Swarup A geminivirus AYVV-derived shuttle vector for tobacco BY2 cells Received: 21 January 2004 / Revised: 1 March 2004 / Accepted: 3 March 2004 / Published online: 7 April 2004 Spring
Trang 1Plant Cell Rep (2004) 23:81–90
DOI 10.1007/s00299-004-0792-0
G E N E T I C S A N D G E N O M I C S
D Tamilselvi · G Anand · S Swarup
A geminivirus AYVV-derived shuttle vector for tobacco BY2 cells
Received: 21 January 2004 / Revised: 1 March 2004 / Accepted: 3 March 2004 / Published online: 7 April 2004
Springer-Verlag 2004
Abstract We have developed a plant-Escherichia coli
pASV shuttle vector from the essential elements of the
Ageratum yellow vein virus (AYVV) The geminivirus
vector contains the AYVV genome with the coat-protein
deletion, the E coli vector backbone of pUC19, a unique
cloning site and gene expression cassettes for plant
se-lection and reporter gene activity The replication of
pASV vectors was compared in Nicotiana benthamiana
and N tabacum BY2 cells, and the latter were found to be
suitable for long-term maintenance of the vectors in
cul-ture The vector DNA was detected at regular intervals by
PCR, b-glucuronidase expression analysis and plasmid
rescue during a 4-month culture period A novel
meth-ylation-based PCR assay was carried out to show de novo
replication for pASV-derived vectors in 2-month-old
to-bacco BY2 cell lines This is the first report of the
ex-trachromosomal replication of monopartite begomovirus
with stability and foreign gene expression in long-term
cell cultures
Keywords Ageratum yellow vein virus · Geminivirus ·
Shuttle vector · Replication · Foreign gene expression
Abbreviations ACMV: African cassava mosaic virus ·
AYVV: Ageratum yellow vein virus · CP: Coat protein ·
GUS:b-Glucuronidase · TGMV: Tomato golden mosaic
virus · Tobacco BY2: Tobacco L cv Bright Yellow 2
Introduction
Whole plants and plant cells are emerging as viable and competitive expression systems for large-scale protein production as a means of obtaining biologically active and safe biopharmaceutical proteins at affordable prices Consequently, there is renewed interest in developing novel vectors to express foreign proteins in these systems While the most widely used method for producing foreign proteins is via stably transformed plants, plant cell cul-tures provide an alternative The main advantages of the latter lie in the large-scale production of proteins in in-dustrially sized bioreactors under sterile, defined, and controllable conditions, all of which makes the plant cell culture system amenable to standard biomanufacturing practices As a result of recent commercial interest, there have been many advances in the areas of transgenic plants (Fischer and Emans 2000; Ma et al 2003), production in suspension cultures (Fischer et al 1999a) and the appli-cation of plant vectors for foreign protein expression (Fischer et al 1999b) Vast choices of plant transforma-tion vectors are currently available due to the early start researchers have made in this field Compared to the stable transformation vectors based on T-DNA, however, there are only a few reports on extrachromosomal shuttle vectors for expressing foreign genes
In plants, single-stranded DNA geminiviruses have been used as potential sources of extrachromosomal vector replicons to enable multiplication in the nuclei of infected cells (reviewed by Davies and Stanley 1989; Mullineaux et al 1992; Stanley 1993; Timmermans et al 1994) Geminiviruses have small single-stranded circular DNA genomes of 2.5–3.0 kb They are characterized by twinned (geminate) icosahedral capsids and can replicate using plant host machinery via double-stranded DNA intermediates The geminiviridae family has three sub-groups, which can be distinguished based on their genetic organization, plant host, and insect vector The genera Mastrevitesus and Curtovirus have a single genomic component and infect monocot and dicot species, re-spectively Begomovirus members, on the other hand,
Communicated by P.P Kumar
D Tamilselvi · G Anand · S Swarup ())
Department of Biological Sciences,
National University of Singapore,
Singapore, 117 543
e-mail: dbsss@nus.edu.sg
Tel.: +65-6874-7933
Fax: +65-6779-2486
Present address:
G Anand, Temasek Capital (Private) Limited,
Temasek Tower, Singapore, 068811
Trang 2exclusively infect dicot species and have a genome
comprising two similar-sized DNA components (DNA A
and DNA B) (reviewed by Gutierrez 2000) DNA A
en-codes a replication-associated protein (Rep), coat-protein
(CP), and proteins that participate in the control of
rep-lication and gene expression The DNA B component
encodes proteins required for nuclear trafficking and
cell-to-cell movement of the viral DNA Relatively few
be-gomoviruses have been described to possess a
monopar-tite genome, which resembles DNA A Those include the
tomato leaf curl virus (TLCV), Ageratum yellow vein
virus (AYVV), and cotton leaf curl virus (CLCuV) (Dry
et al 1993; Tan et al 1995; Briddon et al 2000)
AYVV can replicate and form infectious symptoms in
