Transgenic Res

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BRIEF COMMUNICATION

Enhanced foreign protein accumulation in Nicotiana

benthamiana leaves co-infiltrated with a TMV vector
and plant cell cycle regulator genes
Lilya Kopertekh . Joachim Schiemann

Received: 24 October 2018 / Accepted: 11 May 2019

Ó Springer Nature Switzerland AG 2019

Abstract In this short communication, we report
that the cell cycle checkpoint genes At-CycD2 and At-
CDC27a from Arabidopsis thaliana enhance the
transient heterologous protein expression in Nicotiana
benthamiana. We selected a well-studied and widely
used virus expression vector based on TMV for the
delivery of recombinant proteins into the host plant.
Co-infiltration of TMV-gfp and binary expression
vectors carrying the At-CycD2 and At-CDC27a genes,
respectively, resulted in enhanced GFP fluorescence in
agroinoculated leaves. These findings corresponded
with the observation of (1) higher mRNA levels for
TMV and gfp and (2) increased GFP protein accumu-
lation. Furthermore, by co-delivery of the TMV-scFv-
TM43-E10 and At-CycD2/At-CDC27a expressing
constructs we observed an enhanced amount of the
scFv-TM43-E10 antibody fragment compared to the
delivery of the TMV-scFv-TM43-E10 alone. We
anticipate that this finding might be adapted for
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s11248-019-00128-3) con-
tains supplementary material, which is available to authorized
users.
L. Kopertekh (&)
J. Schiemann
Julius Kuehn Institute - Federal Research Centre for
Cultivated Plants (JKI), Institute for Biosafety in Plant
Biotechnology, Erwin-Baur-Str. 27, 06484 Quedlinburg,
Germany
e-mail: lilya.kopertekh@julius-kuehn.de
enhancing foreign protein production in N. benthami-
ana as the host plant.
Keywords Cell cycle check point genes
Nicotiana
benthamiana
Recombinant protein
TMV
Transient
expression
Introduction
Transient expression has become a standard technol-
ogy for the production of recombinant proteins in
plants. The yield of foreign proteins depends on both
the expression vector and the plant cell physiology.
Different host cell engineering strategies including
preventing unintended proteolysis (Robert et al. 2013),
modulating the pH in cell secretory compartments
(Jutras et al. 2015), leaf proteome rebalancing (Robert
et al. 2015), and suppression of RNA silencing
(Garabagi et al. 2012) have been developed to design
favorable environments for recombinant protein
production.
The basis for high accumulation of foreign proteins
provided by plant virus vectors is an efficient multi-
plication of the virus genetic material. Viruses utilize a
great number of host proteins for their replication and
can modify the function and composition of the host
cell to benefit their proliferation (Ahlquist et al. 2003;
Whitham and Wang 2004). Some viruses can target
specific phases of the host cell cycle, particularly the

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transition from G1 to S stage. For instance, Human
papillomavirus (HPV), Chicken anemia virus (CAV),
Adenovirus (Ad) and Human cytomegalovirus
(HCMV) target the anaphase promoting complex
(APC) regulating both the cell division and the timing
of re-entry into S phase for their own replication
benefit (Mo et al. 2012). Begomoviruses (Tomato
Golden Mosaic Virus, Tomato Yellow Leaf Curl Virus)
and mastreviruses (Maize Streak Virus, Wheat Dwarf
Virus, Bean Yellow Dwarf Virus) are able to induce a
G1/S step transition through interaction with a plant
retinoblastoma-related protein (Hanley-Bowdoin et al.
2004).
Several publications described the interaction of the
Tobacco mosaic virus (TMV) with the plant cell cycle
machinery. Early experiments on TMV accumulation
in protoplasts of Nicotiana sylvestris demonstrated
that the attachment efficiency of the virus virions
depends on the host cell’s position in the cell cycle
(Gould et al. 1981). Microarray analysis of the TMV
infected tobacco plants revealed that five transcripts
related to the cell cycle checkpoint control proteins
were upregulated. Interestingly, the transcript of the
cell division checkpoint control protein RAD9A
accumulated to 138 fold more when compared to
non-infected plants (Jada et al. 2014). Furthermore,
Niehl et al. (2012) observed a negative regulation of
TMV accumulation by the cell-division-cycle protein
48 from Arabidopsis thaliana.
