The Plant Journal (2009) 59, 150–162
doi: 10.1111/j.1365-313X.2009.03850.x
TECHNICAL ADVANCE
Transient expression in Nicotiana benthamiana fluorescent
marker lines provides enhanced definition of protein
localization, movement and interactions in planta
Kathleen Martin1, Kristin Kopperud1, Romit Chakrabarty2, Rituparna Banerjee1, Robert Brooks1 and Michael M. Goodin1
Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA, and
2
Department of Biological Sciences, 2500 University Drive, N.W. University of Calgary, Calgary, AB T2N 1N4, Canada
1
Received 12 November 2008; revised 4 February 2009; accepted 12 February 2009; published online 30 March 2009.
For correspondence (fax +1 859 323 1961; e-mail mgoodin@uky.edu).
SUMMARY
Here, we report on the construction of a novel series of Gateway-compatible plant transformation vectors
containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and
Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have
determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and
have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments.
Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein
markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular
fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to
histone 2B provides enhanced data quality and content over assays conducted without the benefit of a
subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that
express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or
endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for
studying intracellular protein interaction, localization and movement, in conjunction with our transgenic
marker lines, constitute powerful tools for the plant biology community.
Keywords: Agrobacterium, agroinfiltration, live-cell imaging.
INTRODUCTION
Defining plant protein interaction networks and the accurate determination of the subcellular localization of the
proteome are fundamental requirements for plant cell research in the post-genomics era. To assist in such studies,
Nicotiana benthamiana is increasingly being used to
conduct protein localization and bimolecular fluorescence
complementation (BiFC) assays in live plant cells (Citovsky
et al., 2006; Goodin et al., 2007a,b, 2008a,b; Ohad et al.,
2007; Waadt et al., 2008). In order to enhance the utility of
this plant in cell biology studies, we, and others, have
developed enhanced vector systems primarily for the
expression of autofluorescent protein (AFP) fusions
derived from the modular pSAT vectors (Tzfira et al., 2005;
Citovsky et al., 2006; Goodin et al., 2007a; Lee et al., 2008).
Although these vectors have been of great utility for
150
steady-state localization experiments, we report here an
assessment of novel AFPs that can be used for monitoring
protein movement, or which are brighter or more photostable than the AFPs used in the currently available
Gateway-compatible plant binary vectors (Tzfira et al.,
2005; Earley et al., 2006; Chakrabarty et al., 2007; Goodin
et al., 2007b; Nelson et al., 2007; Dubin et al., 2008; Zhong
et al., 2008).
In addition to the requirement for appropriate vectors,
generating accurate protein localization data often necessitates the use of marker dyes or proteins in order to provide a
subcellular reference in micrographs. For example, one of
the most commonly used markers is the DNA-selective dye
4¢, 6-diamidino-2-phenylindole (DAPI) used to counterstain
nuclei (Goodin et al., 2007a). In addition to being highly toxic
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd
Live-cell protein interaction and localization 151
or mutagenic, the use of dyes adds to the time and expense
of experiments, and limits high-throughput analyses in plant
tissues. To circumvent such problems, we have generated a
series of transgenic plants that express fluorescent markers
targeted to the endoplasmic reticulum, actin filaments or
nuclei.
In order to rigorously test the utility of our new vectors, we
conducted protein localization and interaction studies using
soluble and membrane-associated proteins encoded by
Sonchus yellow net virus (SYNV) or Potato yellow dwarf
virus (PYDV), as well as two isoforms of N. benthamiana
importin-a (NbImpa1 and NbImpa2; Kanneganti et al., 2007).
We demonstrate how the data content of BiFC experiments
is increased when conducted in transgenic plants expressing a subcellular reference. Additionally, we have evaluated
our marker lines in the context of virus-induced changes in
nuclear membranes. These technically challenging experi-
Figure 1. Construction of pSAT6-AFP-DEST, a
Gateway-compatible derivative of pSAT6-DESTFS in which an EGFP ‘stuffer’ sequence was
engineered with flanking FseI and SpeI restriction sites. Replacement of the stuffer with any
appropriately cloned gene (autofluorescent protein, AFP) resulted in the facile construction of
intermediates required for pSITEII-C1 vectors. A
similar strategy was used to generate pSAT6DEST-AFP intermediates for constructing pSITEII-N1 vectors (see Experimental procedures).
(a)
Figure 2. Construction of pSITEII vectors.
(a) New pSITEII vectors were constructed by
subcloning pSAT6-DEST-AFP derivatives into
the PI-Psp1 site of the plant gene expression
vector pRCS2-nptII, which carries the nptII-selectable marker and the Ti plasmid left and right
borders.
(b) Seven pSITEII-C1 vectors were constructed
with FRET-optimized autofluorescent proteins
(AFPs) such as Cerulean, Midoriishi Cyan (MiCy),
Venus and monomeric Kusabira Orange (mKO).
Additionally, TagRFP was used as a brighter
alternative to mRFP1. Finally, DRONPA was used
to enable the tracking of proteins in plant cells
using photoactivation or photoconversion.
(c) For the construction of fusions to the N termini of AFPs we have produced and validated a
series of pSITEII-N1 vectors. The AFP cloning
intermediates for constructing the C1 series can
also be used for the construction of N1 vectors.
(a)
ments provide confidence that the marker lines reported
here will be of utility in the demanding experiments required
to elucidate protein and membrane dynamics in plant cells.
RESULTS
Construction of pSAT6-DEST-FS and derivative pSITEII
vectors
As we were interested in enhancing the ease by which AFPs
can be exchanged to create new vectors, we constructed
pSAT6-DEST-FS so that sites for rare cutting restriction endonucleases were included at the 5¢ and 3¢ termini of EGFP,
respectively (Figure 1a), which permitted rapid subcloning
to replace the EGFP ‘stuffer sequence’ to generate a variety
of pSAT6-DEST-AFP derivatives (Figure 1b). Using this
strategy, a diverse set of pSITEII vectors were constructed
(Figure 2; Table 1).
