TECHNICAL ADVANCE
In vivo visualization of RNA in plants cells using the kN22
system and a GATEWAY-compatible vector series for
candidate RNAs
Johannes Scho¨ nberger†
, Ulrich Z. Hammes†,*
and Thomas Dresselhaus
Cell Biology and Plant Biochemistry, University of Regensburg, Universita¨tsstrasse 31, D-93053 Regensburg, Germany
Received 30 November 2011; revised 19 January 2012; accepted 20 January 2012; published online 13 April 2012.
*For correspondence (email: ulrich.hammes@biologie.uni-regensburg.de)
†
These authors contributed equally.
SUMMARY
The past decade has seen a tremendous increase in RNA research, which has demonstrated that RNAs are
involved in many more processes than were previously thought. The dynamics of RNA synthesis towards their
regulated activity requires the interplay of RNAs with numerous RNA binding proteins (RBPs). The localization
of RNA, a mechanism for controlling translation in a spatial and temporal fashion, requires processing and
assembly of RNA into transport granules in the nucleus, transport towards cytoplasmic destinations
and regulation of its activity. Compared with animal model systems little is known about RNA dynamics and
motility in plants. Commonly used methods to study RNA transport and localization are time-consuming, and
require expensive equipment and a high level of experimental skill. Here, we introduce the kN22 RNA stem-loop
binding system for the in vivo visualization of RNA in plant cells. The kN22 system consists of two components:
the kN22 RNA binding peptide and the corresponding box-B stem loops. We generated fusions of kN22 to
different fluorophores and a GATEWAY vector series for the simple fusion of any target RNA 5¢ or 3¢ to box-B
stem loops. We show that the kN22 system can be used to detect RNAs in transient expression assays, and that
it offers advantages compared with the previously described MS2 system. Furthermore, the kN22 system can
be used in combination with the MS2 system to visualize different RNAs simultaneously in the same cell. The
toolbox of vectors generated for both systems is easy to use and promises significant progress in our
understanding of RNA transport and localization in plant cells.
Keywords: fluorescence microscopy, GATEWAY technology, RNA binding protein, RNA transport, technical
advance, kN22.
INTRODUCTION
During the past decade innumerable studies have shown
that RNA molecules and their functions are more prevalent,
diverse and tightly controlled than were previously thought.
In the case of endogenous mRNAs, it became evident that
the tight regulation of localized mRNAs provides cells with a
molecular mechanism to post-transcriptionally control gene
expression of master cellular regulators, both spatially and
with precise timing (for a review, see Martin and Ephrussi,
2009). Animal oocytes, stem cells and specialized somatic
cells, such as nerve cells, are highly polarized, and it was
estimated that up to 10% of all mRNAs are polarly localized
in these cells as messenger ribonucleoprotein particles
(mRNPs) (King et al., 2005). Large-scale fluorescent in situ
hybridization experiments of more than three thousand
distinct mRNAs in early Drosophila embryos recently
showed that as much as 70% of mRNAs are localized,
suggesting that mRNA localization is not an exception but
rather the rule (Lecuyer et al., 2007). Furthermore, this
localization broadly correlates with protein localization and
function, implicating mRNA localization pathways in the
coordination of many cellular processes. The localization of
mRNAs thus seems to play a major role in organizing
cellular networks by targeting ribosomes, as well as other
RNPs, and regulating their activity to specific cellular subdomains,
thereby controlling translation spatially (St Johnston,
2005). Very recently it was shown that localization of
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mRNA also occurs in prokaryotes, indicating that RNA
localization may be a very ancient evolutionary feature
(Nevo-Dinur et al., 2011).
Polar localization of mRNA can be achieved by three
different mechanisms: (i) local stabilization and regulated
degradation of mRNA; (ii) local trapping of an RNA that is
diffusing through the cytoplasm; and (iii) active and directed
transport of mRNA. Although there are examples for all
three mechanisms, the latter is the most common mechanism
employed. The transported mRNP complexes form
larger structures, which are referred to as RNA transport
granules. These granules are transported by motor proteins
along microtubules and/or the actin cytoskeleton to their
final destination. In addition to the association with the
cytoskeleton there is also evidence that mRNA transport and
ER trafficking may be coupled (Schmid et al., 2006; Aronov
et al., 2007). Before mRNPs reach their final subcellular
destination, translation is usually delayed by the recruitment
of translational repressors (for a review, see Besse and
Ephrussi, 2008). It is currently still impossible to predict the
structural requirements or ‘zip codes’ within a transported
RNA molecule. In most cases mRNA localization is mediated
by stem-loop structures, found in the 3¢ untranslated region
(3¢-UTR) (Macdonald and Struhl, 1988; Macdonald et al.,
1993; Macdonald and Kerr, 1997; Bullock and Ish-Horowicz,
2001). It also appears that RNA binding proteins (RBPs) bind
to these stem loops with low affinity, and often a large
number of low-affinity interactions, none of which are
absolutely essential for localization, are involved in mRNA
localization (Arn et al., 2003).
