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Journal of Virology, September 1999, p. 7823-7829, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid Delivery of Foreign Genes into Plants by
Direct Rub-Inoculation with Intact Plasmid DNA of a Tomato Bushy
Stunt Virus Gene Vector
Herman B.
Scholthof*
Department of Plant Pathology and
Microbiology, Texas A&M University, College Station, Texas 77843
Received 8 March 1999/Accepted 17 May 1999
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ABSTRACT |
Tomato bushy stunt virus (TBSV) cDNA, positioned between a modified
cauliflower mosaic virus 35S promoter and the hepatitis delta virus
antigenomic ribozyme with a downstream nopaline synthase gene
polyadenylation signal, established infections upon rub-inoculation of
plants with intact plasmids. Application of this methodology produced a
TBSV DNA-based gene vector which yielded readily detectable levels of
localized foreign gene expression in inoculated leaves. This is the
first demonstration of an infectious DNA from a member of the
Tombusviridae which permits rapid TBSV-mediated
foreign-gene expression upon direct rub-inoculation of miniprep DNA
onto a variety of plant species.
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TEXT |
In the past decade, molecular
genetic studies of plant RNA viruses have benefited tremendously from
the methodology introduced by Ahlquist et al. (1), which
permits the generation of infectious RNA upon in vitro transcription of
full-length cDNA clones. This technique has since been applied to many
different RNA plant viruses (4), including tomato bushy
stunt virus (TBSV) (12). Infectious cDNA clones have
expedited studies expanding our knowledge of molecular virus genetics
and have stimulated the use of RNA plant viruses as foreign gene
vectors for research and commercial purposes (13, 14, 26).
Gene delivery systems based on TBSV have several advantages
(26), which include the broad experimental host range of the
virus and rapidly occurring, high levels of replication and gene
expression. These features have contributed to the successful use of
TBSV gene vectors to quickly assess the biological activity of
heterologous foreign proteins and/or RNA in protoplasts or inoculated
leaves (3, 25, 26, 32).
While the reactions for in vitro transcription are generally
straightforward, the costs associated with these procedures may be
considerable if large numbers of constructs are tested, for example,
when virus vectors are used to screen cDNA expression libraries in
plants (15). The objective of this study was to optimize the
usefulness of the TBSV vector system by alleviating the necessity of in
vitro linearization or transcription of cDNA and to allow direct
delivery without the need for agroinoculation or biolistics. For this
purpose, plant and animal virus transcription and processing signals
were tested to optimize the infectivity of intact, untreated plasmid
DNA on several plant species.
Constructs were generated with full-length TBSV cDNA positioned
downstream of a cauliflower mosaic virus (CaMV) 35S promoter for the
initiation of transcription at the authentic 5' end of the viral RNA in
the plant nucleus (8). The nopaline synthase poly(A) signal
[nos-poly(A)] (28) was used to permit the in vivo
termination of transcription through polyadenylation, and the hepatitis
delta virus antigenomic ribozyme (HDVagrz) (21) was
incorporated to generate 3' termini resembling those of the native TBSV
RNA. These modifications permitted the simple rub-inoculation of
untreated plasmid DNA onto leaves to infect different plants. Application of this technology to a TBSV construct in which a multiple-cloning region (MCR) replaces the coat protein (CP) gene permitted the convenient introduction of foreign genes which were rapidly expressed to yield detectable levels of proteins in the inoculated leaves of various plants.
Infectivity of TBSV DNA.