(b)
(b)
(c)
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
152 Kathleen Martin et al.
Table 1 Characteristics of the autofluorescent proteins (AFPs) used in the construction of pSITE and pSITEII vectors. The nomenclature for pSAT
and pSITE vectors has been maintained, as described in the Experimental procedures. Note that all vectors are available in the C1 orientation.
Not all vectors have been converted to their N1 equivalents
Vector
Protein
Structure
Ex. max.a
(nm)
pSITEII-1C1
pSITE-1C1/N1
pSITEII-2C1
pSITEII-3C1/N1
pSITE-3C1/N1
pSITEII-4C1
pSITEII-5-C1
pSITE-4C1/N1
pSITEII-6C1
pSITEII-7C1/N1
pSITEII-8C1
Mi-Cy
ECFP
Cerulean
EGFP
EYFP
Venus
mKO
mRFP1
TagRFP
Dendra2
DRONPA
Dimer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
472
439
433
484
514
515
548
584
555
490/553
503
Em. max.b
(nm)
Mol ex. coeff.c
(M)1 cm)1)
Quantum
yield
Brightnessd
495
476
475
507
527
528
559
607
584
507/573
518
27 300
32 500
43 000
56 000
83 400
92 200
51 600
50 000
52 000
45 000/35 000
57 000
0.90
0.40
0.62
0.60
0.61
0.57
0.60
0.25
0.48
0.50/0.55
0.62
25
13
27
34
51
53
31
13
25
23/19
35
a
Excitation maximum.
Emission maximum.
c
Molar extinction coefficient.
d
Brightness = (molar extinction coefficient · quantum yield)/1000.
b
To provide a consistent series of Gateway-compatible vectors for the study of both localization and protein interaction,
we converted several of the previously reported pSAT-BiFCN1 and pSAT-BiFC-C1 vectors (Citovsky et al., 2006) to their
pSITE derivatives (Figure 3). Note that these new Gateway
vectors do not have the restriction-site modifications of the
pSITEII vectors; therefore, we will refer to these vectors as
pSITE-BiFC vectors, according to the established convention
(Chakrabarty et al., 2007).
or pSITEII vectors. Expressing the AFPs via agroinfiltration in
N. benthamiana leaves showed that GFP spectral variants
and TagRFP accumulated in the nuclei, but that they were
excluded from nucleoli. In contrast, both Midoriishi Cyan
(MiCy) and monomeric Kusabira Orange (mKO) appeared to
accumulate in both loci, such that nucleoli could not be discerned in cells expressing these proteins (Figure S1).
The nine AFPs examined differed greatly in their photostability, with the relative order from most to least stable
being: EYFP > EGFP = Venus > mKO > Cerulean > ECFP >
mRFP1 > TagRFP > MiCy (Figure S1).
In planta photostability of nine different AFPs
Comparative brightness of AFP fusions in plant cells
To our knowledge, the relative photostability of a large
number of AFPs expressed from a similar vector backbone
has not been compared in planta. However, these data are
critical for evaluating the suitability of AFPs for use in various
biological assays. Therefore, we examined the localization
and photostability of nine AFPs expressed from either pSITE
Based solely on published calculations of brightness
(Table 1), the expected utility of AFPs in plants was predicted
to follow the order Venus ‡ EYFP > EGFP > mKO >
Cerulean > MiCy = TagRFP > ECFP = mRFP1. Except for
mKO and MiCy, this prediction generally holds under conventional imaging conditions (Figures S2 and S3). For
Construction of pSITE vectors for BiFC
(a)
(b)
Figure 3. pSITE-BiFC-C1 vectors were constructed by subcloning the AgeI/BglII fragment
of pSAT4-BiFC (a) into pSAT6-DEST (b). As for all
pSITE vectors, the pSAT6 derivatives were
subcloned into RCS2-nptII to produce Gatewaycloning compatible binary vectors. (c) pSITEBiFC-N1 vectors were constructed by subcloning
the AgeI/ApaI fragment of pSAT6-DEST into
pSAT4-BiFC. The four available vectors are designated pSITE-BiFC-C1nec, pSITE-BiFC-C1cec,
pSITE-BiFC-N1nen and pSITE-BiFC-N1cen.
(c)
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
Live-cell protein interaction and localization 153
SYNV-N fusions, we found that Venus fusions, more so than
any other AFP, were prone to aggregation (Figure S2, and
data not shown).
sizes that researchers should be aware that not all AFPs
provide the same localization information when fused to the
same protein.
Photostability of DRONPA fusions in plant cells
Dendra2 for protein tracking in plant cells
DRONPA is a reversibly photoactivatable AFP that has been
used for protein tracking experiments in a manner similar to
EosFP, or the more popular PA-GFP (Ando et al., 2004; Lippincott-Schwartz and Patterson, 2008; Schenkel et al., 2008).
To extend the range of functionality of the pSITEII vector
series, we tested DRONPA expression, photoactivation and
stability in N. benthamiana leaf epidermal cells. DRONPA
and DRONPA fusions proved to be photoactivatable in plant
cells (Figure 4a,b). We also found that fusions to DRONPA
were more resistant to photobleaching than the unfused AFP
(Figure 4c). Unexpectedly, DRONPA-SYNV-N localized
exclusively to the nuclear periphery, in marked contrast to
GFP-SYNV-N or RFP-SYNV-N fusions, which accumulated in
the nucleus proper, and were excluded from the nucleolus
(Goodin et al., 2001, 2007a). Although we do not know why
DRONPA-SYNV-N fusions mislocalize, this result empha-
We verified the pSITEII-7-C1 and pSITEII-7-N1 vectors for the
expression of Dendra2 fusions in plant cells (Figure 5).