In this study, we co-expressed the CycD2 and
CDC27a genes from Arabidopsis thaliana with a
TMV-based vector in Nicotiana benthamiana to
investigate possible effects of these genes on the
accumulation of recombinant proteins provided by the
TMV vector. The At-CycD2 gene belongs to the
D-type cyclin gene family and promotes the transition
from G1 to S phase of the cell cycle (Soni et al. 1995).
The anaphase promoting complex subunit CDC27a
also takes part in the stimulation of G1/S transition
through increased expression of the cyclin-dependent
kinases and G1/S specific cyclins (Rojas et al. 2009).
Stable expression of both genes in Nicotiana tabacum
resulted in morphologically normal transgenic plants
with accelerated growth and increased biomass pro-
duction (Cockcroft et al. 2000; Rojas et al. 2009).
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Transgenic Res
Materials and methods
Plasmid construction
The At-CycD2 gene was amplified from A. thaliana
cDNA using the At-CycD2-forw and At-CycD2-rev
primers, cloned first into the pGEM-T vector (Pro-
mega) and then by NcoI-XbaI digestion into the pCK-
gfp plasmid (Reichel et al. 1996). The HindIII DNA
fragment from the pCK-35S-At-CycD2 intermediate
construct comprising of CaMV 35S promoter, At-
CycD2 gene and CaMV 35S terminator was ligated
with the similarly restricted pLH7000 binary vector
(Hausmann and Töpfer 1999) yielding the final pLH-
35S-At-CycD2 construct.
The At-CDC27a gene was isolated from the
pGSV435SAtCDC27a vector (Rojas et al. 2009) with
NcoI-BamHI digestion and cloned into the equally
restricted pCK-gfp construct between the CaMV 35S
promoter and terminator. The HindIII restriction
fragment of the pCK-35S-At-CDC27a plasmid con-
taining the At-CDC27a expression cassette was then
sub-cloned into the HindIII digested pLH7000 creat-
ing the final pLH-35S-At-CDC27a vector.
The pICH17344 expression vector harboring the
TMV-gfp was provided by Icon Genetics GmbH
(Halle, Germany). To design the TMV-scFv-TM43-
E10 construct a scFv-TM43-E10 sequence was ampli-
fied from the pOPE101-TM43-E10 plasmid (Meyer
et al. 2011) using XhoI-TMV43-E10-forw and SwaI-
TM43-E10-rev primers, digested with XhoI and SwaI
and ligated into the pICH17344 restricted with the
same enzymes. The construction of the pLH-35S-gfp
plasmid was described previously (Yelina et al. 2005).
The cloning primers are shown in Table 1S. All
DNA fragments designed by PCR were sequenced.
Plant agroinfiltration
Agrobacterium tumefaciens cultures containing the
expression constructs were grown overnight in LB
medium supplemented with appropriate antibiotics,
pelleted by centrifugation and resuspended in MMA
solution (10 mM MES, pH 5.6, 10 mM MgCl2,
150 lM acetosyringone). The final optical density of
agrobacteria carrying the TMV-gfp, TMV-scFv-
TM43-E10 and pLH-35S-gfp vectors was adjusted to
OD600 of 0.1. The cultures of A. tumefaciens carrying
the pLH-35S-At-CDC27a and pLH-35S-At-CycD2
Transgenic Res
plasmids were brought to a final OD600 of 0.3.
Agroinfiltration was performed according to Maril-
lonnet et al. (2004) on leaves of 5–6 week-old N.
benthamiana plants grown in the greenhouse at
24/22 °C day/night temperature with a 16 h light and
8 h dark photoperiod.
qPCR analysis
RNA extraction was performed by the RNeasy Mini
Kit (Qiagen) according to manufacturer’s specifica-
tions. cDNA was synthesized using the Maxima
Reverse Transcriptase and random hexamer primer
following manufacturer’s instructions (Thermo Sci-
entific). Quantification of target gfp and virus RNA
relative to ubiquitin transcripts was performed in
MastercyclerÒ EP realplex (Eppendorf) using Maxima
SYBR Green qPCR Master Mix (Thermo Scientific).