Localized mRNAs often appear in mRNP granules, which
are heterogeneous in size and composition. Four types of
mRNP granules can be distinguished in higher eukaryotes:
(i) germ cell (polar) granules (GGs); (ii) stress or stored
granules (SGs); (iii) RNA processing bodies (P bodies or PB
granules); and (iv) mRNA transport granules (Moser and
Fritzler, 2010). Apart from animal model systems with large
and easily accessible egg cells or oocytes, as well as polar
cells that can be cultivated such as fibroblasts and neurons,
surprisingly little is known about the occurrence, composition
and function of localized mRNAs/mRNPs in higher
organisms. The study of post-transcriptional events associated
with localized RNAs/RNPs and specialized ribosomes
have received even less attention in plants, mainly because
of inefficient plant-derived in vitro systems (Bailey-Serres
et al., 2009; Lorkovic, 2009). Most progress in plants has
been made on the understanding of the transport of plant
virus-derived mRNPs (Sambade et al., 2008). The majority of
plant mRNPs reported to date are involved in stress
response and mRNA degradation processes in P bodies
(reviewed by Xu and Chua, 2011), but little is known about
RNA localization in plant cells. Localized protein synthesis at
the ER, involving cytoskeleton-associated RBPs, has recently
been reported for seed storage proteins in Oryza sativa (rice)
endosperm (Hamada et al., 2003; Wang et al., 2008; Doroshenk
et al., 2009; Crofts et al., 2010), and for actin-organizing
factor profilin in growing maize root hairs (Baluska et al.,
2000). The first mRNA, encoding an interleukin-1 receptorassociated
kinase (IRAK)/Pelle-like kinase, was reported to
be stored in pollen and only activated for translation in the
egg cytoplasm after fertilization (Bayer et al., 2009). It is very
likely that mRNAs encoding IRAK/Pelle-like kinases and
other proteins in pollen are transported and localized as
mRNPs, for example, to the tube tip or cytoplasmic sperm
cell microdomains. In summary, molecular mechanisms
regulating the localization and subsequent distribution of
mRNAs/mRNPs exist in plants, but their identity and composition
is largely unknown.
In situ imaging techniques have been applied successfully
to visualize and identify such mRNAs/mRNPs, subsets of
small non-coding RNAs (sncRNAs) containing RNPs or
specialized ribosomal RNPs (rRNPs), such as ribosomes
using fixed animal cells. However, these techniques are
difficult to achieve with most plant tissues, as they require
proper sectioning and are therefore not amenable for
high-throughput studies. Moreover, due to tissue fixation
in situ approaches don‘t allow to study RNP dynamics, RNA
trafficking and localization processes. The methods, which
were successfully applied in the animal field, are in principle
also available to visualize RNA dynamics in plants (for a
review, see Christensen et al., 2010). However, because of
the rigid cell wall and turgor pressure, microinjection of
fluorescent probes such as molecular beacons or directly
labelled RNA is more difficult to achieve in plant cells than in
animal cells. In any case these are mechanically invasive
methods that require both specialist equipment and experimental
skill, and therefore do not allow for the highthroughput
screening of localized RNAs/RNPs in plants.
Until now, directly labelled RNA was successfully used to
visualize the attachment of Tobacco Mosaic Virus RNA to the
plant actin/ER network (Christensen et al., 2009). The Pumillio-BiFC
system employs genetically encoded reporters that
target stem-loop structures, visualized by bimolecular fluorescence
complementation (BiFC) (Ozawa et al., 2007). The
great advantages of this method are that it is not invasive
and its sequence remains unmodified because the reporter
proteins are engineered to recognize the target RNA molecule
(Cheong and Hall, 2006). The Pumillo-BiFC method has
recently been used successfully in plants (Tilsner et al.,
2009). The drawback of this method is that it requires a great
deal of knowledge about the target RNA, the design of
Pumillo variants and additionally is time consuming. Similar
drawbacks apply to a very recently published method that
requires the design of RNA aptamers that mimic the
fluorophore in GFP (Paige et al., 2011).
