Transgenic Res

https://doi.org/10.1007/s11248-019-00128-3 (0123456789().,-volV)
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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 enhancing foreign protein production in N. benthami-
that the cell cycle checkpoint genes At-CycD2 and At- ana as the host plant.
CDC27a from Arabidopsis thaliana enhance the
transient heterologous protein expression in Nicotiana Keywords Cell cycle check point genes
Nicotiana
benthamiana. We selected a well-studied and widely benthamiana
Recombinant protein
TMV
Transient
used virus expression vector based on TMV for the expression
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 Introduction
agroinoculated leaves. These findings corresponded
with the observation of (1) higher mRNA levels for Transient expression has become a standard technol-
TMV and gfp and (2) increased GFP protein accumu- ogy for the production of recombinant proteins in
lation. Furthermore, by co-delivery of the TMV-scFv- plants. The yield of foreign proteins depends on both
TM43-E10 and At-CycD2/At-CDC27a expressing the expression vector and the plant cell physiology.
constructs we observed an enhanced amount of the Different host cell engineering strategies including
scFv-TM43-E10 antibody fragment compared to the preventing unintended proteolysis (Robert et al. 2013),
delivery of the TMV-scFv-TM43-E10 alone. We modulating the pH in cell secretory compartments
anticipate that this finding might be adapted for (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
Electronic supplementary material The online version of production.
this article (https://doi.org/10.1007/s11248-019-00128-3) con- The basis for high accumulation of foreign proteins
tains supplementary material, which is available to authorized
users.
provided by plant virus vectors is an efficient multi-
plication of the virus genetic material. Viruses utilize a
L. Kopertekh (&)
J. Schiemann great number of host proteins for their replication and
Julius Kuehn Institute - Federal Research Centre for can modify the function and composition of the host
Cultivated Plants (JKI), Institute for Biosafety in Plant
cell to benefit their proliferation (Ahlquist et al. 2003;
Biotechnology, Erwin-Baur-Str. 27, 06484 Quedlinburg,
Germany Whitham and Wang 2004). Some viruses can target
e-mail: lilya.kopertekh@julius-kuehn.de specific phases of the host cell cycle, particularly the

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transition from G1 to S stage. For instance, Human Materials and methods

papillomavirus (HPV), Chicken anemia virus (CAV),
Adenovirus (Ad) and Human cytomegalovirus Plasmid construction
(HCMV) target the anaphase promoting complex
(APC) regulating both the cell division and the timing The At-CycD2 gene was amplified from A. thaliana
of re-entry into S phase for their own replication cDNA using the At-CycD2-forw and At-CycD2-rev
benefit (Mo et al. 2012). Begomoviruses (Tomato primers, cloned first into the pGEM-T vector (Pro-
Golden Mosaic Virus, Tomato Yellow Leaf Curl Virus) mega) and then by NcoI-XbaI digestion into the pCK-
and mastreviruses (Maize Streak Virus, Wheat Dwarf gfp plasmid (Reichel et al. 1996). The HindIII DNA
Virus, Bean Yellow Dwarf Virus) are able to induce a fragment from the pCK-35S-At-CycD2 intermediate
G1/S step transition through interaction with a plant construct comprising of CaMV 35S promoter, At-
retinoblastoma-related protein (Hanley-Bowdoin et al. CycD2 gene and CaMV 35S terminator was ligated
2004). with the similarly restricted pLH7000 binary vector
Several publications described the interaction of the (Hausmann and Töpfer 1999) yielding the final pLH-
Tobacco mosaic virus (TMV) with the plant cell cycle 35S-At-CycD2 construct.
machinery. Early experiments on TMV accumulation The At-CDC27a gene was isolated from the
in protoplasts of Nicotiana sylvestris demonstrated pGSV435SAtCDC27a vector (Rojas et al. 2009) with
that the attachment efficiency of the virus virions NcoI-BamHI digestion and cloned into the equally
depends on the host cell’s position in the cell cycle restricted pCK-gfp construct between the CaMV 35S
(Gould et al. 1981). Microarray analysis of the TMV promoter and terminator. The HindIII restriction
infected tobacco plants revealed that five transcripts fragment of the pCK-35S-At-CDC27a plasmid con-
related to the cell cycle checkpoint control proteins taining the At-CDC27a expression cassette was then
were upregulated. Interestingly, the transcript of the sub-cloned into the HindIII digested pLH7000 creat-
cell division checkpoint control protein RAD9A ing the final pLH-35S-At-CDC27a vector.
accumulated to 138 fold more when compared to The pICH17344 expression vector harboring the
non-infected plants (Jada et al. 2014). Furthermore, TMV-gfp was provided by Icon Genetics GmbH
Niehl et al. (2012) observed a negative regulation of (Halle, Germany). To design the TMV-scFv-TM43-
TMV accumulation by the cell-division-cycle protein E10 construct a scFv-TM43-E10 sequence was ampli-
48 from Arabidopsis thaliana. fied from the pOPE101-TM43-E10 plasmid (Meyer
In this study, we co-expressed the CycD2 and et al. 2011) using XhoI-TMV43-E10-forw and SwaI-
CDC27a genes from Arabidopsis thaliana with a TM43-E10-rev primers, digested with XhoI and SwaI
TMV-based vector in Nicotiana benthamiana to and ligated into the pICH17344 restricted with the
investigate possible effects of these genes on the same enzymes. The construction of the pLH-35S-gfp
accumulation of recombinant proteins provided by the plasmid was described previously (Yelina et al. 2005).
TMV vector. The At-CycD2 gene belongs to the The cloning primers are shown in Table 1S. All
D-type cyclin gene family and promotes the transition DNA fragments designed by PCR were sequenced.
from G1 to S phase of the cell cycle (Soni et al. 1995).
The anaphase promoting complex subunit CDC27a Plant agroinfiltration
also takes part in the stimulation of G1/S transition
through increased expression of the cyclin-dependent Agrobacterium tumefaciens cultures containing the
kinases and G1/S specific cyclins (Rojas et al. 2009). expression constructs were grown overnight in LB
Stable expression of both genes in Nicotiana tabacum medium supplemented with appropriate antibiotics,
resulted in morphologically normal transgenic plants pelleted by centrifugation and resuspended in MMA
with accelerated growth and increased biomass pro- solution (10 mM MES, pH 5.6, 10 mM MgCl2,
duction (Cockcroft et al. 2000; Rojas et al. 2009). 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