Dendra2-SYNV-N, in contrast to DRONPA fusions, localized
exclusively to nuclei, in a pattern similar to that for GFP or
RFP fusions (Goodin et al., 2001, 2007a,b). It was possible to
selectively photoconvert subnuclear regions of interest from
green (Figure 5a1–a5) to red (Figure 5b1–b5, c1–c5) without
affecting the nuclei in adjacent cells. The Dendra2-SYNV-N
fusion in these selected regions was undetectable within
seconds after photoconversion (Figure 5c2,c3). Further
investigation is required to determine if the rapid disappearance of red Dendra2-SYNV-N is related to diffusion,
degradation or some combination thereof. Interestingly,
the red form of SYNV-P-Dendra2 (Figure 5d1–d5; e1–e5)
diffused more slowly than the SYNV-N fusion. Additionally, we were unable to photoactivate SYNV-P-Dendra2
Figure 4. Photoactivation of DRONPA (a) and
DRONPA-SYNV-N (b) in agroinfiltrated Nicotiana
benthamiana leaf epidermal cells. DRONPA was
photoactivated (1.1 s) with a 50-ms pulse from a
405-nm laser following the acquisition of a preactivation image (0 s). Cells were imaged continuously for another 23 s using a 488-nm laser
line for excitation. (c) Normalized fluorescence
intensity showing the relative photostability of
DRONPA-SYNV-N and DRONPA. Each curve
represents the average of three independent
assays. Scale bars: 5 lm (a); 10 lm (b). For (c),
the average fluorescence intensity of DRONPA
prior to photoactivation was taken as the zero
point.
(a)
(b)
(c)
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
154 Kathleen Martin et al.
(a2)
(a3)
(a4)
(a5)
(b1)
(b2)
(b3)
(b4)
(b5)
(c1)
(c2)
(c3)
(c4)
(c5)
(d1)
(d2)
(d3)
(d4)
(d5)
(e1)
(e2)
(e3)
(e4)
(e5)
(f1)
(f2)
(f3)
(f4)
(f5)
Overlay
Red
Green
Overlay
Red
Green
(a1)
Pre-activation
Time post-photoactivation (sec)
Figure 5. Photoconversion of Dendra2 fusions expressed from pSITEII-C1 (a1–c5) or pSITEII-N1 (d1–f5) vectors. Dendra2 fusions were transiently expressed in
Nicotiana benthamiana leaf epidermal cells. ROIs within selected nuclei (arrowheads) were photoconverted using a 50-ms pulse from a 405-nm laser set at 78% of
full power. Micrographs were acquired immediately prior to photoactivation, and were continuously acquired for 11 s thereafter. Shown are the green (a1–a5; d1–
d5), red (b1–b5; e1–e5) and overlain (c1–c5; f1–f5) micrographs of Dendra2-SYNV-N and SYNV-P:Dendra2, respectively. The inserts in panels c2 and c3 highlight the
photoconverted region in a nucleus in which Dendra2-SYNV-N was localized. Scale bar: 10 lm.
with the precision observed for SYNV-N (compare Figure
5c2, f2). Unlike the SYNV-N fusion, SYNV-P-Dendra2 was
still detectable at 5 min post-photoconversion (data not
shown). The quantification of relative fluorescence
intensities showed that the green and red forms of
SYNV-P-Dendra2 were equilibrated in nuclei within 15 s of
photoconversion (Figure S4). These results suggest that
each protein of interest may have unique rates of diffusion,
which must be determined empirically.
Validation of BiFC vectors
In order to be broadly useful, BiFC vectors must permit
protein associations to be assayed in a variety of cellular loci.
The simplest associations to determine are those formed by
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
Live-cell protein interaction and localization 155
proteins in soluble complexes, such as those between the
phosphoprotein (P) and nucleocapsid (N) proteins of SYNV
(Goodin et al., 2001). However, testing interactions of
membrane-associated proteins, such as the SYNV glycoprotein (SYNV-G; Goldberg et al., 1991) is technically more
challenging, given the requirement for the YFP fragments to
be on the same side of the membrane in order for them to
associate. Additionally, conventional wisdom holds that
AFPs should be fused to the C termini of membrane-associated proteins in order to prevent interference of the function of signal peptides at their N termini. Therefore, the
pSITE-BiFC vectors were validated in the contexts of both
soluble (Figure 6a–d) and membrane-associated (Figure
6e–h) viral protein complexes. Converting the pSAT-BiFC
vectors to their Gateway-compatible pSITE derivatives
resulted in insignificant background fluorescence when
the two non-fused halves of YFP were co-expressed (Figure 6a,e), or when non-fused halves were co-expressed with
protein fusions (Figure 6b,c,f and g). Thus, bona fide interactions could be scored easily, such as in the case of the
soluble SYNV-N/SYNV–P (Figure 6d) complex or the selfassociation of SYNV-G (Figure 6h). We note that, contrary to
conventional wisdom, the SYNV-G interaction was only
detected when this protein was expressed from the BiFC-C1
vectors, which places the YFP fragments in front of the
SYNV-G signal peptide. When expressed from the BiFC-N1
vectors, which places the YFP fragments at the C terminus of
SYNV-G, no fluorescent signal was observed, despite the
fact that fusions could be detected by immunoblotting (data
not shown).
A further reduction of the background in BiFC experiments
was achieved when nYFP or cYFP fragments were expressed
as fusions to glutathione-S-transferase (GST) or maltosebinding protein (data not shown). We now routinely use
these GST:YFP fragment fusions as negative controls in BiFC
assays (Figure 7).
In addition to enhancing the utility of the pSITE-BiFC
vectors, we succeeded in further increasing the data
content and quality of micrographs by conducting BiFC
experiments in transgenic N. benthamiana plants that
expressed CFP fused to histone 2B (CFP-H2B; Figure 6i–q).