The methods that appear to be most suitable for highthroughput
studies of localized RNAs/RNPs are those that
take advantage of RNA stem-loop binding RBPs. These
174 Johannes Scho¨ nberger et al.
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methods are mechanically non-invasive but require the
introduction of foreign RNA sequences, often in multiple
repeats to increase sensitivity, which may interfere with
target RNA function. The two methods, which are the subject
of this paper, are the MS2 system and the kN22 system. The
coat protein of phage MS2 (MS2-CP) binds a 19-nucleotide
(19-nt) RNA stem-loop structure with high specificity and
affinity (Kd = 6.2 nM) (LeCuyer et al., 1995). When fused to a
fluorescent tag this protein can be used to visualize RNAs
(Bertrand et al., 1998). The MS2 system has already been
used successfully to study RNA transport in plants (Hamada
et al., 2003; Zhang and Simon, 2003; Sambade et al., 2008).
The same principle was adapted recently to optimize GFPbased
RNA stem-loop reporter systems. kN22 consists of a
22 amino-acid peptide ligand from the lambda phage fused
to a fluorescent tag (Daigle and Ellenberg, 2007). The kN22
protein recognizes a 15-nt RNA stem loop, termed box-B, to
which the kN22 peptide binds with slightly lower affinity
(Kd = 22 nM) than the MS2 CP (Daigle and Ellenberg, 2007).
Until now, the kN22/ box-B system has been successfully
used in animal cells and fungi (Daigle and Ellenberg, 2007;
Lange et al., 2008; Konig et al., 2009), but not in plants.
Here we introduce a two-component GATEWAYTM
technology
based vector series for the use of both systems –
MS2-CP and kN22 – in plant cells. These RBPs were fused to
CFP, GFP, mVenus or mCherry for high versatility, and to
perform co-localization studies with each other or any other
RBP, allowing simultaneous visualization of various RNPs.
The target RNAs under investigation can easily be introduced
by GATEWAY recombination, either in the 5¢ or in the
3¢ position of corresponding stem-loop structures. We
further show that kN22/ box-B can successfully be used to
visualize RNPs in plants and demonstrate that kN22/ box-B
has considerable advantages over the MS2 system, and is
therefore well suited for low-background and high-throughput
RNP studies in plants.
RESULTS AND DISCUSSION
Generation of the two-component GATEWAYTM
vector series
In order to be able to monitor RNA trafficking in vivo, and to
set the basis for high-throughput RNA localization screening
tools and the simultaneous visualization of multiple RNP
components, we set out to adopt the kN22 system as an
additional tool and to compare it with the MS2 system for
use in plants. To be able to fully use both RBPs we created
fusions of both proteins with CFP, eGFP1, mVenus or
mCherry (Figure 1a). These different tags allow the localization
of different RNAs using these RBPs in conjunction
with other RBPs within the same cell, and will enable
downstream applications like fluorescence resonance
energy transfer (FRET) analyses. The SV40 nuclear localization
sequence (Kalderon et al., 1984) was introduced at the
C-terminal position in all cases to ensure nuclear localization
of the fluorescent RBP when it is not bound to a target RNA,
thus enabling background-free visualization of target RNAs
in the cytoplasm. These fusion proteins are expressed under
control of the ubiquitin 10 promoter of Arabidopsis (UBQ10),
which allows strong expression when transiently expressed
and which can also be used for constitutive expression after
stable transformation (Grefen et al., 2010). In order to enable
stable transformation of plants the fluorescent RBP vectors
are available with a kanamycin selection marker (Figure 1b).
The RBP target RNA stem loops were cloned in either the
5¢ or the 3¢ position of the GATEWAYTM
cassette (Figure 1b).
In order to increase the signal strength we used six repeats of
the MS2 stem loops and 16 repeats of the box-B stem loops.
Candidate RNAs can easily be introduced by GATEWAY
recombination cloning. The transcription of target RNAs is
controlled by the cauliflower mosaic virus 35S promoter
(Benfey and Chua, 1989), and the stable transformation of
plants using this vector series is possible by BASTA selection.
As it was shown that the nuclear history of an RNA
Figure 1. Schematic representation of the two-component RNA visualization
systems.
(a) Both systems take advantage of the specific binding of a viral RNA binding
protein (RBP, brown) to its corresponding stem-loop structure in the target
RNA (here 5¢ as an example). The RBPs used are kN22 and MS2-CP. These
RBPs are fused to a fluorescence protein (FP: CFP, GFP, mVenus or mCherry)
containing the SV40 NLS.
(b) Schematic representation of the two-component system vector series. The
T-DNA region between left border (LB) and right border (RB) is shown. The
RBP-FP-NLS fusion proteins are under the control of the UBQ10 promoter of
Arabidopsis. Stable transformands can be identified by kanamycin selection.
Candidate RNAs can be recombined by GATEWAY technology either in the 5¢
or 3¢ position of the corresponding target RNA stem loops. To enhance the
signal strength, six MS2 repeats and 16 box-B repeats were used, respectively.