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plasmids were brought to a final OD600 of 0.3. alkaline phosphatase (Sigma-Aldrich) and BCIP/NBT
Agroinfiltration was performed according to Maril- reagent (Sigma-Aldrich) and analyzed with Image-
lonnet et al. (2004) on leaves of 5–6 week-old N. Quant TL7.0 software (GE Healthcare). Three middle
benthamiana plants grown in the greenhouse at leaves were agroinfiltrated. Three plants for each
24/22 °C day/night temperature with a 16 h light and construct were treated and each experiment was
8 h dark photoperiod. repeated at least three times.

qPCR analysis
Results and discussion
RNA extraction was performed by the RNeasy Mini
Kit (Qiagen) according to manufacturer’s specifica- We selected a transient expression assay to combine
tions. cDNA was synthesized using the Maxima two different expression vectors (pLH7000 and
Reverse Transcriptase and random hexamer primer TMV) for recombinant proteins and constructs con-
following manufacturer’s instructions (Thermo Sci- taining the At-CDC27a and At-CycD2 genes in N.
entific). Quantification of target gfp and virus RNA benthamiana. The T-DNA regions of the pLH-35S-
relative to ubiquitin transcripts was performed in gfp, pLH-35S-At-CDC27a and pLH-35S-At-CycD2
MastercyclerÒ EP realplex (Eppendorf) using Maxima plasmids are schematically presented in Fig. 1a. In
SYBR Green qPCR Master Mix (Thermo Scientific). the gfp, At-CDC27a and At-CycD2 transcription units
Primer sequences for the gfp, TMV coat protein and the gfp, At-CDC27a and At-CycD2 sequences are
reference ubiquitin (ubi) genes are listed in Table 1S. fused to the CaMV 35S promoter and terminator
PCR thermal cycles were as follows: initial denatura- providing their constitutive expression. A well-studied
tion step for 10 min at 95 °C followed by 45 cycles of and widely used virus vector based on TMV was
denaturation for 15 s at 95 °C, annealing for 30 s at chosen for recombinant protein delivery (Fig. 1a). The
56 °C and extension for 30 s at 72 °C. Relative binary pLH7000 and TMV expression vectors were
quantifications were performed based on the DCT infiltrated alone or co-infiltrated with the pLH-35S-At-
method using the ubi gene as an internal standard. CDC27a or with the pLH-35S-At-CycD2.
To monitor the impact of the cell cycle checkpoint
GFP imaging genes on the expression of foreign proteins provided
by TMV and pLH7000 expression vectors we started
For visual detection of GFP fluorescence N. benthami- with the GFP model protein. As expected, GFP
ana leaves were illuminated with a hand-held UV fluorescence could be observed under UV light at 5
lamp and photographed with the Canon digital camera dpi in N. benthamiana leaves agroinfiltrated with
EOS 300D. pLH-35S-gfp, pLH-35S-gfp:pLH-35S-At-CDC27a,
pLH-35S-gfp:pLH-35S-At-CycD2, TMV-gfp, TMV-
Protein detection and quantification gfp:pLH-35S-At-CDC27a and TMV-gfp:pLH-35S-
At-CycD2. The presence of the At-CDC27a and At-
GFP and scFv-TM43-E10 proteins were detected and CycD2 genes resulted in stronger GFP fluorescence
quantified by western blot. Agroinfiltrated leaves were when the TMV expression vector was applied. In
harvested at 5 dpi, prepared, separated and blotted contrast, co-infiltration of pLH-35S-gfp with the pLH-
according to standard procedures as described previ- 35S-At-CDC27a and pLH-35S-At-CycD2 constructs
ously (Kopertekh et al. 2004). Total soluble proteins did not result in enhancement of gfp expression
(TSP) were measured by BCA Protein Assay Kit (Fig. 1b).
(Thermo Scientific). 12 lg of TSP for each sample Our visual observation was confirmed at RNA level
was loaded together with defined concentrations of by qPCR using TMV and gfp specific primers. qPCR
GFP and scFv-TM43-E10 proteins extracted from of RNA samples extracted from N. benthamiana
E. coli. The membranes were probed with an anti-GFP leaves treated with pLH-35S-gfp, pLH-35S-gfp:pLH-
antibody (ABIN153484) for GFP and an anti-c-myc 35S-At-CDC27a, and pLH-35S-gfp:pLH-35S-At-
antibody (Sigma-Aldrich) for scFv-TM43-E10, CycD2 did not reveal any enhancement of gfp
detected with an anti-mouse lgG antibody linked with expression (Fig. 2a). On the contrary, the presence