To obtain clones with the 5' end of
TBSV cDNA inserted immediately downstream of the CaMV 35S promoter
sequence, a PCR product was obtained with a 5' primer identical to the
5' terminus of TBSV (12) and a 3' primer covering the TBSV
StuI site (Fig. 1A)
(29). Standard molecular biology protocols were used, as described by Sambrook et al. (23) or as provided by the
suppliers of the reagents. The PCR product was cleaved at the internal
AvrII site (Fig. 1A), which yielded a fragment with a blunt
5' end and a 3' AvrII terminus, which was ligated into the
pUC-35S (8) vector between the StuI and
XbaI (compatible with AvrII) sites in the MCR to
yield pHST7. Subsequently, a TBSV cDNA segment from the
BssHII site to the BamHI site (Fig. 1) was
inserted between the BssHII and BamHI sites of
pHST7. Lastly, this intermediate was digested with BamHI and
SmaI, to introduce the BamHI to SmaI fragment of pHST2-14 in which an MCR replaces the CP gene
(26). The resulting plasmid, pHST8, was digested with
NotI (present inside the MCR) and HpaI (Fig. 1),
treated with DNA polymerase Klenow fragment to create blunt ends, and
religated. This created pHST9, in which the internal SacI
and EcoRI sites were removed to permit the following cloning
steps. The nos-poly(A) element of p3'NT (28) was released
with SacI and EcoRI, and the resulting ca. 270-bp
nos-poly(A) fragment was inserted between the SacI and
EcoRI sites at the 3' end of the TBSV cDNA insert of pHST9 to give pHST10. Subsequently, the ca. 90-bp HDVagrz cDNA fragment was
removed with SmaI and SacI from plasmid 2.0 (a
generous gift from A. Ball) and inserted into pHST10, which was opened
with the same restriction enzymes, to generate pHST11. To obtain a cloning intermediate without the nos-poly(A) signal, pHST11 was digested with SacI and EcoRI, treated with Klenow
fragment, and religated to generate pHST15. Sequence analyses were
performed at all intermediate steps to ensure the proper positioning
and sequence composition of the cloned fragments.
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FIG. 1.
Plasmids and regulatory sequences. (A) Infectious cDNA
of TBSV (12). The open reading frames are indicated by
boxes, and encoded proteins (numbers show molecular masses, in
kilodaltons) are provided with their function; thick lines represent
untranslated sequences. Selected restriction enzyme sites that are
relevant for this study are shown. (B) The cDNA cloning vector (pHST40)
for in vivo transcription. Two alternative transcriptional start sites
are indicated, and the HDVagrz RNA cleavage site is shown at the
SmaI site on the DNA. Any cDNA can be cloned between the
StuI and SmaI sites, which will facilitate in
vivo transcription of RNAs with authentic 5' and 3' termini. Thin lines
represent pUC18 sequences. (C) The plasmids pHST17 through pHST20 show
the DNA-based TBSV constructs with the CaMV 35S promoter and the
combinations of nos-poly(A) and/or HDVagrz at the 3' end. The asterisks
denote the positions of primers utilized to amplify the PCR products
used in the assays described for Fig. 3.
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The
EcoRI-to-
SacI fragment from pHST11,
comprising the HDVagrz and nos-poly(A), was transferred into pUC-35S to
yield pHST40.
This potential universal cloning vector has been
constructed to
permit the insertion of plant viral cDNAs between the
StuI and
SmaI sites (Fig.
1B). To obtain pHST17
through pHST20 (Fig.
1C),
the
BamHI-to-
SalI
fragment from the kanamycin-resistant plasmid
pHS24, which harbors a
TBSV cDNA fragment with the intact CP gene
(
27), was used to
replace the
BamHI-to-
SalI fragments from pHST9,
pHST10, pHST15, and pHST11,
respectively.
As reported for cucumoviruses (
8,
9,
30) and cowpea mosaic
virus (
8), the infectivity of DNA-based systems may
benefit
strongly from the linearization of plasmids. To test this
possibility
for TBSV, pHST17 (Fig.
1C) was inoculated onto plants
either as
untreated supercoiled DNA or after linearization at
the 3' end of the
TBSV cDNA insert. The results in Fig.
2A show
that
infectivity was substantially improved by linearization prior
to
inoculation. Because the treatment of DNA with a restriction
enzyme
affected infectivity, this experiment provided indirect
evidence that
the infections were initiated by DNA rather than
contaminant RNA. This
was further confirmed by the observation
that incubation of plasmid DNA
with DNase I destroyed infectivity,
whereas RNase A had no effect (Fig.
2B). The enzymatic treatments
were followed by phenol-chloroform
extractions, ethanol precipitation,
and resuspension in water or
Tris-EDTA buffer prior to inoculation.
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FIG. 2.
Infectivity assays of plasmids. (A) C. quinoa leaves 5 days after inoculation with pHST17 DNA that was
SmaI linearized (left) or supercoiled (right). (B) C. quinoa leaves 6 days after inoculation with pHST20 DNA, treated
with RNase A (left) or DNase I (right). (C) C. quinoa leaves
9 days after inoculation with supercoiled DNA of pHST17 (top left),
pHST18 (top right), pHST19 (bottom left), or pHST20 (bottom right).