The expression of the resulting RNA molecule is controlled by the
cauliflower mosaic virus 35S promoter. Stable transformands can be selected
by BASTA.
RNA imaging using the kN22 two-component system 175
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 173–181
molecule may play an important role in subsequent transport
and localization processes (Giorgi and Moore, 2007), it is
advisable to use genomic DNA including both UTRs and
introns for the generation of candidate RNA constructs,
especially if this information is known for an RNA of interest
or is available, e.g. from TAIR (Lamesch et al., 2010).
The different selection markers of RBP and target stem
loops enable the combination of the two components by
transformation or by the crossing and direct selection of the
offspring on both selective agents. All constructs were
tested and are functional after transient expression in
Nicotiana benthamiana (tobacco), as described below. Upon
email request all vectors described in this manuscript will be
made available for academic research purposes, without
charge.
Expression of fluorescent RBPs in tobacco leaves
In order to test the functionality of the systems, Agrobacterium
tumefaciens C58C1 harbouring constructs were used to
transiently express kN22-GFP-NLS and MSCP-mVenus-NLS
by infiltration into N. benthamiana leaves. Fluorescence was
visible 2 days after infiltration. In the absence of a target
RNA both proteins were exclusively localized to the nuclei
(Figure 2a, b, e and f). Stronger fluorescence was always
observed in the nucleoli. This localization was background
free and is the prerequisite to studying the cytosolic path of
candidate RNAs. Upon co-infiltration of target RNAs the
fluorescence was still strongest in nuclei, but could be
additionally observed in the cytoplasm of the cells (Figure
2c, d, g and h), and was frequently seen in cytoplasmic
foci (see below). The same pattern was observed for all
fluorescent tags used (not shown). In order to demonstrate
the specificity of the RNA-binding proteins, we co-expressed
kN22-GFP-NLS with an RNA encoding a scorable cytoplasmic
marker (tagRFP) lacking stem loops (Figure S1a–c) or
containing the MS2 stem loops (Figure S1d–f). Neither
mRNA had any effect on the localization of kN22-GFP-NLS.
The same results were obtained when we co-expressed
MS2-CP with the tagRFP mRNA lacking stem loops (Figure
S1g–i) or the tagRFP mRNA containing box-B stem loops
(Figure S1j–l). These data are in agreement with previous
studies using GFP coupled to the MS2 system in plants
(Hamada et al., 2003; Zhang and Simon, 2003), and are
comparable with the results obtained for kN22 in mammalian
cell culture or yeast (Daigle and Ellenberg, 2007; Lange
et al., 2008).
In order to rule out that the different distribution of
fluorescence within the cell after co-expression of a target
RNA was the result of degradation or any other form of
modification of the fluorescent RBPs, total protein was
extracted from infiltrated leaf discs and analysed by western
blotting (Figure 2i). In the absence of a target RNA, kN22
could be identified as a single band corresponding to the
predicted molecular weight of 31 kDa. MS2-CP was identi(a)
(b)
(c) (d)
(e) (f)
(g)
(i)
(h)
Figure 2. Transient expression of both two-component systems in Nicotiana
benthamiana leaves.
(a–d) kN22-GFP-NLS.
(e-h) MS2CP-mVenus-NLS. In the absence of a target RNA (a, b, e and f) the
fusion proteins localized to the nuclei of epidermis cells. In the presence of a
target RNA containing the corresponding target stem loops, fluorescence is
readily observed in the cytoplasm (c, d, g and h). a, c, e and g are
fluorescent light images. b, d, f and h are overlays of fluorescent light
channel and the corresponding bright-field image, so as to highlight the
typical jigsaw shape of the N. benthamiana epidermis cells. Scale bars:
10 lm.
(i) Western blot analysis of RBP-FP-NLS fusion proteins in the presence (+)
or absence ()) of a target RNA. In both cases kN22-GFP-NLS can be detected
as a monomer at the predicted molecular weight (31 kDa). In contrast,
MS2CP-mVenus-NLS was detected at the predicted molecular weight
(43 kDa) in the absence of a target RNA, whereas an additional band
corresponding to the size of the free fluorophore was detected in the
presence of a target RNA (arrowhead). In the absence or presence of a target
RNA an additional band corresponding to the molecular weight of the dimer
($90 kDa) was observed in all samples studied. GFP: free GFP, positive
control.