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Fig. 1 Effect of the At-CDC27a and At-CycD2 cell cycle genes are fused with the 35S promoter and terminator. LB, RB,
regulators on GFP transient expression. a Schematic diagram of left and right border of T-DNA, respectively. (B) GFP
the TMV, pLH-35S-gfp, pLH-35S-At-CDC27a and pLH-35S- fluorescence in agroinfiltrated leaves. N. benthamiana leaves
At-CycD2 expression constructs. TMV vector. Open boxes were agroinfiltrated with Agrobacterium strains containing the
indicate the following genes: Rep, viral replicase; MP, TMV-gfp, TMV-gfp:pLH-35S-At-CDC27a, TMV-gfp:pLH-
movement protein; GOI, gene of interest (gfp/scFv-TM43- 35S-At-CycD2, pLH-35S-gfp, pLH-35S-gfp:pLH-35S-At-
E10); CP, coat protein. Arrows denote the subgenomic RNA CDC27a, and pLH-35S-gfp:pLH-35S-At-CycD2 constructs as
coat protein promoter. The pLH-35S-gfp, pLH-35S-At-CDC27a described in ‘‘Materials and methods’’ section. Pictures were
and pLH-35S-At-CycD2 expression constructs are based on the taken under UV light at 5 dpi. The agroinfiltration experiments
pLH7000 vector backbone. The bar expression cassette includes were repeated at least three times with three agroinfiltrated
the bar selection marker gene between the 35S promoter and plants per treatment each time. The representative picture is
terminator. In the 35S-gfp-ter, 35S-At-CDC27a-ter and 35S-At- shown
CycD2-ter expression units the gfp, At-CycD2 and At-CDC27a

of cell cycle regulators At-CycD2 and At-CDC27a was about 5 times higher than that in TMV-gfp
affected the accumulation of both virus and gfp RNAs infiltrated tissue. For the TMV-gfp:pLH-35S-At-
when the TMV virus vector was used. Virus RNA CDC27a mixture the estimated virus RNA level was
accumulation in leaves inoculated with the combina- increased up to 2.6 fold (Fig. 2a). Furthermore, the
tion of TMV-gfp and the pLH-35S-At-CycD2 plasmid amount of gfp RNA positively correlated with the

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Fig. 2 Analysis of TMV, GFP and scFv-TM43 accumulation. represent the mean of at least six biological replicates reported
N. benthamiana plants were agroinfiltrated with the pLH-35S- with SEM. Each replicate is a pooled sample from three middle
gfp, TMV-gfp and TMV-scFv-TM43 virus vectors either in the leaves of one plant. b Protein accumulation (lg/g FW). GFP and
absence or presence of the pLH-35S-At-CDC27a and pLH-35S- scFv-TM43-E10 proteins were quantified by analyzing western
At-CycD2 expression constructs. Leaf samples were harvested blot images with ImageQuant TL7.0 software (GE Healthcare).
at 5 dpi and investigated for TMV, gfp and scFv-TM43 Values represent the mean of nine biological replicates reported
accumulation. a Q-PCR analysis. Relative quantification of the with SEM. Each replicate is a pooled sample from three middle
TMV and gfp RNA in agroinfiltrated samples was performed leaves of one plant
using virus and gfp specific primers and DCT method. Values

virus RNA accumulation in both TMV-gfp:pLH-35S- with TMV-gfp, TMV-gfp:pLH-35S-At-CDC27a and