To validate this approach we used BiFC to confirm the
homo- and heterologous interactions of the SYNV-N and
SYNV-P proteins, which have been shown previously using
GST pull-downs and yeast two-hybrid assays (Goodin et al.,
2001; Deng et al., 2007). Consistent with previous reports
(Goodin et al., 2001, 2002), the SYNV-N protein localized to
the nucleus exclusively (Figure 6i–k), whereas the co-expression of SYNV-N and SYNV-P resulted in the relocalization of
both proteins to a subnuclear locale (Figure 6i–n). The
SYNV-P protein complex showed accumulation in both the
nucleus and cytoplasm (Figure 6o–q). Given these results, it
is important that researchers conducting similar experiments should be aware that co-expression may alter the
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
(q)
Figure 6. Validation of the pSITE-BiFC vectors for detecting interactions
between soluble (a–d) or integral (e–h) membrane protein complexes.
Identical detector settings were used to acquire all images. Expression was
conducted in leaf epidermal cells of Nicotiana benthamiana using agroinfiltration. Micrographs taken 48 h post-infiltation show results of co-expressed
nYFP+cYFP (a and e); nYFP+cYFP-SYNV-P (blue) (b); nYFP-SYNV-N (red)+cYFP (c); and nYFP-SYNV-N+cYFP:SYNV-P (d). (f) nYFP+cYFP:SYNV-G (purple); (g) nYFP-SYNV-G+cYFP. (h) nYFP-SYNV-G+cYFP:SYNV-G. (i–q) Single
section confocal micrographs demonstrating the use of CFP-H2B tagged
N. benthamiana plants for marking nuclei in bimolecular fluorescence
complementation (BiFC) experiments. (i, k and o) Fluoresence from CFP
localized to nuclei in CFP-H2B plants. (j) YFP fluorescence in a nucleus of a cell
expressing nYFP:SYNV-N+cYFP:SYNV-N. (k) Overlay of images (i) and (j). (m)
YFP fluorescence in a nucleus of a cell expressing nYFP-SYNV-N+cYFP-SYNVP. (n) Overlay of images (l) and (m). (p) YFP fluorescence in a cell expressing
nYFP-SYNV-P+cYFP-SYNV-P. (q) Overlay of images (o) and (p). Scale bars:
10 lm (a–h; o–q); 5 lm (i–k); 3 lm (l–m).
subcellular localization of proteins, relative to the patterns
formed when they are expressed individually.
Differential interaction of cargo with importin-a
Prior to conducting BiFC experiments, it is important to be
aware that isoforms encoded by a particular multigene
family might interact with different subsets of proteins.
To demonstrate such differential binding, we examined
the interactions of two isoforms of importin-a
(NbImpa1 and NbImpa2) with multiple cargo proteins of
viral origin.
The import of proteins into the nucleus is commonly
mediated, in part, by importin-a proteins, which form
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
156 Kathleen Martin et al.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
(q)
(r)
(s)
(t)
(u)
(v)
(w)
(x)
(y)
(z)
(a1)
Figure 7. Single-section confocal micrographs of bimolecular fluorescence
complementation (BiFC), showing differential interactions of NbImpa1 and
NbImpa2 with SYNV-N. Importin-a proteins were expressed as fusions to the
C-terminal half of YFP (Impc). SYNV-N was expressed as a fusion to the
N-terminal half of YFP (Nn). (a–f) Interaction of SYNV-N with NbImpa1. (a–c)
Whole-cell views showing fluorescence from CFP-H2B, BiFC interaction of Nn
and NbImpa1, and the resulting overlain images, respectively. (d–f) Confocal
sections of nuclei in cells expressing the same fusions shown in a–c. (g–i)
Whole-cell views showing fluorescence from CFP-H2B, BiFC interaction of Nn
and NbImpa2, and the resulting overlain images, respectively. (j–l) Confocal
sections of nuclei in cells expressing the same fusions shown in (g–i). (m–o)
Lack of interaction between SYNV-P and NbImpa1. (p–r) Lack of interaction
between SYNV-P and NbImpa2. (s–x) Control reactions showing a lack of
interactions between GST and NbImpa1 or NbImpa2. (y–a1) Control reactions
showing a lack of interactions between GST and NbImpa1 or SYNV-N. Scale
bars: 10 lm, except for (d–f) and (j–l), 5 lm.
oligomeric complexes with cargo proteins and importin-b,
which then translocate from the cytoplasm through
nuclear pore complexes into the nucleus (Xu et al.,
2008). The Arabidopsis isoforms of importin-a have
recently been shown to differ in their selectivity for
particular cargo proteins that are imported into the
nucleus (Bhattacharjee et al., 2008; Lee et al., 2008). To
determine if this phenomenon holds for N. benthamiana,
we tested the ability of NbImpa1 and NbImpa2 to interact
with SYNV and PYDV proteins as test cargo (Palma et al.,
2005; Kanneganti et al., 2007). The SYNV-N protein
contains an arginine/lysine-rich nuclear localization signal
(NLS) at its C terminus, which has been shown to mediate
its interaction with importin-a in vitro (Goodin et al., 2001;
Deng et al., 2007). In contrast, SYNV-P does not contain a
predictable NLS, and does not bind importin-a in vitro
(Deng et al., 2007). Moreover, although the cognate proteins from PYDV lack predictable NLSs, both are localized
exclusively to the nucleus (Ghosh et al., 2008). However,
the nuclear import of proteins lacking canonical NLSs has
been shown, in some cases, to be mediated via an
importin-a-dependent pathway (Wolff et al., 2002). These
experiments showed that, consistent with in vitro binding
data, SYNV-N interacted with both NbImpa1 and NbImpa2
(Figure 7). However, interactions of these proteins
localized to different loci, with the SYNV-N–NbImpa1
interaction being distinctly subnuclear, and with SYNVN–NbImpa2 interacting on the periphery of the nucleus. In
contrast to SYNV, we observed that PYDV-N interacted
with NbImpa1, but not with NbImpa2 (Figure 8). No
detectable fluorescence was produced when PYDV-P and
SYNV-P were tested for interaction with either NbImpa1 or
NbImpa2 (Figure 8).