Ageratum, tomato, French bean and Nicotiana
ben-thamiana (Tan et al 1995) Transmission of AYVV is by
whiteflies (Tan et al 1995), and its monopartite genome
has two overlapping virion-sense open reading frames
(ORFs), V1 and V2 that encode the CP and movement
protein, respectively ORF C1 encodes the
replication-associated protein, ORFs C2 and C3 regulate virion-sense
gene expression and DNA replication, respectively,
ORF C4 is a pathogenicity determinant that may affect
the host cell, and there is an additional ORF C5 of
un-known function The intergenic region (IR) contains the
initiation site of rolling circle DNA replication
Irrespective of the monopartite or bipartite nature of
geminiviruses, only their intergenic and the
complemen-tary strand ORFs are necessary for replication
(Lazaro-witz et al 1989; Kammann et al 1991; Ugaki et al 1991)
Hence, a popular strategy used in developing geminiviral
vector backbone is the replacement of the CP gene with
reporter genes Such vectors have been developed using
monopartite viruses such as wheat dwarf virus (WDV),
maize streak virus (MSV) and bean yellow dwarf virus
(BeYDV) (Ward et al 1988; Lazarowitz et al 1989;
Topfer et al 1989; Mor et al 2003) and DNA A genomes
of bipartite virus TGMV (Kanevski et al 1992) Various
vectors have used both the native CP promoter and
foreign promoters such as the cauliflower mosaic virus
(CaMV) 35S promoter to express foreign genes or
re-porter genes (Ugaki et al 1991; Mor et al 2003) for
transient expression Stable maize and tobacco cell lines
containing the replicating viral episome for MSV or
TGMV, respectively, have also been reported (Kanevski
et al 1992; Palmer et al 1999) WDV, a monopartite
mastrevirus, replicon-based shuttle vectors with bacterial
replicons, namely ColE1 and p15A, have been used
(Kammann et al 1991; Ugaki et al 1991) for
extrachro-mosomal replication study However, no shuttle vectors
have been developed and evaluated based on the
monopartite begomoviruses
We report here the development and evaluation of a
plant-Escherichia coli shuttle vector using the replicon of
the monopartite begomovirus AYVV and a bacterial
replicon derived from pUC19 to ensure a high copy
number in E coli Two different methods, namely
elec-troporation and biolistic bombardment, were evaluated
for their efficiency to transform the plant cells A novel
method based on the unique DNA methylation-specificity
of the plant and E coli cells was used to study de novo replication of the shuttle vector in the plant cells The successful rescue and maintenance of structural integrity
of the shuttle vector from plant cells into E coli and the expression of the reporter gene was demonstrated in 4-month-old cultures of tobacco BY2 cell suspension cul-tures
Materials and methods
Construction of plasmids Plasmid pASV82, a shuttle vector for Escherichia coli and tobacco cells, was constructed based on the AYVV geminiviral and E coli pUC19 backbone The genealogy of vector construction is detailed
in Fig 1 A 2.7-kb AYVV DNA fragment was released from pHN419 (Tan et al 1995) following BamHI digestion This frag-ment was self-ligated to obtain a circular AYVV DNA template, which was then used to amplify a 2.3-kb fragment using primers extending away from the CP gene, thereby creating its deletion The CP sense primer (50 -AATTCGTACTCATGCCAG-TAATCC-AGTGTATGC-30) and Mlu antisense primer (50 -AATTCATTAC-CACGCGTGACATCACTAACAC-3 0 ) were used with the proof-reading Vent DNA polymerase PCR cycling parameters were 95C for 1 min, 50C for 1 min and 72C for 2 min The number of PCR cycles was kept to a maximum of 20 to further minimize proof-reading errors EcoRI-compatible ends were generated using T4 DNA polymerase and the fragment ligated to the unique EcoRI site of the vector pNKA210.2, a derivative of pIBT210.1 (Haq et al 1995) pNKA210.2 has a pUC19 (2.68 kb) backbone and a plant gene expression cassette consisting of a 35S CaMV promoter,
50UTR of the tobacco etch virus (TEV-50UTR), a translational enhancer, a replaceable stuffer fragment, and a vspB terminator sequence This shuttle vector was named pASV A 1.68-kb neo-mycin phosphotransferase II (NPTII) expression cassette from pNGI (Klien et al 1989) was ligated into the unique HindIII site of pASV to generate pASVNPT To facilitate cloning of the foreign gene in this shuttle vector, we created a unique enzyme site There were two HindIII sites in pASVNPT A single HindIII site was generated in pASVNPT by destroying one of the two HindIII sites
by partial digestion, followed by blunt-ending and self-ligation to yield pASV82 Foreign gene expression cassettes can be inserted in pASV82 (8.2 kb) at its unique HindIII site The constructs were selected in E coli using ampicillin (50 mg/ml) and in plant cells using kanamycin (50 mg/ml) Plasmid pASVGUS, a derivative of pASV82 (8.2 kb) containing the GUS expression cassette, was constructed by inserting the 3-kb HindIII fragment of pRTL2-GUS (Carrington et al 1991) into the HindIII site of pASV82.