176 Johannes Scho¨ nberger et al.
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 173–181
fied as a band corresponding to its calculated molecular
weight (43 kDa) and an additional broad band, which was
also detected when a target RNA was co-expressed, corresponding
to the calculated molecular weight of the MS2-CP
dimer. This band was neither observed in protein isolated
from kN22 leaf discs nor observed in positive control discs
(GFP alone), indicating that MS2-CP-mVENUS-NLS may
form higher order complexes, preferentially very stable
dimers, in the absence and presence of target RNA. This
hypothesis is supported by the finding that the broad band
was always present even after treating the protein extract
with strong reducing agents (data not shown). In the
presence of a target RNA we always observed an additional
weaker band corresponding to the molecular weight of free
GFP (arrowhead in Figure 2i), indicating that MS2-CP-mVenus-NLS
is subject to proteolytic degradation in the cytoplasm,
which does not occur in the case of kN22 in the
presence of a target RNA.
Taken together our findings suggest that data obtained
with the MS2 system must be interpreted with caution
because not all the fluorescence observed in the cytosol is
the result of MS2-CP bound to its target RNA. Additionally,
higher molecular structures observed with the MS2 system
might be artificial, and a result of di-/multimerization of the
fluorescent RBP. The fact that these issues do not occur with
the kN22 system indicates that kN22 may be more reliable
than the MS2 system, and therefore be used when labelling
a single candidate RNA under investigation.
Influence of the position of the stem-loop structure
To further establish the system as a tool to study plant RNPs
we wanted to investigate the influence of the position of the
stem-loop structures within the target RNA construct on
the redistribution of fluorescence and the translation of the
target mRNA. To this end we co-infiltrated kN22-eGFP1-NLS
or MS2-CP-mVenus-NLS with constructs comprising their
corresponding stem-loop structure either in the 5¢ or in the 3¢
position of the target RNA. In order to easily visualize the
translation of target RNAs we used the open reading frame
of tagRFP, which results in orange-red fluorescence derived
from the translated and active protein. As shown in Figure 3,
comparable results were obtained with both the kN22 and
MS2 systems. Regardless of the position of the stem loops
we observed fluorescence in the cytosol resulting from RBPs
bound to tagRFP mRNA. Frequently signals accumulated in
cytoplasmic foci (Figure 3a, d, g and j). However, in both
systems translation of the target mRNA, indicated by the
orange-red fluorescence of tagRFP, was observed only when
the stem loops were in the 3¢ position of the tagRFP gene
(Figure 3e and k). As shown in Figure S1 the co-expression
of the marker proteins with non-specific tagRFP mRNAs did
not lead to a redistribution of fluorescence. Therefore, the
presence of marker proteins, as indicated by the fluorescence
in the cytoplasm, results from RNA binding and not
from non-specific interaction of the marker proteins with
tagRFP mRNA. We never detected fluorescence of the
translation product when the stem loops were in the 5¢
position of the tagRFP gene (Figure 3b and h). To rule out the
possibility that the failure to observe tagRFP fluorescence
was caused by the lack of transcription of the target RNA, we
isolated RNA from infiltrated sectors, treated these with
DNase and performed oligo dT primed reverse transcriptase
PCR. As shown in Figure S2, transcripts were present demonstrating
that the absence of tagRFP fluorescence originated
from a translation block occurring when the stem
loops were in the 5¢ position of the tagRFP open reading
frame. Translation is probably inhibited because ribosome
entry is blocked by the presence of multiple RBP-FP fusion
proteins. Alternatively, the 5¢ MS2 and box-B stem loops
contain several ATG start codons that are not in frame, and
may thus serve as translation initiation sites leading to the
translation of non-sense peptides.
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j) (k) (l)
Figure 3. Transient co-expression of kN22-GFP-NLS and MSCP-mVenus-NLS
with a target RNA encoding tagRFP as a scorable marker to study the influence
of the 5¢ and 3¢ position of the corresponding stem loops (box-B or MS2) on
the translation of the co-expressed RNA.
(a–c) kN22-GFP-NLS and 16 box-B -tagRFP; (d–f) kN22-GFP-NLS and tagRFP-
16boxB; (g–i) MS2-CP-mVenus-NLS and 6MS2-tagRFP; (j–l) MS2-CP-mVenusNLS
and tagRFP-6MS2. In all cases the co-expression led to the presence of
fluorescence in the cytosol (a, d, g and j). The translation of tagRFP only
occurred when the stem loops were in the 3¢ position of the tagRFP RNA (e
and k), leading to the yellow fluorescence resulting from co-localization in f
and l. Scale bars: (d–f) and (j–l), 20 lm; (a–c) and (g–i) or 10 lm.