RFP-marker N. benthamiana plants for supporting
localization projects
In addition to the CFP-H2B plants, the use of which clearly
improves BiFC experiments, we have also developed
N. benthamiana lines that express RFP-H2B or RFP with an
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
Live-cell protein interaction and localization 157
(a)
(b)
(c)
(a)
(b)
(c)
(d)
(e)
(f)
(d)
(e)
(f)
(g)
(h)
(i)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
(q)
(r)
Figure 8. Single-section confocal micrographs of bimolecular fluorescence
complementation (BiFC), showing differential interactions of NbImpa1 and
NbImpa2 with PYDV-N. Importin-a proteins were expressed as fusions to the
C-terminal half of YFP (Impc). SYNV-N was expressed as a fusion to the
N-terminal half of YFP (Nn). Agroinfiltration was used to express proteins in
transgenic Nicotiana benthamiana plants expressing the nuclear marker CFPH2B. (a–c) Whole-cell views showing the interaction between PYDV-N and
NbImpa1 (Nn/Imp1c). (d–f) Whole-cell views showing the lack of interaction
between PYDV-N and NbImpa2 (Nn/Imp2c). (g–i) Whole-cell views showing
the self-interaction of PYDV-N (Nn/Nc). Scale bars: 10 lm.
endoplasmic reticulum (ER) retention signal. In order to
demonstrate their utility, we took advantage of the ability of
SYNV to selectivity induce the intranuclear accumulation of
the inner nuclear membrane (INM), as has been reported
previously (Martins et al., 1998; Goodin et al., 2007a). We
postulated that in SYNV-infected plants, the GFP fused to the
human LaminB receptor (LBR-GFP), which is targeted to the
INM in plants, should therefore accumulate on the periphery
of SYNV-induced intranuclear membranes. The perinuclear
space in such nuclei should then ‘fill in’ with RFP in transgenic plants that express RFP targeted to the ER (Collings
et al., 2000; Irons et al., 2003; Goodin et al., 2007a). Conversely, GFP fused to WIP1, which is anchored to RanGAP1
on the outer nuclear membrane (ONM) in Arabidopsis (Xu
et al., 2007), should not accumulate on intranuclear membranes in SYNV-infected cells, as electron micrograph
studies suggest that the ONM remains largely unaffected in
SYNV-infected plants (Martins et al., 1998). Consistent with
its localization in Arabidopsis thaliana, WIP1-GFP localized
exclusively to the nuclear rim in N. benthamiana RFP-H2B
plants (Figure 9). In contrast to the results predicted for LBRGFP (Figure 9m–o; Goodin et al., 2007a), WIP1-GFP did not
accumulate on intranuclear membranes in SYNV-infected
RFP-ER transgenic lines, as expected for a protein that is
indeed anchored to the ONM (Figure 9j–l). Taken together,
Figure 9. (a–l) Single-section confocal micrographs showing localization of
AtWIP1:GFP in transgenic Nicotiana benthamiana plants expressing RFP:H2B.
(a–c) Whole-cell views showing the GFP channel (a), the RFP channel (b) and
the overlay (c) of WIP1:GFP in mock-inoculated plants. (d–f) As for (a–c),
showing a view of a single nucleus. (g–i) As for (a–c), except that WIP1:GFP
expression was conducted in SYNV-infected plants. (j–l) Similar to (d–f),
showing WIP1:GFP localization in SYNV-infected transgenic N. benthamiana
plants expressing RFP-ER. (m–o) Expression of the inner nuclear marker LBRGFP in SYNV-infected cells. (p–r) GFP channel, RFP channel and overlay
micrographs of a single nucleus in mock-inoculated transgenic plants
expressing RFP-ER agroinfiltrated with the LBR-GFP marker. Scale bars:
2 lm, except (a–c) and (g–i), 10 lm.
these experiments provide confidence that our transgenic
plant lines are suitable for use in experiments where complex changes in protein or membrane localization are to be
studied.
Finally, for situations where researchers may need to
localize proteins to the cytoskeleton, we produced plants
that express the actin-binding protein, Talin, fused to EGFP
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Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
158 Kathleen Martin et al.
(a)
(b)
(c)
(Figure 10). Despite potential problems with Talin as a
marker for labeling actin, which includes cytotoxicity, it
remains a widely utilized and effective marker (Ketelaar
et al., 2004; Yoneda et al., 2007; Schenkel et al., 2008). Plant
lines that are transgenic for GFP-Talin, which were indistinguishable from wild-type plants under glasshouse conditions, were screened for their ability to provide high-contrast
views of actin filaments (Figure 10a). We also produced
transgenic plants expressing Talin fused to RFP, but were
unsatisfied with these lines with respect to producing
high-contrast micrographs (data not shown).
DISCUSSION
The pSITE-BiFC and pSITEII vectors described here offer a
greatly expanded range of functionality of AFPs that were
previously unavailable in Gateway-compatible binary vectors (Earley et al., 2006; Chakrabarty et al., 2007; Coutu et al.,
2007; Dubin et al., 2008; Goodin et al., 2007b; Nelson et al.,
2007; Tzfira et al., 2005; Zhong et al., 2008). By characterizing
a wide variety of AFPs in planta, we hope to prevent the
costs, frustration and time delays often associated with
acquiring and screening novel AFPs. It is abundantly clear
from our experience that AFPs deemed suitable for use in
animal or bacterial systems may or may not be similarly
useful in plant cells (Teerawanichpan et al., 2007). Additionally, the restriction site modifications to the pSITEII
vectors make it very efficient to replace and test novel AFPs,
which are being reported at rates that greatly outpace the
ability to rigorously validate them in plant-based assays
(Merzlyak et al., 2007; Teerawanichpan et al., 2007; Schenkel
et al., 2008; Shaner et al., 2008; Subach et al., 2008;
Tasdemir et al., 2008).