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To examine if 3' processing signals enhanced infectivity and thereby
eliminated the necessity for in vitro linearization,
pHST18, pHST19,
and pHST20 DNA were rub-inoculated onto
Chenopodium quinoa
(Fig.
2C). Approximately 10 µg of CsCl gradient-purified
supercoiled
plasmid DNA was mixed with RNA inoculation buffer
(
25) to a
total volume of 50 to 100 µl for the inoculation of
two to four
leaves per plant. Irrespective of the construct, local
lesions became
visible ca. 3 to 4 days postinoculation. Although
the total number of
local lesions varied between inoculation experiments,
the trend shown
in Fig.
2C was consistent. The results of four
experiments in which the
number of lesions per leaf was tabulated
yielded a range of 1 to 10 lesions for pHST17 and pHST19, 10 to
15 lesions for pHST18, and 30 to
40 lesions for pHST20. The same
trend was supported by comparative
tests on cowpea (
Vigna unguiculata),
another sensitive
local-lesion host. Combined, these results illustrated
that the
combination of the HDVagrz and nos-poly(A) signals provided
the highest
level of
infectivity.
Hosts which are very susceptible to inoculations with in
vitro-generated transcripts, e.g.,
Nicotiana benthamiana,
Nicotiana clevelandii,
C. quinoa, cowpea, and
Spinacia oleracea (spinach),
were also susceptible to direct
DNA inoculation. Five days prior
to DNA inoculation,
N. benthamiana and
N. clevelandii plants were
transferred
from the greenhouse or growth chambers to the laboratory,
where
presumably the low light conditioned the leaves, resulting
in improved
inoculation efficiency (unpublished results). Compared
to inoculations
with transcripts, inoculation with pHST20 DNA
would result in a 1- to
2-day delay in the appearance of symptoms.
The substitution of virus
inoculation buffer (1% Celite, 50 mM
KH
2PO
4
[pH 7.0]) for RNA inoculation buffer (pH 9.3) had no obvious
effect
on
infectivity.
The CaMV 35S promoter has been used by itself and in combination with a
3' poly(A) signal to generate infectious cDNA plasmids
of several plant
virus RNAs (
5,
6,
8-10,
17-20,
30,
31,
35). The results in
this study demonstrate that the modified
CaMV 35S promoter
(
8) is very effective in promoting the transcription
of
infectious TBSV RNA in vivo. Furthermore, the polyadenylation
of TBSV
RNA from pHST18, which is predicted to add ca. 300 extra
bases to the
3' end of the viral RNA, does not abolish infectivity
(Fig.
1 and
2).
This is in agreement with observations by Dalmay
et al. (
7),
who also observed that cymbidium ringspot tombusvirus
containing extra
sequences at the 3' end of the RNA maintained
the ability to replicate.
This phenomenon may also explain the
infectivity of intact circular
pHST17, which lacks any 3' processing
signals (Fig.
1 and
2).
Ribozyme activity.
The bioassays showed that the presence of
the HDVagrz at the 3' end of TBSV cDNA, upstream of the nos-poly(A)
signal, improved infectivity. To confirm the activity of the ribozyme
within the TBSV and nos-poly(A) context, transcripts were generated
directly from PCR products containing the 3' proximal 950 bp of the
TBSV cDNA and the nos-poly(A) sequence either alone or in combination with the HDVagrz. PCR fragments were amplified with TaqI
polymerase (Promega, Madison, Wis.) or Vent DNA polymerase (New England
Biolabs, Beverly, Mass.), with either pHST18 or pHST20 as a template
(Fig. 1C). For this purpose, a 5' primer was used which contained the T7 promoter sequence attached to the transcriptional start site sequence of subgenomic RNA2 (sgRNA2) on the cDNA (Fig. 1C). The 3'
universal reverse-sequencing primer annealed immediately downstream of
the nos-poly(A) signal (Fig. 1C).