RNA imaging using the kN22 two-component system 177
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 173–181
Presence of higher order mRNPs and transport granules
In addition to the presence of fluorescence in the cytosol,
upon co-expression of a corresponding target RNA cytoplasmic
foci were observed using both systems (Figure 4;
Video Clips S1 and S2). These foci were always observed,
and formed independently of the type of RNA, i.e. from
foreign RNA such as tagRFP or a plant mRNA encoding a
cytosolic or secreted protein (data not shown), and thus
were independent of translation at free ribosomes or at
ribosomes associated with the rough ER. These cytoplasmic
foci were rather uniform in size. Their true diameter is
difficult to evaluate in fluorescence images; however,
based on the images the size of the foci is 800–1200 nm
and their motility indicated that they represent mRNP
transport granules. These transport granules were highly
motile and moved directionally, occasionally in a stopand-go
fashion, indicating an association with a cytoskeletal
component rather than simple diffusion (Video Clips 1
and 2). kN22 granules moved with a velocity of 0.98
Æ 0.1 lm s)1
(n = 5, time series taken in different cells),
and MS2-CP particles moved at an average velocity of
0.31 Æ 0.05 lm s)1
(n = 5 time series taken in different
cells). These data are in good agreement with velocities
observed for both systems in yeast, filamentous fungi or
the Drosophila egg cell, in which the velocities ranged from
400 nm s)1
for MS2-CP granules to 1.6 Æ 0.7 lm s)1
for
kN22 granules (Bertrand et al., 1998; Becht et al., 2006;
Lange et al., 2008; Zimyanin et al., 2008; Konig et al., 2009).
Because we detected extremely stable dimers in our western
blot analysis (Figure 2i) we limited the detailed
evaluation of the transport granules to the kN22 system.
When kN22-GFP-NLS and tagRFP- box-B were transiently
co-expressed within the same cell, we observed green
fluorescence (Figure 3d) resulting from the fluorescent RBP
and orange-red fluorescence resulting from the translation
of the tagRFP mRNA (Figure 3e) within the same cell.
Merging both channels resulted in yellowish fluorescence
arising from co-localization. Figure 4a shows a section of
the cell shown in Figure 3f. It is readily visible that the colocalization
was not absolute, i.e. there were domains
within the cytoplasm that were exclusively highlighted by
red fluorescence, and other domains exclusively highlighted
by green fluorescence. The most prominent green
foci are labelled with arrowheads, and represent motile
transport granules. Fluorescence intensity values of both
channels were measured for each pixel in Figure 4a, and
were plotted against one another to quantify the green and
red fluorescence (Figure 4b). From this illustration it is
obvious that there are pixels that are either only red or only
green. Among the pixels that display the highest intensities
in the green channel is a subset of pixels contained in the
circles of Figure 4b. These pixels constitute transport
granules marked by arrowheads in Figure 4a. The granules
thus did not co-localise with the translation product. In
summary, these findings indicate that mRNA is transported
within granules. The translation product could be laid
down, whereas the particle continues to move or, similar to
the situation in yeast or animal cells, translation does not
occur in these structures. In order to be translated the
granules need to be disassembled, which contributes or
gives rise to all of the green fluorescence, which in the case
of the epidermal cells appears to be evenly distributed. It
will be interesting to see if polar localization within certain
cells exists in plants, similar to animal cells, fungi and even
bacteria, and how this polarity is brought about. In this
case the aforementioned observations, and especially the
positioning of the stem loops, has several important
implications. If a protein encoded by a target mRNA plays a
role in the assembly, transport or polar localization of its
own mRNA, a true picture can only be obtained in transient
assays once the mRNA can be translated. However, overexpression,
especially in transient assays or systems using
strong promoters may also result in the accumulation of
target RNA-encoded proteins, leading to artefacts. Moreover,
if a target RNA under investigation was localized by a
‘localized translation mechanism’ (for a review, see St
Johnston, 2005), the use of a construct with the stem loops
(a) (b) Figure 4. Transient co-expression of kN22-GFPNLS
with a target RNA encoding tagRFP as a
scorable marker leads to the presence of green
fluorescence in the cytoplasm and the formation
of transport granules [arrowheads in (a)].
Fluorescence intensities of each pixel in (a) in the
green and the red channel were plotted against
each other (b). The four circles highlighted in (b)
represent pixels, which are high in green fluorescence
and almost free of red fluorescence.
These pixels constitute the fluorescent foci/
transport granules in (a). These data show that
the transport granules are composed exclusively
of tagRFP mRNA and RBP. Scale bar: 20 lm
178 Johannes Scho¨ nberger et al.
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 173–181
in the 5¢ position would fail to detect such a mechanism.
On the other hand, if localization signals were contained
within the 3¢-UTR of the RNA under investigation, the
additional stem loops in the 3¢ position might mask the
correct transport and localization of this RNA. In order to
circumvent these obstacles we suggest cloning stem loops
in both 5¢ and 3¢ positions with respect to the RNA under
study.