Primer sequences for the gfp, TMV coat protein and
reference ubiquitin (ubi) genes are listed in Table 1S.
PCR thermal cycles were as follows: initial denatura-
tion step for 10 min at 95 °C followed by 45 cycles of
denaturation for 15 s at 95 °C, annealing for 30 s at
56 °C and extension for 30 s at 72 °C. Relative
quantifications were performed based on the DCT
method using the ubi gene as an internal standard.
GFP imaging
For visual detection of GFP fluorescence N. benthami-
ana leaves were illuminated with a hand-held UV
lamp and photographed with the Canon digital camera
EOS 300D.
Protein detection and quantification
GFP and scFv-TM43-E10 proteins were detected and
quantified by western blot. Agroinfiltrated leaves were
harvested at 5 dpi, prepared, separated and blotted
according to standard procedures as described previ-
ously (Kopertekh et al. 2004). Total soluble proteins
(TSP) were measured by BCA Protein Assay Kit
(Thermo Scientific). 12 lg of TSP for each sample
was loaded together with defined concentrations of
GFP and scFv-TM43-E10 proteins extracted from
E. coli. The membranes were probed with an anti-GFP
antibody (ABIN153484) for GFP and an anti-c-myc
antibody (Sigma-Aldrich) for scFv-TM43-E10,
detected with an anti-mouse lgG antibody linked with
alkaline phosphatase (Sigma-Aldrich) and BCIP/NBT
reagent (Sigma-Aldrich) and analyzed with Image-
Quant TL7.0 software (GE Healthcare). Three middle
leaves were agroinfiltrated. Three plants for each
construct were treated and each experiment was
repeated at least three times.
Results and discussion
We selected a transient expression assay to combine
two different expression vectors (pLH7000 and
TMV) for recombinant proteins and constructs con-
taining the At-CDC27a and At-CycD2 genes in N.
benthamiana. The T-DNA regions of the pLH-35S-
gfp, pLH-35S-At-CDC27a and pLH-35S-At-CycD2
plasmids are schematically presented in Fig. 1a. In
the gfp, At-CDC27a and At-CycD2 transcription units
the gfp, At-CDC27a and At-CycD2 sequences are
fused to the CaMV 35S promoter and terminator
providing their constitutive expression. A well-studied
and widely used virus vector based on TMV was
chosen for recombinant protein delivery (Fig. 1a). The
binary pLH7000 and TMV expression vectors were
infiltrated alone or co-infiltrated with the pLH-35S-At-
CDC27a or with the pLH-35S-At-CycD2.
To monitor the impact of the cell cycle checkpoint
genes on the expression of foreign proteins provided
by TMV and pLH7000 expression vectors we started
with the GFP model protein. As expected, GFP
fluorescence could be observed under UV light at 5
dpi in N. benthamiana leaves agroinfiltrated with
pLH-35S-gfp, pLH-35S-gfp:pLH-35S-At-CDC27a,
pLH-35S-gfp:pLH-35S-At-CycD2, TMV-gfp, TMV-
gfp:pLH-35S-At-CDC27a and TMV-gfp:pLH-35S-
At-CycD2. The presence of the At-CDC27a and At-
CycD2 genes resulted in stronger GFP fluorescence
when the TMV expression vector was applied. In
contrast, co-infiltration of pLH-35S-gfp with the pLH-
35S-At-CDC27a and pLH-35S-At-CycD2 constructs
did not result in enhancement of gfp expression
(Fig. 1b).