Ribozyme activity was routinely analyzed with ca. 0.2 µg of in
vitro-generated transcripts in reaction buffer with a final
concentration of 1 mM MgCl
2, 5 M urea (optional), and 1.2%
sodium
dodecyl sulfate to prevent RNase activity. The mixture was
incubated
for 2 h at room temperature, followed by 30 min of
incubation
on ice and centrifugation at 9,000 ×
g for ca. 1 min. The entire
sample was electrophoresed through a 2% agarose gel in
Tris-borate-EDTA
buffer (
23). After electrophoresis, the
gels were incubated
in water to remove excess urea, followed by
standard Northern
blotting and hybridization
procedures.
The results in Fig.
3 demonstrate that no
specific cleavage product was obtained when the ca. 1,280-nucleotide
(nt) transcript
was derived from pHST18 which lacks the HDVagrz.
However, the
ca. 1,370-nt transcript from pHST20, containing the
HDVagrz, was
processed into two RNA products of ca. 430 and 940 nt. As
predicted,
the larger cleavage product was the same size as the
transcripts
that were obtained when cDNA templates were digested with
SmaI,
which cleaves DNA at the position where the ribozyme
cleaves RNA
(Fig.
1 and data not shown). The larger RNA product
hybridized
with TBSV but not with the nos-poly(A) segment (Fig.
3B),
whereas
the smaller RNA fragment hybridized only with the nos-poly(A)
DNA (Fig.
3C), which is in agreement with the anticipated position
of
the ribozyme cleavage site.
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FIG. 3.
In vitro HDVagrz activity. (A) Two percent agarose gel
with RNA transcribed from ca. 1,300- and 1,400-bp PCR products obtained
from the 3' proximal fragments of pHST18 (lane 18) or pHST20
(lane 20), respectively (see Fig. 1 for the locations of
primer annealing sites). The upper band represents uncleaved RNA, and
the lower bands in the lane with RNA from pHST20 (lane 20)
represent the HDVagrz cleavage products. The positions of
double-stranded DNA size markers are indicated in basepairs. (B) RNA
hybridization assay with pHS49 (24) as a specific probe for
detection of 3' proximal TBSV RNA sequences. (C) RNA hybridization of a
similar Northern blot with p3'NT (28) as a probe for
detection of nos-poly(A) sequences.
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Since the proper parameters for HDVagrz activity are not predictable
(
2), it was determined if ribozyme activity was maintained
under varying conditions. For this purpose, RNA was incubated
by using
the following combinations: room temperature or 55°C;
0, 1, or 10 mM
MgCl
2; and 0 or 5 M urea. Although complete cleavage
was
not observed under any of these conditions, compared to the
results
shown in Fig.
3, ca. threefold less ribozyme activity
was obtained upon
incubation of ca. 0.2 µg of RNA and 1 mM MgCl
2 for 2 h at room temperature. However, in vitro ribozyme activity
was again
improved twofold by denaturation of the RNA at 55°C
with or without 1 mM MgCl
2. Regardless of the slight quantitative
influences,
the expected ratio and sizes of cleavage products
were readily obtained
under a variety of incubation conditions.
These results strongly
suggest that the improved infectivity on
plants upon inclusion of the
HDVagrz in pHST20 results from ribozyme
activity.
A positive influence of ribozymes on the infectivity of in
vivo-transcribed RNAs was previously demonstrated for tobacco mosaic
virus (TMV) (
6,
34). Compared with these reports, an
ancillary
finding with the TBSV DNA system is that the bioassays on
local-lesion
hosts revealed that the highest levels of infectivity were
obtained
with plasmid DNA containing the HDVagrz in addition to a
poly(A)
signal. Omission of the nos-poly(A) signal resulted in a
reduced
number of lesions, suggesting that the termination of
transcription
by polyadenylation either increases the efficiency of the
HDVagrz
activity or properly positions the transcript for nuclear
export.
Comparison of TBSV DNA inoculations with those of other DNA-based
plant RNA viruses.
CaMV 35S promoter-mediated in vivo-transcribed
cDNAs of TMV (35) and brome mosaic virus (19)
were infectious only on Chenopodium species, and TMV DNA was
not infectious on its natural host, Nicotiana tabacum
(31). Although cucumber mosaic virus cDNA was infectious on
a variety of plant species, for high levels of infectivity the DNA
cassette needed to be released with restriction enzymes prior to
inoculation (9, 30). Subsequent studies have shown that the
infectivity of cDNA plasmids of two potyviruses (10, 11),
two luteoviruses (16, 22), or TMV on N. tabacum
(6) requires either biolistic delivery or agroinoculation.