Simultaneous visualization of different RNAs
within the same cell
We further investigated whether it is possible to use the kN22
and the MS2-CP system simultaneously to visualize two
different RNA populations within the same cell. To this
end we transiently transformed N. benthamiana epidermis
cells with four different constructs: kN22-CFP-NLS, MS2CP-mVENUS-NLS
and two different target RNAs containing
the corresponding stem loops. As a target for kN22 we used
an mRNA encoding a plasma membrane localized protein
which is translated at the rough ER. As a target for MS2-CP
we used an mRNA encoding a cytoplasmic protein, which is
translated at free ribosomes. In both cases we used the entire
genomic region including UTRs and introns based on
the annotations in TAIR 10 (Lamesch et al., 2010). As shown
in Figure 5a–c, distinct transport granules could be observed,
which indicates that the two systems can be used to
simultaneously to monitor two different populations of
mRNA. However, it should be mentioned that because of the
fairly quick movement of the granules simultaneous tracking
of two populations of granules requires patience and
sophisticated equipment.
In order to demonstrate that the motile granules observed
represent RNA transport granules and to show the use of the
kN22 system in conjunction with endogenous cytoplasmic
RBPs, we co-expressed kN22-mCherry-NLS, a target mRNA
and the cytoplasmic RBP At4g17520 fused to GFP. This
protein is a member of the hyaluronan/mRNA binding
protein family. Members of this family have been shown to
be involved in stabilizing mature mRNA in the cytoplasm of
animal cells (Heaton et al., 2001). Upon co-expression of the
target mRNA encoding a plasma membrane-localized protein
(see above) and box-B stem loops in the 3¢ position, we
observed more green granules derived from cytoplasmic
RBP-GFP fusion protein compared with red granules, simply
because an RNA-binding protein from plants with low
specificity has more targets than the heterologous and
highly sequence-specific kN22 RBP. We also observed red
granules not joined by green granules (arrowheads in
Figure 5d–f), which are transported independently. Interestingly,
often cytoplasmic foci were found to be composed of
green and red granules, which moved together at an average
velocity of about 0.5 Æ 0.1 lm s)1
. This was insignificantly
slower than that of the individual red or green granules
(0.9 Æ 0.1 and 1.4 Æ 0.5 lm s)1
, respectively; n = 3 individual
cells). In all cases the ‘tandem’ granules moved in
an oriented, probably cytoskeleton-mediated directional
fashion (arrows Figure 5d–f), indicating that the two foci
are co-ordinately transported. This suggests that the
(a) (b) (c)
(d) (e) (f)
Figure 5. Transient co-expression of kN22-CFP-NLS and MSCP-mVenus-NLS with their target RNAs containing the corresponding stem loops in the same epidermis
cell of Nicotiana benthamiana.
(a) CFP channel; (b) YFP channel; and (c) overlay of panels (a) and (b). A distinct higher order transport granule containing kN22-CFP-NLS is marked by an arrow in (a)
and (c), and a transport granule of MSCP-mVenus-NLS is marked by an arrowhead in (b) and (c). The outline of the cell is represented by a dotted line.
(d–f) Co-expression of kN22-mCherry-NLS, a GFP-labelled cytoplasmic RNA binding protein (RBP) and a target RNA containing box-B stem loops. RNA transport
granules consisting of kN22-mCherry-NLS are labelled with an arrowhead. Transport granules consisting of kN22-mCherry-NLS, its mRNA target plus GFP-labelled
RBP (arrow) move co-ordinately and directionally together. The times indicated are the real-time observations relative to (d) as the starting point. Scale bars: 10 lm.
RNA imaging using the kN22 two-component system 179
ª 2012 The Authors
The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 173–181
coordinated movement is mediated by RNA binding
because in the absence of a box-B-containing target, RNA
kN22 remained in the nucleus, whereas RBP1-GFP granules
could be observed at comparable frequencies (arrowheads
in Figure S3). The series of pictures shown in Figure 5d–f is
also provided as Video Clip S3. The joint movement of viral
RBP and the endogenous RBP is very similar to the movement
of Staufen RBP and its target oskar mRNA tagged with
MS2 stem loops and visualized by MS2-CP and co-localized
with the Staufen RBP (Zimyanin et al., 2008). In summary,
these data show that the kN22 system can be used to monitor
RNA transport and movement in vivo, and additionally could
be used to visualize the movement of RNAs in combination
with endogenous RBPs.
Conclusion and outlook
In this technical paper we introduced the kN22 system as an
additional tool to visualize RNA/RNPs and to study their
trafficking and dynamics in plant cells in vivo. We
demonstrated in vitro that kN22 does not display the possible
pitfalls that may be encountered using the MS2-CP system,
and may therefore be more reliable. These features may
essentially result from the smaller size of the RNA binding
component (22 compared with 117 amino acids) of the RBP.