Importantly, AFPs such as DRONPA can affect the
localization of their fusion partners, as demonstrated here
by fusion to the SYNV-N protein. However, given that this
protein is brighter than PA-GFP, and that DRONPA fusions
are reasonably photostable compared with the native
protein, it is worthwhile to develop DRONPA-based
assays.
In contrast to DRONPA, we showed that the fusion of
SYNV proteins to Dendra2 resulted in localization patterns
consistent with those observed for conventional AFPs.
Moreover, the efficient photoconversion of these fusions
from green to red suggests that the pSITEII-7-C1 and
Figure 10. Transient expression of RFP-H2B (a)
in a leaf epidermal cell of transgenic Nicotiana
benthamiana expressing GFP-Talin, an actin
binding protein (b). The overlain image of (a)
and (b) is presented in (c).
pSITEII-7-N1 vectors might be of significant utility in protein tracking experiments, as has been demonstrated for
mEosFP (Schenkel et al., 2008).
Another important finding relevant to selecting the appropriate AFP for localization studies was that both mKO and
MiCy accumulated in nucleoli, from which GFP variants,
TagRFP and RFP variants were excluded. This raises the
possibility that some AFPs may negatively impact the
subcelluar localization of fusion partners. In addition to its
accumulation in nucleoli, the extreme sensitivity of MiCy to
photobleaching and the low brightness of mKO prevent us
from recommending these fluors for use in planta. However,
the problems encountered here may be overcome by using
variants of AFPs selected for greater photostability or
enhanced spectral characteristics (Ai et al., 2008; Shaner
et al., 2008).
Our BiFC results demonstrating the differential interactions of plant nuclear importers with the -N and -P proteins
of SYNV and PYDV, support the contention that the various
isoforms of plant importin-a proteins differ in their cargo
specificities (Jiang et al., 1998; Palma et al., 2005; Deng
et al., 2007; Bhattacharjee et al., 2008). Interestingly, the
interaction of SYNV-N with NbImpa1 and NbImpa2 on the
periphery of nuclei, or in intranuclear sites, respectively, is
similar to the recent finding that the VirE2 protein of
Agrobacterium tumefaciens interacts with Arabidopsis
importin-a isoform-1 and isoform-4 in the cytoplasm and
nucleus, respectively (Lee et al., 2008). These studies,
together with the discovery that MOS6, an importin-a
homologue required for signaling responses related to
innate immunity (Palma et al., 2005), demonstrate that
importin-a isoforms cannot be entirely functionally
redundant. Collectively, these data underscore the need to
determine and compare the cargo specificities of nuclear
import-receptor isoforms in order to fully appreciate nuclear
transport in plants. More generally, the different sites
of localization and interaction may reflect functional
differences of protein isoforms that are involved in different
physiological processes (Morsy et al., 2008).
Although the CFP-H2B plants were generated to improve
data quality in BiFC experiments, our RFP-H2B- and RFPER-expressing lines proved equally useful for a variety of
localization studies. Both of the H2B lines offer an exceptional alternative to the use of DAPI, which is commonly
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
Live-cell protein interaction and localization 159
used to counterstain nuclei, particularly when many infiltrations need to be conducted (Launholt et al., 2006; MMG,
unpublished data).
Importantly, we demonstrated that marker proteins for
the outer and inner nuclear membranes function in
N. benthamiana as predicted, based upon their function in
A. thaliana or Nicotiana tabacum. These experiments should
help to underscore the functional conservation of proteins,
and indicate that accurate data related to the localization of
heterologous proteins can be obtained in N. benthamiana
(Citovsky et al., 2006; Goodin et al., 2007a,b; Ohad et al.,
2007; Tardif et al., 2007).
One potential criticism of the present vectors is their
utilization of a double 35S promoter to drive the expression
of the AFP fusions. It is often speculated that this will lead to
artifacts resulting from protein overexpression. Although
this is potentially the case with some protein fusions, the
data presented here strongly suggest that biologically relevant interactions can be easily scored with these vectors. One
such comparison made here is the localization of RFP-H2B in
transient assays and transgenic plants. This nuclear marker
accumulated in both nuclei and nucleoli in transient assays,
but was excluded from nucleoli in transgenic plants. Such
differences do not obviate the use of RFP-H2B as a nuclear
marker, per se. However, it does suggest that proteins of
interest for which the localization was initially determined
under transient conditions may need to be studied further in
transient plants. However, given the great expense and time
required for generating transgenic plant lines, it is infinitely
more practical to first determine protein localization in
transient assays. Should weaker promoters be required, the
pSAT vectors from which the present series were derived are
conveniently modular, which permits the facile replacement
of promoters (Chung et al., 2005). Users of these vectors are
therefore encouraged to select promoter/AFP combinations
relative to the specifics of their research objectives.
Taken together, the combination of binary vectors and
transgenic plants reported here provides a novel set of tools
to probe membrane and protein dynamics in potentially
many areas of plant biology.
EXPERIMENTAL PROCEDURES
Construction of modified pSAT6 vectors
In order to permit expression of protein fusions with AFPs at the C
termini of the proteins of interest, a modified pSAT6 vector that
included FseI and SpeI sites flanking a stuffer GFP was constructed.
We used GFP primers with the following modifications: NcoI and
FseI were added to the 5¢ end, and SpeI and BglII were added to the
3¢ end. These primers were designed using VECTOR NTI ADVANCE v.10
(Invitrogen, http://www.invitrogen.com). Forward primer sequence:
5¢-CCATGGGGCCGGCCGCTATGGTGAGCAAGGGCGAGGAGCTGTTCACC-3¢. Reverse primer sequence: 5¢-AGATCTACTAGTCCCGGCGGCGGTCACGAACTCCAGCAGGACCATG-3¢.