Within this context, the novel or attractive features of the new TBSV DNA system include the following: (i) linearization of plasmid DNA is
not required; (ii) infectivity is obtained upon simple rub-inoculation;
(iii) DNA-mediated infections are established on a wide variety of
plants; and (iv) although inoculations with TBSV DNA were initially
performed with CsCl-purified DNA, inoculation of less-pure miniprep DNA
also yields very efficient infections, which provides a practical level
of convenience.
The reasons for the ability of the DNA-based TBSV constructs to
efficiently establish infections after simple rub-inoculation
of many
plant species are unknown. This capacity is probably not
due to a more
infectious nature of the DNA, since the ca. 10 µg
of TBSV DNA used
for our inoculations (final concentration of
50 to 125 ng/µl) is
within the range reported for other systems
(
6,
30).
However, it is conceivable that the combination
of the high infectivity
of TBSV transcripts and the nonselective
invasion of different tissues
contributes to the effectiveness
of the DNA
constructs.
Rub-inoculation of TBSV vector DNA for expression of foreign
genes.
Previously, it was shown that TBSV-mediated
-glucuronidase (GUS) expression could be readily detected in
inoculated leaves following inoculation with in vitro-generated
transcripts (25, 27). In those experiments, the GUS gene was
fused to the 5' end of the CP gene, which resulted in translational
initiation from the authentic CP start codon and the production of an
enzymatically active fusion protein. Plasmid pHST12 (Fig.
4A) was designed to allow the transcription of an sgRNA1 with an elongated leader sequence,
on which translational initiation occurs from the start codon of the
introduced foreign gene. To generate the pHST12 gene vector (Fig. 4A),
pHST11 was digested with SnaBI and SalI (Fig. 1),
and the released fragment was replaced with the compatible fragment of
pHST2-14 (26). To obtain pHST34 (Fig. 4A), a DNA-based vector with an intact p19 gene, the BamHI-to-NcoI
fragment of pHST20 was replaced with that of pHST2-14 (26).
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FIG. 4.
Design and implementation of TBSV-based DNA gene
vectors. (A) Diagram of pHST12, p12-Max9, and pHST32 (not to scale).
The stippled box for p19 indicates that this gene is active in pHST32
but inactive in pHST12 and p12-Max9. Nucleotide sequence details are
provided for the region containing the MCR, starting at the 5' end for
sgRNA1, which is transcribed from the position indicated by the arrow.
The mutated methionine codons (Met*) are indicated, and selected
restriction enzyme sites are provided (sites displayed in bold and
underlined are unique in the whole plasmid); those sites used for
cloning of PMV CP and GUS genes are indicated. (B) Histochemical
visualization of GUS expression in N. clevelandii and cowpea
leaves inoculated with pHST32 DNA 5 or 4 days previously, respectively.
(C) Immunodetection of PMV CP with alkaline phosphatase (left panel) or
by horseradish peroxidase chemiluminescence (middle and right panels).
The panel on the left shows that PMV CP expressed upon inoculation of
C. quinoa with pMax9 transcripts migrates at the same
position as 35 ng of purified PMV CP (the lower bands presumably
represent CP breakdown products). The two panels on the right show
results obtained with C. quinoa and cowpea, inoculated with
different concentrations of pMax9 transcripts or p12-Max9 miniprep DNA.
Leaves inoculated with RNA or DNA were harvested 4 or 7 days after
inoculation, respectively, and samples containing ca. 10 µg of leaf
protein were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, followed by Western blot analysis. Protein
concentrations were determined with a Micro BCA protein assay reagent
kit (Pierce, Rockford, Ill.). The results for the middle and right-hand
panels can be compared because these images originate from the same
immunoblot.
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Although the TBSV DNA-based vectors pHST12 and pHST34 can be directly
used for the insertion of foreign genes (Fig.