Our visual observation was confirmed at RNA level
by qPCR using TMV and gfp specific primers. qPCR
of RNA samples extracted from N. benthamiana
leaves treated with pLH-35S-gfp, pLH-35S-gfp:pLH-
35S-At-CDC27a, and pLH-35S-gfp:pLH-35S-At-
CycD2 did not reveal any enhancement of gfp
expression (Fig. 2a). On the contrary, the presence
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Fig. 1 Effect of the At-CDC27a and At-CycD2 cell cycle
regulators on GFP transient expression. a Schematic diagram of
the TMV, pLH-35S-gfp, pLH-35S-At-CDC27a and pLH-35S-
At-CycD2 expression constructs. TMV vector. Open boxes
indicate the following genes: Rep, viral replicase; MP,
movement protein; GOI, gene of interest (gfp/scFv-TM43-
E10); CP, coat protein. Arrows denote the subgenomic RNA
coat protein promoter. The pLH-35S-gfp, pLH-35S-At-CDC27a
and pLH-35S-At-CycD2 expression constructs are based on the
pLH7000 vector backbone. The bar expression cassette includes
the bar selection marker gene between the 35S promoter and
terminator. In the 35S-gfp-ter, 35S-At-CDC27a-ter and 35S-At-
CycD2-ter expression units the gfp, At-CycD2 and At-CDC27a
of cell cycle regulators At-CycD2 and At-CDC27a
affected the accumulation of both virus and gfp RNAs
when the TMV virus vector was used. Virus RNA
accumulation in leaves inoculated with the combina-
tion of TMV-gfp and the pLH-35S-At-CycD2 plasmid
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Transgenic Res
genes are fused with the 35S promoter and terminator. LB, RB,
left and right border of T-DNA, respectively. (B) GFP
fluorescence in agroinfiltrated leaves. N. benthamiana leaves
were agroinfiltrated with Agrobacterium strains containing the
TMV-gfp, TMV-gfp:pLH-35S-At-CDC27a, TMV-gfp:pLH-
35S-At-CycD2, pLH-35S-gfp, pLH-35S-gfp:pLH-35S-At-
CDC27a, and pLH-35S-gfp:pLH-35S-At-CycD2 constructs as
described in ‘‘Materials and methods’’ section. Pictures were
taken under UV light at 5 dpi. The agroinfiltration experiments
were repeated at least three times with three agroinfiltrated
plants per treatment each time. The representative picture is
shown
was about 5 times higher than that in TMV-gfp
infiltrated tissue. For the TMV-gfp:pLH-35S-At-
CDC27a mixture the estimated virus RNA level was
increased up to 2.6 fold (Fig. 2a). Furthermore, the
amount of gfp RNA positively correlated with the
Transgenic Res
Fig. 2 Analysis of TMV, GFP and scFv-TM43 accumulation.
N. benthamiana plants were agroinfiltrated with the pLH-35S-
gfp, TMV-gfp and TMV-scFv-TM43 virus vectors either in the
absence or presence of the pLH-35S-At-CDC27a and pLH-35S-
At-CycD2 expression constructs. Leaf samples were harvested
at 5 dpi and investigated for TMV, gfp and scFv-TM43
accumulation. a Q-PCR analysis. Relative quantification of the
TMV and gfp RNA in agroinfiltrated samples was performed
using virus and gfp specific primers and DCT method. Values
virus RNA accumulation in both TMV-gfp:pLH-35S-
At-CycD2 and TMV-gfp:pLH-35S-At-CDC27a infil-
trated samples (Fig. 2a).
After having identified the positive impact of the
At-CDC27a and At-CycD2 genes on virus and gfp
RNA accumulation, we determined the concentration
of the GFP protein in the plant tissue. N. benthamiana
leaves agroinfiltrated with TMV-gfp:pLH-35S-CycD2
cultures accumulated 3 fold more GFP protein than
samples agroinfiltrated with TMV-gfp. Treatment of
tobacco plants with the TMV-gfp:pLH-35S-CDC27a
mixture enhanced the amount of GFP protein up to two
fold (Fig. 2b).
The time course analysis of GFP concentration in
extracts from N. benthamiana leaves agroinfiltrated
represent the mean of at least six biological replicates reported
with SEM. Each replicate is a pooled sample from three middle
leaves of one plant. b Protein accumulation (lg/g FW). GFP and
scFv-TM43-E10 proteins were quantified by analyzing western
blot images with ImageQuant TL7.0 software (GE Healthcare).
Values represent the mean of nine biological replicates reported
with SEM. Each replicate is a pooled sample from three middle
leaves of one plant
with TMV-gfp, TMV-gfp:pLH-35S-At-CDC27a and
TMV-gfp:pLH-35S-At-CycD2 further confirmed the
positive impact of the At-CDC27a and At-CycD2 cell
cycle regulators on GFP accumulation at 4, 6 and 8 dpi
(Figure 1S).