At-CycD2 and TMV-gfp:pLH-35S-At-CDC27a infil- TMV-gfp:pLH-35S-At-CycD2 further confirmed the
trated samples (Fig. 2a). positive impact of the At-CDC27a and At-CycD2 cell
After having identified the positive impact of the cycle regulators on GFP accumulation at 4, 6 and 8 dpi
At-CDC27a and At-CycD2 genes on virus and gfp (Figure 1S).
RNA accumulation, we determined the concentration Taken together, these data indicate that the
of the GFP protein in the plant tissue. N. benthamiana enhanced GFP protein accumulation in the presence
leaves agroinfiltrated with TMV-gfp:pLH-35S-CycD2 of the At-CDC27a and At-CycD2 cell cycle checkpoint
cultures accumulated 3 fold more GFP protein than genes is based on the increased levels of virus and gfp
samples agroinfiltrated with TMV-gfp. Treatment of RNA. The mechanism of this phenomenon remains to
tobacco plants with the TMV-gfp:pLH-35S-CDC27a be elucidated. We suppose that the co-expression of
mixture enhanced the amount of GFP protein up to two cell cycle checkpoint genes creates an intracellular
fold (Fig. 2b). environment supportive for enhanced TMV replica-
The time course analysis of GFP concentration in tion. Cell-cycle analysis of At-CycD2-expressing
extracts from N. benthamiana leaves agroinfiltrated transgenic tobacco meristems demonstrated a

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reduction in the length of cell cycle due to a reduction subsequent foreign protein accumulation. A number
in the G1 phase (Boucheron et al. 2005). Flow- of commercial transient expression systems such as
cytometry analysis of cells isolated from At-CDC27a GENEWAREÒ (Shivprasad et al. 1999), TRBO
N. tabacum transgenic plants revealed the enhance- (Lindbo 2007b), magnICONÒ (Gleba et al. 2007),
ment of both G1/S and G2/M transition as well as the TMV launch vector (Musiychuk et al. 2007) are based
exit from mitosis. Additionally the levels of S-phase on full or deconstructed versions of TMV. Therefore,
specific cyclins were upregulated (Rojas et al. 2009). this finding might be useful for transient foreign
To examine whether the At-CDC27a and At-CycD2 protein production using TMV as the vector backbone.
genes can improve the expression of other recombi- Further studies are required to determine the effect
nant proteins, we designed the TMV-scFv-TM43-E10 of cell cycle checkpoint genes on the expression of
expression vector containing the scFv-TM43-E10 recombinant proteins provided by other virus vectors,
antibody fragment with a molecular mass of 29 kDa particularly those that are based on plant DNA viruses.
(Fig. 1a). Five days after the introduction of A. To advance our finding to a practical level the
tumefaciens strains harboring the TMV-scFv-TM43- accumulation of foreign proteins with high molecular
E10, TMV-scFv-TM43-E10:pLH-35S-At-CDC27a, weight in the presence of the At-CDC27a and At-
and TMV-scFv-TM43-E10:pLH-35S-At-CycD2 in N. CycD2 genes should be estimated.
benthamiana, leaf samples were investigated for scFv-
TM43-E10 protein accumulation. In the TMV-scFv- Acknowledgements We thank Cornelia Freyer for technical
support, Icon Genetics GmbH (Halle, Germany) and Dr.
TM43-E10 agroinfiltrated leaves expression levels of Ferreira (Universidade Federal do Rio de Janeiro, Brazil) for
231 lg/g fresh weight (FW) were obtained, while in providing pICH17344 and pGSV435sAtCDC27a plasmids,
the presence of the At-CycD2 gene the scFv-TM43- respectively. The financial support provided by the German
E10 expression levels increased 2-fold to 464 lg/g Federal Ministry of Food and Agriculture (BMEL) is greatly
acknowledged.
FW. In the case of the TMV-scFv-TM43-E10:pLH-
35S-At-CDC27a, levels of 373 lg/g FW were Author contributions LK designed the study and performed
achieved, 1.8 times higher than those observed in the the experiments. LK and JS analyzed the data, wrote and
absence of the At-CDC27a gene (Fig. 2b). reviewed the manuscript.
Several publications describe the importance of the
interplay between the plant cell biology and TMV Compliance with ethical standards
virus vector for efficient recombinant protein produc- Conflict of interest The authors declare that there is no con-
tion. One of the most interesting strategies is based on flict of interest.
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
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