A replication-defective control plasmid, pASVDIR, was con-structed by partial digestion of pASVNPT with BamHI, followed
by inverse PCR amplification using primers flanking the IR region with a mixture of KlenTaq (Fermentas, Hanover, Md.) and Pfu polymerase (Promega, Madison, Wis.) The primers IRD (50 -TA-CTCTCCTGATACGATTGGGC-30) and C1 (50 -AATTCCCAAA-GTGCCATTCGG-30) were used for PCR with the following pa-rameters: 25 cycles of 1 min 30 s at 94C, 1 min at 55C, and 8 min
at 72C The PCR product lacking the IR (pASVDIR) was first blunted and then self-ligated to circularize it.
All plasmids were constructed and propagated in E coli strain DH5a and key junctions of the fragments sequenced after each construction Large-scale amplification and purification of plasmids was performed by the alkaline lysis method followed by CsCl ul-tracentrifugation (Sambrook et al 1989) or purified with a Nucle-obond DNA purification AX500 column (Clontech laboratories, Palo Alto, Calif.) and compared.
82
Trang 3Optimization of electroporation conditions
for N benthamiana mesophyll-derived protoplasts
Protoplast isolation, electroporation medium and culture conditions
for stable transformation were as described by Sala et al (1989) To
determine the optimum electroporation conditions for introducing
plasmid DNA into protoplasts, we used two electroporation
de-vices, namely T820, a square wave pulse generator (BTX, San
Diego, Calif.) and a Gene Pulser II exponential wave pulse
gen-erator (Bio-Rad, Hercules, Calif.) The optimization methods were
based on Trypan blue uptake and fluorescein diacetate staining to
determine viability according to Saunders et al (1995) with minor
modifications A suspension of 1106 protoplasts in 600 ml of
HeNa/F buffer (10 mM HEPES, pH 7.1, 5 mM CaCl 2 , 150 mM
NaCl, 0.2 M mannitol) was used for electroporation in 4-mm gap
cuvettes obtained from the manufacturers of the two electropora-tors The electroporation of protoplasts with the BTX T820 was optimized with a single pulse of 80 ms and with varied levels of field strength under low and high voltage modes The Bio-Rad Gene Pulser II was operated with the capacitance set at 1,000 mF, the resistance set at 100–200 W and the time constant at approxi-mately 18–26 ms with varied field strength Trypan blue uptake was observed under bright field microscopy, and the FDA staining was observed under UV fluorescence in a dark field with the in-verted fluorescent Leitz Fluovert FU microscope (Leitz, Wetzlar, Germany) equipped with a UV lamp.
Fig 1 The Ageratum yellow
vein virus (AYVV) genome and
cloning strategy of the
AYYV-derived plant-Escherichia coli
shuttle vector In the AYVV
genome, ORF V1 encodes
coat-protein, V2 encodes movement
protein, C1 encodes
replication-associated protein, C2 regulates
virion-sense gene expression,
C3 regulates DNA replication,
C4 encodes pathogenicity
de-terminant, C5 encodes an
un-known function Exp cas1
Ex-pression cassette 1, IR
inter-genic region The recombinant
plasmids described in the
Re-sults are indicated in boxes The
unique HindIII in pASV82 can
be used to clone expression
cassettes with the gene of
in-terest
83
Trang 4Transformation of N benthamiana mesophyll-derived protoplasts
with plasmid DNA and confirmation using PCR
For transformation using our optimized conditions, we generally
electroporated 50 mg of purified closed circular plasmid DNA into
1106 protoplasts using the BTX electroporator To confirm the
transformation, we extracted the DNA according to Townsend et al.