Taken together, these data indicate that the
enhanced GFP protein accumulation in the presence
of the At-CDC27a and At-CycD2 cell cycle checkpoint
genes is based on the increased levels of virus and gfp
RNA. The mechanism of this phenomenon remains to
be elucidated. We suppose that the co-expression of
cell cycle checkpoint genes creates an intracellular
environment supportive for enhanced TMV replica-
tion. Cell-cycle analysis of At-CycD2-expressing
transgenic tobacco meristems demonstrated a
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reduction in the length of cell cycle due to a reduction
in the G1 phase (Boucheron et al. 2005). Flow-
cytometry analysis of cells isolated from At-CDC27a
N. tabacum transgenic plants revealed the enhance-
ment of both G1/S and G2/M transition as well as the
exit from mitosis. Additionally the levels of S-phase
specific cyclins were upregulated (Rojas et al. 2009).
To examine whether the At-CDC27a and At-CycD2
genes can improve the expression of other recombi-
nant proteins, we designed the TMV-scFv-TM43-E10
expression vector containing the scFv-TM43-E10
antibody fragment with a molecular mass of 29 kDa
(Fig. 1a). Five days after the introduction of A.
tumefaciens strains harboring the TMV-scFv-TM43-
E10, TMV-scFv-TM43-E10:pLH-35S-At-CDC27a,
and TMV-scFv-TM43-E10:pLH-35S-At-CycD2 in N.
benthamiana, leaf samples were investigated for scFv-
TM43-E10 protein accumulation. In the TMV-scFv-
TM43-E10 agroinfiltrated leaves expression levels of
231 lg/g fresh weight (FW) were obtained, while in
the presence of the At-CycD2 gene the scFv-TM43-
E10 expression levels increased 2-fold to 464 lg/g
FW. In the case of the TMV-scFv-TM43-E10:pLH-
35S-At-CDC27a, levels of 373 lg/g FW were
achieved, 1.8 times higher than those observed in the
absence of the At-CDC27a gene (Fig. 2b).
Several publications describe the importance of the
interplay between the plant cell biology and TMV
virus vector for efficient recombinant protein produc-
tion. One of the most interesting strategies is based on
the modification of TMV cDNA to meet the require-
ments of the plant cell RNA processing machinery.
Removal of the cryptic splice sites and addition of
introns in the TMV sequence allowed a 712-fold
increase in the efficiency of initiation of viral repli-
cation (Marillonnet et al. 2005). The second remark-
able example explores silencing suppressors to
modulate plant cell defense against TMV. The ectopic
expression of p19 silencing suppressor from Tomato
bushy stunt virus increased the efficiency of TMV-gfp
expression at least 100-fold (Lindbo 2007a). An
alternative approach uses the inhibition of plant genes
involved in antiviral response. For example, silencing
of the 33 K subunit of the oxygen-evolving complex
of photosystem II resulted in a 10-fold increase of
TMV accumulation (Abbink et al. 2002).
Our results show that co-expression of a TMV-
based vector with plant genes affecting cell cycle
progression resulted in an increased virus and
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Transgenic Res
subsequent foreign protein accumulation. A number
of commercial transient expression systems such as
GENEWAREÒ (Shivprasad et al. 1999), TRBO
(Lindbo 2007b), magnICONÒ (Gleba et al. 2007),
TMV launch vector (Musiychuk et al. 2007) are based
on full or deconstructed versions of TMV. Therefore,
this finding might be useful for transient foreign
protein production using TMV as the vector backbone.
Further studies are required to determine the effect
of cell cycle checkpoint genes on the expression of
recombinant proteins provided by other virus vectors,
particularly those that are based on plant DNA viruses.
To advance our finding to a practical level the
accumulation of foreign proteins with high molecular
weight in the presence of the At-CDC27a and At-
CycD2 genes should be estimated.
Acknowledgements We thank Cornelia Freyer for technical
support, Icon Genetics GmbH (Halle, Germany) and Dr.
Ferreira (Universidade Federal do Rio de Janeiro, Brazil) for
providing pICH17344 and pGSV435sAtCDC27a plasmids,
respectively. The financial support provided by the German
Federal Ministry of Food and Agriculture (BMEL) is greatly
acknowledged.
Author contributions LK designed the study and performed
the experiments. LK and JS analyzed the data, wrote and
reviewed the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that there is no con-
flict of interest.
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