging experiments in co-transformed protoplasts, we created pLCS108 containing the FP mCherry [9]. Additionally, we created pLCS109 containing a gene encoding mTagBFP2, the brightest blue fluorophore currently available [10]. mTagBFP2 can be used to serve as a donor for Förster resonance energy transfer experiments (FRET) when paired with mNeonGreen, such as that carried by pLCS107.
To enable tracking of specific pools of protein and super resolution microscopy (e.g., PALM), we created pLCS105 and pLCS106 by inserting the genes encoding the green-to-red photoswitchable proteins DENDRA2 or moxMaple3, respectively [11, 12] (Fig. 3C). Similarly, pLCS103 and pLCS104 have a SNAP-tag or HaloTag respectively. SNAP-tag and HaloTag combine the advantage of being genetically encoded with the exceptional brightness of synthetic dyes, at the expense of labeling time [13]. Both the SNAP-tag and HaloTag, as well as various ligands, have been used successfully in plants (Fig. 3A) [14, 15].
pLCS111 carries a gene encoding a tandem fluorescent protein timer (tFT) consisting of mScarlet-I fused to mNeonGreen. mNeonGreen matures very rapidly (~ 10 mins), whereas mScarlet-I, a fast-maturing variant of mScarlet, matures more slowly (~ 26 mins) [16]. By fusing the mScarlet-I-mNeonGreen tFT to a protein of interest, it is possible to determine the age of the fusion protein based on the ratio of red-to-green fluorescence [17, 18] (Fig. 3E). It is important to note that the N-terminus of a protein is often a major determinant of protein stability due to the N-end rule [19–21]. As the default orientation for fusions in pLCS111 are N-terminal fusions it is prudent to test in the opposite orientation. Care should be taken when interpreting these data and appropriate controls should be used [22].
Finally, pLCS99 has no fluorescent protein to enable construction of custom protein fusions driven by the UBQ10 promoter. The pLCS series was designed to be modular, thus the UBQ10 promoter in each vector is flanked by a NruI and XhoI restriction site allowing the UBQ10 promoter to be swapped easily using Gibson assembly or restriction ligation cloning. Similarly, the fluorescent protein in each vector is flanked by XhoI and HindIII restriction sites.
pJRA Binary Vectors
The original pMCS-GW and pUBQ10-YFP-GW vectors [1] while immensely useful, both contain kanamycin resistance genes. As most Gateway entry vectors, such as pENTR/D-TOPO, are also kanamycin resistant, it was necessary to linearize such entry vectors before performing an LR reaction. To remedy this problem, we replaced the kanamycin resistance gene with a spectinomycin/streptomycin resistance gene to create pJRA-GW. Taking advantage of the protoplast vectors previously created, the UBQ10 promoter, FP, and gateway cassette from each pLCS vector was inserted into the pJRA-GW backbone to create the pJRA series of vectors with the same fluorophores as each pLCS vector (Fig. 2). The pJRA series of vectors are well-suited to transient expression in tobacco and for creation of stable transgenic lines. In the latter case, transformed plants can be screened based on resistance to glufosinate/phosphinothricin (commonly referred to as BASTA™).
Each vector in the pJRA series was used for transient expression in Nicotiana benthamiana to test their functionality (Fig. 3). Though fluorescence was visible for all constructs, high background was observed in the case of pJRA103 due to unbound SNAP-Cell TMR-Star, even after multiple washes. Curiously, this problem was not observed in Arabidopsis protoplasts transformed with pLCS103 (Fig. 3A), nor in tobacco transformed with pJRA104 when labeled with HALO-TMR (Fig. 3B). This discrepancy suggests that the SNAP-Cell TMR-Star ligand may have difficulty penetrating the cell wall or cuticle of intact plant cells. Further optimization of the treatment and washing procedure is likely needed to ensure the ligand enters the cells and all unbound dye is removed, particularly when dealing with intact plant tissues.