(1986) at various intervals during protoplast culture Linear PCR
for the NPTII gene and NAD5 gene was carried out for 15 cycles
of 1 min at 96C, 1 min at 55C, and 1 min at 72C with primers
NPTfor (5 0 -GAAGGCGATAGAAGGCGA-3 0 ) and NPTrev (5 0
-GGGTGGAGAGGCTATTCGGC-30) To amplify the NAD5 gene,
we used the PCR primers NAD5for (50
-TAGCCCGACCGTAGT-GATGTTAA-30) and NAD5rev (50
-ATCACCGAACCTGCACT-CAGGAA-30) with the following parameters: 15 cycles of 30 s at
96C, 1 min at 55C, 1 min at 72C.
Transformation of tobacco BY2 by particle bombardment
and PCR detection of transformed lines
Cells of N tabacum L cv BY2 were maintained in MS medium
(Murashige and Skoog 1962) supplemented with 0.18 mg/l
K 2 HPO 4 , 100 mg/l myoinositol, 1 mg/l thiamine HCl, 0.5 mg/l
MES, 30 g/l sucrose in the dark at 120 rpm and 25C A biolistic
particle gun (model PDS-1000/He; Bio-Rad) was used for biolistic
bombardment of the tobacco BY2 cells as described by Kikkert
(1993) Conditions were optimized for various rupture disks (900,
1,100, 1,300 psi), and 1-mm gold particles as microcarriers coated
with pRTL2-GUS vector DNA were used The BY2 cells were
bombarded and assayed for transient GUS expression 2 days after
bombardment Under the optimized conditions the biolistic
bom-bardment was carried out for the tobacco BY2 cells using the
shuttle plasmids, and the transformed cell lines were screened on
selection medium containing 50 mg/ml kanamycin Total DNA was
extracted according to Dellaporta et al (1983) Transformed
to-bacco cells were confirmed by PCR amplification of the AYVV
region with the primers C1 and CP sense PCR cycling parameters
were 25 cycles of 1 min at 96C, 1 min at 57C, and 2 min at 72C.
The same DNA extract used for the AYVV primers was also
used for a tobacco chromosomal gene, SAMDC (S-adenosyl
me-thionine decarboxylase) The SAMDC gene was amplified using
the primers samdcfor (50
-CGGCTGCTCACATGACTGTTAGTT-CTGGC-3 0 ) and samdcrev (5 0
-AACATGCAAGCACCTTCTCAA-CCAG-30) with the following PCR cycling parameters: 25 cycles of
1 min at 95C, 30 s at 50C, 30 s at 72C.
Rescue of the pASVNPT and pASVGUS shuttle vectors
from tobacco BY2 cells in E coli
Total DNA was isolated from tobacco BY2 transformed cells as
described by Dellaporta et al (1983) Five micrograms of total
plant DNA was transformed into E coli strain DH5a to rescue the
shuttle vector Transformed cells were selected on LB plates
con-taining ampicillin (50 mg/ml) Plasmid DNA was isolated for
fur-ther analysis using the alkaline lysis method.
Analysis of replicating DNA by coupled restriction enzyme
digestion-random amplification PCR (CREDRA-PCR)
and Southern blot analysis
CREDRA-PCR has been used previously to identify DNA
meth-ylation in plants (Cai et al 1996; Prakash and Kumar 1997) We
modified CREDRA-PCR to study plasmid replication in plant cells
as follows Total DNA (5 mg) from DH5a, DNA from the tobacco
transformed cell line, and rescued DNA from DH5a were restricted
with m6A methylation-sensitive (DpnI) and methylation-resistant
(BclI) enzymes for 5 h in a 20-ml reaction volume The digested
DNA was re-purified and amplified using AYVV primers (C1 and
CP sense) under the conditions described earlier Five-microliter
aliquots of the PCR products were fractionated by agarose gel electrophoresis and analyzed by Southern blot hybridization ac-cording to manufacturer’s protocol (Boehringer Mannheim, Ger-many) The 1.7-kb AYVV gene probe was generated by PCR amplification and was labeled with digoxygenin-labeled dUTP by random priming.
Histological GUS assays Putative transformed cell lines and control BY2 cells were tested for histochemical localization of GUS (Gallagher 1992) The cells were incubated overnight at 37C in an assay buffer consisting of
100 mM NaPO 4 (pH 7.0), 1 mM X-Gluc (5-bromo-4-chloro-3-in-dolyl glucuronide cyclohexylammonium salt), examined for fluo-rescence, and photographed under a bright field using an inverted Lietz Fluovert FU microscope (Leitz, Wetzlar, Germany).