Moreover, we provide a vector series and seeds of marker
plants for numerous studies involving all kinds of RNAs,
such as mRNAs, sncRNAs as well as rRNAs, to investigate,
for example, RNP assembly, RNA/RNP transport and
movement, translational repression as well as polar RNA
localization. Moreover, by combining both systems it is now
possible to distinguish between two different RNAs in the
same cell including functionally different RNPs such as
ribosomes and mRNP granules. It will be interesting to see if
these methods can be used to identify polar-localized
mRNAs in plants.
EXPERIMENTAL PROCEDURES
Generation of the two component vector series for
the kN22 and MS2 systems
Open reading frames of kN22 and MS2-CP, as well as the corresponding
stem loops box-B (16 repeats) and MS2 (6 repeats), were
amplified by PCR from the original vectors provided by Michael
Feldbru¨ gge (University of Du¨sseldorf), Jan Ellenberg (EMBL Heidelberg)
and Tom Okita (Washington State University). Details of
the cloning procedure and primer sequences are available upon
request. All constructs were confirmed by DNA sequencing and
were functionally tested in transient transformation assays in
tobacco (N. benthamiana).
Infiltration of tobacco leafs
Infiltration of N. benthamiana epidermis cells by A. tumefaciens
C58C1 was performed essentially as described by Bartetzko et al.
(2009). Briefly, cultures were centrifuged at 3000 g for 5 min and
cells were resuspended in the same volume of infiltration buffer
[10 mM MgCl2, 10 mM 2-(N-morpholine)-ethanesulphonic acid,
pH 5.7, 100 lM acetosyringone], and after incubation for 1 h infiltrated
into N. benthamiana leaves.
Microscopy
Confocal laser scanning microscopy (CLSM) was performed
48–72 h after infiltration using LSM 510 Meta and LSM 710 microscopes
(both Zeiss, http://www.zeiss.com), as well as Leica SP2 and
SP5 microscopes equipped with acousto-optic beam splitters.
Excitation and emission were at: 405 and 450–500 nm, respectively,
for CFP; 488 and 500–530 nm, respectively, for GFP; 514 and
530–560 nm, respectively, for mVenus; 561 and 590–620 nm,
respectively, for Tag-RFP; and 543 and 585–615 nm, respectively, for
mCherry.
RNA isolation and RT-PCR
Leaf material of infiltrated tobacco plants was sampled into liquid
nitrogen and total RNA extracts prepared using the RNeasy Plant
Mini Kit (Qiagen, http://www.qiagen.com), according to the manufacturer’s
instructions. Reverse transcription was performed using
the SuperScriptÒ VILOÔ cDNA Synthesis Kit (Invitrogen, https://
www.lifetechnologies.com).
Protein isolation and immunoblotting
Leaf material from N. benthamiana was sampled in liquid nitrogen:
1 g of material was ground in 3 ml of grinding buffer (20 mM
HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1· Complete
Protease Inhibitor; Roche, http://www.roche.com). Crude cell debris
was removed by centrifugation at 400 g for 4°C. Protein levels were
determined by a Bradford assay and 20 lg of protein from the
supernatant was used for western blot analysis using a GFP
monoclonal antibody (Roche) in a 1 : 1000 dilution and a 1 : 5000
dilution of horseradish peroxidase-coupled anti-mouse secondary
antibody (Santa Cruz Biotechnology, http://www.scbt.com).
ACKNOWLEDGEMENTS
This project was funded by the German Research Council DFG
grants DR 334/7-1 and SFB 960. We are grateful to Norbert Sauer
and Uwe Sonnewald (University of Erlangen) for granting access
to their confocal microscopes. We thank two anonymous
reviewers for their constructive and valuable input to improve the
article.
SUPPORTING INFORMATION
Additional supporting information may be found in the online
version of this article:
Figure S1. Co-expression of the markers with non-target RNAs.
Figure S2. Oligo-dT primed RT-PCR was performed to detect tagRFP
transcripts (top). Actin (bottom) was used a positive control.
Figure S3. Co-expression of kN22-mCherry-NLS and GFP-RBP1 in the
absence of a target RNA containing box-B stem loops.
Video Clip S1. Representative time-lapse film of a cell infiltrated with
MS2-CP-mVenus-NLS and a target RNA.
Video Clip S2. Representative time-lapse film of a cell infiltrated with
kN22-GFP-NLS and a target RNA.
Video Clip S3. Representative time-lapse film of a cell infiltrated with
kN22-mCherry-NLS, RBP-GFP (At4g17520) and a target RNA.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
180 Johannes Scho¨ nberger et al.
ª 2012 The Authors
The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 173–181
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RNA imaging using the kN22 two-component system 181
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