Using these primers, PCR for the amplification of GFP yielded a
band of the predicted size on agarose gel electrophoresis, which
was cloned into pGEM-T (Promega, http://www.promega.com)
following the kit directions. This fragment is hereafter referred to
as the ‘AFP’ fragment.
The pSAT6-MCS (Tzfira et al., 2005) and pGEM-T-AFP clones
were digested with NcoI and BglII. The corresponding backbone
from pSAT6-MCS and the inserted fragment from the pGEM-T clone
were gel-purified, and the AFP insert was ligated into pSAT6-MCS
via T4 ligase (New England Biolabs, http://www.neb.com).
The pSAT-AFP construct was modified to include the DEST
fragment from the Gateway system (Invitrogen), via digestion of
pSAT-AFP and pSAT6-EYFPC1-DEST (Chakrabarty et al., 2007) with
BglII and NdeI. The backbone from pSAT6-EYFPC1-DEST and the
insert from pSAT-AFP were gel purified and ligated together via T4
ligase. The pSAT6-AFP-DEST (Figure 1) constructs were confirmed
via PCR using the same primers designed for cloning and enzyme
digestion with FseI and SpeI (New England Biolabs).
The construct pSAT6-AFP-DEST was digested with PI-PspI (New
England Biolabs) to release the expression cassette insert. This
fragment was ligated to a similarly digested binary vector RCS2nptII (Goderis et al., 2002). This created the vector pSITEII-AFP
(Figure 2). The vector was checked for accuracy using PCR with the
specific primers described above, and enzyme digestion with FseI
and SpeI.
In order to facilitate the expression of fusion proteins with AFPs at
the C termini of the proteins of interest, a modified pSAT6 vector
was constructed using a strategy similar to that reported for
pSAT6-AFP-DEST. The forward and reverse primers used for this
construction were: 5¢-GGGCCCGGGCCGGCCATGGTGAGCAAGGGCGAGGAGCTGTT-3¢ and 5¢-GGATCCACTAGTTTGTACAGCTCGTCCATGCCGAGAGTGATC-3¢, respectively. The modified GFP stuffer
was mobilized into two variants of pSAT6 to create pSAT6-AFP-N1A
and pSAT6-AFP-N1B, which contain or lack Nco1 sites upstream of
the AFP, respectively. The DEST cassette was added to the pSATAFP-N1 vectors using an AgeI/ApaI fragment from pSAT-DEST-GFPNA and pSAT-DEST-RFP-NB (Chakrabarty et al., 2007) to create
pSAT-DEST-AFP-N1A and pSAT-DEST-AFP-N1B.
The two pSAT-DEST-AFP-N1 vectors were digested with PI-PspI
(New England Biolabs) to release the expression cassettes, which
were in turn moved into binary vector RCS2-nptII (Goderis et al.,
2002). This created the vectors pSITEII-AFP-N1A and pSITEII-AFPN1B. Only the pSITEII-AFP-N1B vector was used in the experiments
reported here.
Construction of pSITEII vectors containing different AFPs
In this study, we present data for nine different AFPs, including:
Cerulean (Rizzo et al., 2004); MiCy (Karasawa et al., 2004); Venus
(Nagai et al., 2002); mKO (Karasawa et al., 2004); TagRFP (Merzlyak
et al., 2007); DRONPA (Ando et al., 2004); and Dendra2 (Chudakov
et al., 2007). We also examined photoswitchable CFP (PS-CFP;
Chudakov et al., 2004); monomeric EosFP (Wiedenmann et al.,
2004); PA-GFP (Patterson and Lippincott-Schwartz, 2004); and
Azurite (Mena et al., 2006). However, fluorescence from these latter
three AFPs was not easily detectable in plant cells (data not shown),
and will therefore not be considered further.
Primers for Cerulean, MiCy, Venus, mKO, TagRFP, DRONPA,
Dendra2, PA-GFP, PS-CFP and GST were designed to add FseI to the
5¢ end and SpeI to the 3¢ end. Each fragment was PCR amplified and
ligated into either pGEM-T or pJET-T (Fermentas, http://www.
fermentas.com), following the kit(s) directions. The accuracy of
inserts was determined by PCR amplification from pGEM-T or pJETT. The correct plasmids were then digested with FseI and SpeI. The
pSAT6-DEST-AFP construct was similarly digested with FseI and
SpeI, and the inserts were added to replace the AFP fragment with
each of the fragments: Cerulean, MiCy, Venus, mKO, TagRFP,
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
160 Kathleen Martin et al.
DRONPA, Dendra2, PA-GFP, PS-CFP and GST. These constructs
were moved into RCS2-nptII as described above.
Construction of the pSITE-BiFC vectors
Digestion of the previously constructed pSAT6-EYFP-C1-DEST
(Chakrabarty et al., 2007) with AgeI and BglII was conducted to
release the EYFP fragment. pSAT4-nEYFP-C1 (DQ168994) and
pSAT1-cEYFP-C1 (DQ168996; Citovsky et al., 2006) were also
digested with AgeI and BglII to release the nEYFP and cEYFP fragments. The nEYFP and cEYFP fragments were ligated to the backbone of pSAT6-EYFP-C1-DEST to create pSAT6-nEYFP-DEST-C1
and pSAT6-cEYFP-DEST-C1, respectively. The PI-PspI fragments
from these vectors were mobilized into RCS2-nptII to create pSITEBiFC-C1nec and pSITE-BiFC-C1cec vectors (Figure 3). In a similar
manner, pSITE-BiFC-N1nen and pSITE-BiFC-N1cen vectors were
constructed from pSAT4A-nEYFP-N1 (DQ169002) and pSAT4AcEYFP-N1 (DQ169003; Figure 3).