Results and discussion
Construction of pASV plant-E coli shuttle vectors
We chose a monopartite AYVV clone from pHN419 to construct a pASV series of plant-E coli shuttle vectors pASVNPT, pASV82, pASVGUS and pASVDIR (Fig 1) The vector pASV has all of the essential elements of AYVV except the CP (V1) to allow AYVV viral replicon for extrachromosomal replication in plant cells The pUC19 backbone enables the plasmids to replicate in E coli under ampicillin selection for easy manipulation and recovery of the clones from plants in E coli The NPTII expression cassette [consisting of the CaMV 35S pro-moter and nopaline synthase (NOS) gene terminator] in pASVNPT allows kanamycin selection in plant cells The pASVDIR plasmid, with the deletion of the intergenic region in pASVNPT, serves as a replication-defective control An intermediate cloning vector, pASV82, was derived from pASVNPT and contains a unique HindIII site for subsequent cloning of the expression cassette of genes of interest The GUS expression cassette was cloned at this HindIII site to yield pASVGUS for its ex-pression in plant cells The exex-pression cassette has a dual CaMV 35S promoter, 50UTR translation enhancer of TEV and a CaMV 35S terminator An alternate expression cassette1 with the CaMV 35S promoter, 50UTR of TEV, a stuffer fragment, and vspB terminator is also available in this vector This stuffer fragment can be replaced with the gene of interest between NcoI and KpnI sites However, the unique HindIII site is recommended for the insertion
of foreign gene expression cassette since NcoI and KpnI are not unique sites in the vector
Optimization of electroporation condition
Our objective was to study the replication of pASV-de-rived vectors in N benthamiana and N tabacum proto-plasts We first standardized the electroporation condi-tions for introducing the vectors to the isolated mesophyll protoplasts of N benthamiana using two electroporators
A comparison of the effect of the two different pulse 84
Trang 5types revealed a general trend Freshly isolated
proto-plasts had 50–60% viability, and in both types of pulse
generators the viability dropped below 10% with higher
pulse strength (Fig 2) The number of Trypan
blue-stained protoplasts increased and viability rapidly
de-creased with increasing field strength with both
electro-porators To maintain a protoplast viability of 50%, we
determined that the optimum field strength should be 0.3 kV/cm for the exponential wave pulse generator and 0.5–0.6 kV/cm for the square wave pulse generator The protoplasts showed decreased viability under a low volt-age mode with BTX T820 (data not shown) In subse-quent experiments we used the BTX electroporator with a pulse field strength of 0.55 kV/cm and a single pulse of
80 ms
Transformation of mesophyll-derived protoplasts
of N benthamiana with pASVNPT
Using the optimized electroporation conditions with high-quality (circular form) input DNA (derived from Nucle-obond column-purified kit), we electroporated mesophyll protoplasts with pASVNPT and followed the replication
of vector DNA over a 6-day period Microscopic obser-vation showed no active protoplast division during this period The NPTII PCR products of pASVNPT were de-tectable throughout the period tested, but their levels declined with time in the cells (Fig 3b) As expected, there was no increase in the level of PCR products for the chromosomal gene NAD5 (Fig 3c) The steady-state levels of the pASVNPT vector decreased slightly during the 6-day period; this could be due to either decreased replication or to the same level of replication ability but
an increased turnover of vector DNA In practical terms, either situation would lead to a lower copy number of vector molecules These results show that the pASVNPT vector did not replicate efficiently in the N benthamiana cells Previous reports of transient replication experiments with protoplasts derived from mesophyll cells have shown that host cell division is a prerequisite for the replication
of some types of begomovirus but not for all For ex-ample, replication of the ACMV requires host cell divi-sion (Townsend et al.1986), while TGMV does not (Brough et al 1992) In other geminiviruses also, such dependency on host cell division varies In the Mastre-virus group, WDV replication is dependent on cell
divi-Fig 2 Electroporation efficiency of mesophyll protoplasts
Try-pan-blue uptake and viability as determined by fluorescein
diace-tate staining of mesophyll protoplasts electroporated using: a BTX
model T820 (square wave pulse generator), b Bio-Rad Gene
pulser II (exponential pulse generator) Arrows indicate the
opti-mum conditions used in subsequent experiments
Fig 3a–c PCR detection of
pASVNPT DNA in Nicotiana
benthamiana protoplasts a
pASVNPT construct IR
Inter-genic region b Amplification
products of the vector-borne
NPTII gene L Low-mass DNA
ladder c Amplification
prod-ucts of the chromosomal NAD5
gene L 1-kb ladder
(Fermen-tas) Lanes 1–6 PCR for days 1–
6 in duplicate, C negative
con-trol
85
Trang 6sion of the host Triticum monococcum (Matzeit et al.
1991), while in another study with maize it was shown to
proceed in the absence of cell division (Timmermans et
al 1992) Hence, the dependency of geminiviral
replica-tion on host cell division may be affected by both viral
and host factors
Because there was a lack of replication of pASVNPT
in the N benthamiana system in our studies, we conclude
that AYVV replication is dependent on host cell division
and that a slowly or non-dividing host system such as N
benthamiana would not be suitable for pASV-derived
vectors Consequently, we selected the related N tabacum
BY2 cells, which are known for sustaining rapid division
in long-term cultures, for further testing
Transformation of pASV-derived vectors
in N tabacum BY2 cells by particle bombardment
In order to efficiently transform tobacco BY2 cells we
optimized the particle bombardment conditions using
pRTL2-GUS In transient GUS expression studies with
pRTL2-GUS, the highest number of uniformly
blue-col-ored cells (4,423€225 cells) with 1,300 psi were observed
2 days post-bombardment This is in contrast to the low
number of stained cells with 900 and 1,100 psi (271€11,
1,019€45 cells), respectively, and no stained cells
ob-served in the control mock bombarded cells Subsequent
experiments were conducted under these optimized
con-ditions
With the objective to study the replication of
pASV-derived vectors in tobacco BY2 cells, the pASVNPT,
pASVGUS and a replication-defective pASVDIR DNA
were bombarded into BY2 tobacco cells for clonal
se-lection of transformed lines on antibiotic-containing
me-dium Two hundred putative transformed independent
lines from each vector were further cultured on selection
plates for an extended period of 4 months The calluses
transformed with pASVNPT and pASVGUS appeared as
tiny white clumps between 20 days and 30 days
post-bombardment, thereby allowing clonal selection of transformed cell lines Both non-transformed tobacco calluses and cells transformed with pASVDIR showed no callus proliferation on selection plates (Fig 4e,f), while the growth of control transformed calluses on non-selection plates was normal The absence of colonies with the negative control pASVDIR-transformed cells proved that the intergenic region is necessary for viral replication and that the selection process of transformed cells was efficient This observation is in accordance with deletion analyses of other geminiviruses such as WDV (Ugaki et
al 1991) and MSV (Shen and Hohn 1994), as this region contains the initiation site for rolling circle DNA repli-cation The transformed calluses maintained for 4 months kept their ability to stably replicate the pASV vectors In other analyses cell suspension cultures of TGMV and MSV transformed tobacco and maize lines were main-tained for 6 months and 1 year, respectively (Kanevski et
al 1992; Palmer et al 1999) Because calluses require less frequent transfers than cell suspensions, the methods described here would allow easier maintenance in pro-longed periods
Replication studies of pASV-derived shuttle vectors
As the maintenance of long-term cultures provides only indirect evidence for the stable replication of vectors, replication was studied using four methods to obtain in-dependent validation: (1) detecting the presence of the vector DNA by PCR in long-term cultures of transformed calluses, (2) rescuing pASVGUS from plant cells into E coli, (3) CREDRA-PCR assays based on methylation differences of DNA replicated in plant and E coli cells, and (4) assaying for foreign reporter genes in transformed tobacco BY2 cells
Fig 4a–f Selection of
trans-genic tobacco BY2 calluses
30 days following
bombard-ment with pASV vectors a
pASVNPT-transformed calluses
in selection medium b
pASV-GUS-transformed calluses in
selection medium c, d Control
tobacco calluses in the absence
of kanamycin e Control
tobac-co calluses in selection medium.