Protein fusion construction
Genes of interest were mobilized into pSITE and pSITEII vectors
from pDONR intermediates by recombination-based cloning, as
described in Goodin et al. (2008a).
Transient expression of proteins in leaf epidermal cells
Suspensions of A. tumefaciens strain LBA4404 or C58C1 were
infiltrated into leaves of N. benthamiana as previously described
(Goodin et al., 2002; Tsai et al., 2005). To express proteins in SYNVinfected cells, symptomatic leaves of plants were infiltrated at the
peak of symptom expression, typically 10–14 days post-inoculation.
Following a 48-h incubation of infiltrated plants under constant
illumination at 25C, water-mounted sections of leaf tissue were
examined by confocal microscopy.
Confocal microscopy
All microscopy was conducted using an FV1000 point-scanning/
point-detection laser scanning confocal microscope (Olympus
http://www.olympus-global.com), equipped with lasers spanning
the spectral range of 405–633 nm. Micrographs for dual-color
imaging were acquired sequentially, as described in Goodin et al.
(2007a). The objective used was an Olympus water immersion
PLAPO60XWLSM (NA 1.0), unless otherwise noted. Image acquisition was conducted at a resolution of 512 · 512 pixels, with a scan
rate of 10 ms per pixel. Olympus FLUOVIEW 1.5 was used to control
the microscope, image acquisition and the export of TIFF files.
Figures were assembled using PHOTOSHOP 7.0 (Adobe, http://
www.adobe.com) and CANVAS 8.0 (Deneba Software, http://
www.deneba.com).
Photoactivation/photoconversion of AFPs
All photoactivation/photoconversion experiments were performed
using the Olympus FV1000 microscope described above. Briefly, 25mm2 square sections of tissue were excised from agroinfiltrated
leaves and mounted on glass slides in water, and then covered with
a glass coverslip. Imaging for DRONPA experiments was conducted
using a 40· objective and a 488-nm laser line from a multi-line argon
laser set at 0.3–1.0% of full power. Regions of interest were photoactivated for 50 ms using a 405-nm diode laser, set at 50–80% of full
power, which was delivered via the FV1000 Simultaneous (SIM)
scanner. Images for FRAP analyses were acquired at a resolution of
512 · 512 pixels with a scan rate of 2 ms per pixel, which was
necessary to monitor the fast protein dynamics. Two images were
acquired prior to photobleaching, followed by an additional seven
images to monitor fluorescence recovery. Quantitative fluorescence
data, in EXCEL format (Microsoft, http://www.microsoft.com), and
confocal images, in TIFF format, were exported using the Olympus
FLUOVIEW software.
The photoconversion of Dendra2 was conducted in a similar
manner to settings employed for DRONPA photoactivation. However, following the 405-nm pulse, the fluorescence from photoconverted Dendra2 was imaged using a 543-nm laser set at 20–40% of
full power.
Where applicable, mean and standard deviations for fluorescence
intensity at each time point were calculated and plotted using EXCEL.
Agrobacterium tumefaciens-mediated plant transformation
Transgenic plants were derived from the parental line used to
generate the ‘16c’ line of N. benthamiana mGFP5-ER plants
(Haseloff et al., 1997; Brigneti et al., 1998; Ruiz et al., 1998). The
transformation procedure was an adaptation of the methods
described by Horsch et al. (1985), Kalantidis et al. (2002) and
Chakrabarty et al. (2007). Briefly, A. tumefaciens strain LBA4404
carrying the pSITE vectors for the expression of RFP-H2B, CFP-H2B
or RFP-ER were grown overnight at 28C. Surface-sterilized leaves
from glasshouse-grown N. benthamiana plants were inoculated
with the Agrobacterium culture. The explants were co-cultivated for
2 days on MS media (Murashige and Skoog, 1962), supplemented
with benzylaminopurine (BA; 2 mg l)1) and indole-3-acetic acid
(IAA; 0.5 mg l)1). Putative transgenic shoots from the leaf explants
were induced on the same medium supplemented with Cefotaxime
(500 mg l)1) and Kanamycin (150 mg l)1). Regenerated shoots were
transferred to rooting media that included MS, with Cefotaxime
(250 mg l)1) and Kanamycin (50 mg l)1). After rooting, the plants
were transferred to soil in pots and were kept in a culture room at
25C with a 16-h photoperiod. Later, seeds were collected from T0
plants. Repeated rounds of screening resulted in the selection of
plant lines for which there was no segregation of the fluorescent
markers for five generations. These lines are assumed to be
homozygous.
ACKNOWLEDGEMENTS
We sincerely thank Iris Meier for providing the WIP1:GFP expression
construct, and Jörg Kulda for giving us a preprint of his multicolor
BiFC paper. We also thank Drs Ralf Dietzgen and Anindya Bandyopadhyay for their critical review of this manuscript prior to submission. This research was supported by USDA and Kentucky
Tobacco Research and Development Center and NSF awards to MG.
Upon request, all novel materials described in this publication will
be made available in a timely manner for non-commercial research
purposes, subject to the requisite permission from any third-party
owners of all or parts of the material. Obtaining any necessary
permission will be the responsibility of the requestor.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Photosensitivity of autofluorescent proteins (AFPs)
expressed in leaf epidermal cells of Nicotiana benthamiana.
Figure S2. Comparative brightness of spectrally-related groups of
autofluorescent proteins (AFPs) as a function of photomultiplier
tube-voltage from 300–700 V in 100-V increments.
Figure S3. Comparative brightness of spectrally-related groups of
autofluorescent proteins (AFPs).
Figure S4. Relative fluorescence intensity of the green and red forms
of SYNV-P:Dendra2 photoconverted in panels A1–A5 in Figure 5.
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 150–162
Live-cell protein interaction and localization 161
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
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