f pASVNPTDIR-transformed
replication-defective control on
selection medium
86
Trang 7PCR detection of pASVNPT and pASVGUS vectors
in transformed calluses
Randomly picked healthy calluses at the 6-week stage
growing on selection plates were screened for the
pres-ence of the vector A 1.7-kb AYVV DNA fragment from
the pASVNPT and pASVGUS constructs was amplified
from the DNA of the transformed calluses A 0.3-kb
SAMDC DNA fragment from the chromosomal gene
was amplified as a control (Fig 5b) Twenty transformed
calluses that showed the presence of pASVGUS DNA
were transferred to MS medium with kanamycin for
vector selection in suspension culture
Shuttling ability and rescue of pASVGUS
from plant cells to E coli
Suspensions of transformed cell lines, as described above,
were used for studying shuttle replication of the
con-structs in plant and E coli cells The suspension cell
cultures were maintained by subculturing in selection
medium once a week The morphology and growth rates
were similar in all cell suspension cultures up to 2–
3 months in comparison to the control BY2 cells without
kanamycin The control BY2 cells on kanamycin did not
multiply in suspension cells Subculturing was continued
for a period of 4 months
We hypothesized that presence of the 1.7-kb AYVV
PCR fragment would not directly confirm the shuttling
ability of the vector Hence, we attempted to rescue the
vector from plant cells into E coli at monthly intervals
This also allowed us to study any major rearrangements in
the vector as a consequence of replication in both the
plant and E coli cells The rescue of pASVGUS from
plant cells into E coli was carried out on 1-, 2- and
3-month-old suspension cultures maintained on selection
medium Between 10 and 20 E coli colonies were
ob-tained with 5 mg of total DNA derived from cells of the
tobacco suspension culture, while DNA prepared from the untransformed control BY2 cells did not yield any E coli transformants, as was expected This further confirmed that pASVGUS DNA was propagated along with the chromosomal DNA in transformed plant cells The structural integrity of the AYVV vector was studied by PCR and the sizing of the constructs was determined by restriction profiling Plasmid DNA was prepared from the rescued colonies and screened for the presence of the AYVV and GUS regions, respectively (Fig 6a,b) All of the rescued clones that were screened showed the 1.7-kb AYVV fragment and the 0.7-kb GUS gene fragment upon PCR-based amplification Uncut plasmids were compared
to detect any size differences, but no major differences in the sizes of the plasmids were found Additional restric-tion analyses of the rescued clones were performed to further investigate the possibilities of any vector DNA rearrangements: no significant restriction fragment length polymorphisms were seen between the rescued and con-trol vector DNA (Fig 6c) Similar results were obtained from 1-, 2-, and 3-month-old cultures Taken together, these results confirm that the pASV backbone vector can replicate in both plant and E coli without undergoing any detectable size alterations or rearrangements Previous studies on the rescue of geminiviral shuttle vectors were carried out on 6-day- and 7-day-old cultures with the graminaceous host containing mastrevirus, WDV (Ugaki
et al 1991; Kammann et al 1991) We report here long-term maintenance of the structural integrity of bego-movirus-based pASV vectors
Although, these results show no major rearrangements, minor ones would not have been detected using the methods described here Also, the rearranged vectors could not have been rescued if the rearrangement had affected their ability to replicate in E coli The versatility
of the vectors to replicate in other host plants needs to be tested with respect to broader applications
Fig 5a, b PCR detection of pASVGUS in tobacco BY2
trans-formed calluses a pASVGUS construct b Lanes: L 1-kb ladder
(Fermentas), 1 negative control lacking total DNA from calluses, 2
PCR products of AYVV gene from pASVNPT, 3 pASVGUS-transformed callus DNA, 4, 5 PCR products of SAMDC gene— internal positive control for chromosomal DNA from calluses
87
Trang 8Replication studies based on DNA methylation differences
We reasoned that direct evidence for vector replication in
plant cells would require either distinguishing the
replicative intermediates of the vector or showing the
presence of DNA replication features specific to plants
We chose the latter line of proof and based our
experi-ments on the m5C methylation that occurs during the
replication of DNA in plants but which is absent in E
coli The converse argument that m6A methylation is
found in E coli but is absent in eukaryotic DNA was also
used in these studies Other researchers have proposed
that the extrachromosomal replicons may not be
acces-sible for the host methylases, although the barrier pre-venting access of the methylases was not defined (Brough
et al 1992; Doerfler 1993) Based on these considera-tions, we studied the differences in the methylation status
of the input DNA obtained from E coli and that of the DNA obtained from newly transformed plant cell lines using a combination of methylation-sensitive restriction enzymes followed by PCR
To study the replication of extrachromosomal DNA
of the pASVGUS shuttle vector we used a modified methylation-based PCR method termed CREDRA-PCR (Fig 7a) This method was carried out on total ge-nomic DNA isolated on the third day of subculture from
Fig 6a–c Rescue of pASVGUS shuttle vectors from transformed
BY2 plant cells in E coli PCR detection of pASVGUS plasmid
DNA with: a AYVV primers, b GUS primers Lanes: L 1-kb ladder
(Fermentas), C negative control with non-transformed tobacco cell,
2 PCR with control pASVGUS plasmid DNA, 3–8 PCR with
res-cued pASVGUS clones (RC 3–8) from E coli clones c Restriction
profiles of pASVGUS rescued clones RC 3 (3*) and RC 4 (4*) from panels a and b Lanes: 1, 4, 7 Non-digested DNA controls, 2,
5, 8 BamHI-digested, 3, 6, 9 HindIII-digested The experiment was repeated with 1-, 2-, and 3-month-old suspension cultures Results from the 2-month-old suspension cultures are shown in this figure
Fig 7a–c Verification of
pASVGUS shuttle vector
repli-cation in tobacco cells using
CREDRA-PCR a Experimental
setup for studying
extrachro-mosomal replication b
CRE-DRA-PCR products using
AYVV primers for pASVGUS
detection Lanes: 1–3 Control
input pASVGUS m6A
methyl-ated DNA, 4–6 total genomic
DNA from
pASVGUS-trans-formed tobacco cells, 7–9
res-cued plasmid DNA in DH5a (U
un-digested, BclI BclI-digested,
DpnI DpnI-digested) c
South-ern blot hybridization of the
CREDRA-PCR products from
the gel shown in panel b with
DIG-labeled 1.7-kb AYVV
fragment
88
Trang 92-month-old cell suspension cultures To discriminate
between the input and the newly replicated DNA, both the
DNA from suspension tobacco cells and the DNA rescued
from E coli were digested with either
dam-methylation-dependent restriction enzyme DpnI or BclI followed by
PCR amplification and Southern analyses DpnI is
de-pendent on the methylation of adenine in the recognition
sequence GA6/TC and, consequently, it cleaved the both
the input and rescued DNA that was methylated in E
coli BclI did not cleave the m6A methylated sequence
(Fig 7b,c) In contrast, the replicative DNA in tobacco
cells was resistant to digestion with DpnI since they
lacked the m6A site, consequently giving a PCR product
with the AYVV primers BclI did cleave the de novo
synthesized DNA that lacked m6A methylation in the
tobacco cells Hence, vector DNA was not amplified and
there was no corresponding signal in either the agarose
gel or its Southern blot probed with vector DNA (Fig 7c)
that showed a higher sensitivity of detection Based on
these observations, we conclude that de novo replication
of the pASVGUS vector occurred in plant cells in
2-month-old suspension cells The mastrevirus replicating
shuttle vector in protoplasts of Triticum monococcum
(Kammann et al 1991), maize endosperm-derived
pro-toplasts for WDV (Timmermans et al 1992), and tobacco
NT1 cells for BeYDV (Mor et al 2003) have also been
analyzed by methylation sensitivity In these reports,
methylation-based studies of replication were carried out
during the first week following electroporation In order
to study de novo replication in long-term plant cell
cul-tures, we adopted a methylation-based PCR assay—the
CREDRA-PCR method—which is more sensitive,
re-quires little starting material, and is simpler to perform
than the previous methods
Expression of foreign reporter gene
Once the replication ability of pASVGUS was
estab-lished, we further tested its application as an
extrachro-mosomal expression vector using the GUS reporter gene
Transgenic cell lines and control BY2 cells were tested
for histochemical localization of GUS over an extended
period of time spanning 4 months GUS assays were
performed daily for 7 days beginning with the first day of
subculturing on 2-week- and 1-, 2-, 3,- and 4-month-old
suspension cultures In all cases GUS expression was
detected in 0.001–0.006% of the cells screened GUS
expression was maximal on third day, and expression was
not found when the cells had reached the seventh day, a
time when the tobacco BY2 cells are likely to be in the
stationary stage (Nagata et al.1992) The reason for
maximal expression on third day is not yet clear The
highest percentage of GUS-expressing cells occurred in
the first month, with the percentage declining gradually
later in the fourth month However, we speculate that this
could be due to changes in the copy number of the vector
during the different growth phases of the culture This
variation was also noticed with TGMV (Kanevski et al
1992) and BeYDV in tobacco cells (Mor et al 2003) The difference in copy number could arise from interference
of geminiviral replication with plant cell-cycle machinery
or other host cell pathways (Gutierrez 2000)
Conclusion
We have shown here the utility of pASV-derived shuttle vectors for long-term stable maintenance of constructs and expression of foreign genes in cultured plant cells Using methylation-based PCR assays we have also shown
de novo replication of pASV vectors in long-term cul-tures On the basis of the system we have described here pASV vectors will be suitable for use when researchers are looking to express foreign proteins in a closed sterile system rather than in whole transgenic plants They will
be useful in long-term cell cultures by improving the replication and expression levels through extensively characterizing the host factors in synchronized cell cul-tures and promoters highly active in early cell division of BY2 cells
Acknowledgements The authors would like to thank Dr Wong Sek Man for providing pHN419 containing the full-length AYVV coding sequence, Prof Charles Arntzen and Dr Hugh S Mason for their gift of the pIBT210.1 plasmid, and Dr Jaideep Mathur for providing the tobacco BY2 suspension cells TS and GA have been supported by NUS research scholarships.
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