4A), the
GUS insertion
vectors were obtained via different intermediates,
as is discussed
briefly. The GUS donor in these experiments was
pHS23, which is a
kanamycin-resistant plasmid analogous to pHS24
(
27), but
instead of containing sequences from the wild type
(pTBSV-100), it
harbors the
StuI-to-
SalI fragment from pHS45
(
25),
which includes the GUS gene. The GUS gene was removed
from pHS23
with
SmaI and
SacI and ligated between
the
SnaBI to
SacI sites
in the MCR of pHST2-14
(
26) to give pHST6. To permit GUS expression
from a TBSV DNA
vector with the p19 gene inactivated, an intermediate
clone (pHST16)
was generated by replacing the
BamHI-to-
NcoI
fragment
of pHST12 with the corresponding fragment of pHS24
(
27). The
GUS gene was subsequently transferred to the
DNA-based vectors
by exchanging the
BamHI-to-
NcoI
fragments of pHST20 (wild type)
and pHST16 (p19 mutant) with the
BamHI-to-
NcoI fragment of pHST6.
These transfers
yielded pHST32 (Fig.
4) and pHST33,
respectively.
The inoculation of pHST33 or pHST32 DNA onto
C. quinoa,
N. benthamiana, and spinach, followed by standard
histochemical GUS
assays (
25), resulted in readily
detectable levels of GUS expression
(data not shown), as was previously
demonstrated for the RNA-mediated
delivery of TBSV-GUS constructs on
these hosts (
25,
27). To
illustrate that additional hosts
are available for TBSV DNA-mediated
foreign gene expression, GUS
expression for inoculated leaves
of
N. clevelandii and
cowpea is shown in Fig.
4B. The results
are provided for pHST32, rather
than for pHST33, because in cowpea
the p19 gene assists in effective
local spread (unpublished
data).
To further illustrate the usefulness of the TBSV DNA-based vector
system, p12-Max9 was used to express the panicum mosaic
virus (PMV) CP,
and its accumulation was compared to the levels
obtained with the
RNA-based analog pMax9. Plasmids pMax9 and p12-Max9
(a gift from M. Turina and K.-B. G. Scholthof) contain the PMV
CP gene
(
33) cloned between the
SnaBI and
XhoI
sites of pHST2-14
(
26) and pHST12 (Fig.
4A), respectively.
The results in the
left-hand panel of Fig.
4C show that the TBSV vector
expresses
PMV CP that is the same size as purified PMV CP. The
inoculation
of plants with miniprep p12-Max9 DNA resulted in detectable
levels
of PMV CP expression in
N. benthamiana (data not
shown), which
supports a systemic infection with TBSV, as well as in
the local-lesion
hosts
C. quinoa and cowpea (Fig.
4C). These
results imply that
inoculation of
C. quinoa with 15 ng of
RNA per µl yields severalfold
higher levels of PMV CP than
inoculations with 150 ng of DNA per
µl. This difference in
accumulation correlates with the observation
that 15 to 20 local
lesions were obtained with a concentration
of 100 to 150 ng of p12-Max9
DNA per µl, whereas about twice the
number of lesions (20 to 50)
appeared following inoculation with
3 to 23 ng of pMax9 transcripts per
µl.
These experiments demonstrated that foreign proteins can be expressed
from modified TBSV sgRNA1 that contains an elongated
leader promoting
translational initiation at the authentic start
codon on the foreign
gene. The histochemical GUS assays did not
reveal an obvious difference
between the intensity of blue color
obtained with RNA versus DNA-based
vectors (data not shown). However,
the yield of PMV CP obtained with
TBSV DNA was inferior to that
obtained with the analogous RNA-based
system, based on micrograms
of input nucleic acid. Nevertheless, it is
debatable whether there
is any relevance to a comparison between
results obtained upon
inoculation with single-stranded plus-sense
transcripts that initiate
infections in the cytoplasm and those derived
from experiments
with double-stranded plasmid DNA, which needs to enter
the nucleus.
Irrespective of the intrinsic and quantitative differences
between
RNA-based vectors and DNA-based analogues, the advantage is
that
sufficient amounts of TBSV DNA inoculum are easily obtained with
standard miniprep procedures for the immediate inoculation of
plants.
As reviewed previously, RNA-based tobamo-, potex-, and potyvirus gene
vector systems offer the advantage of systemic expression
of the
foreign gene (
26). Thus far, instability features restrict
the use of TBSV vectors to inoculated leaves (
25), but the
broad
host range and rapid and relatively high levels of gene
expression
permit the convenient and rapid TBSV-mediated transient
introduction
of foreign genes in plants. The usefulness of the TBSV
RNA-based
vector has been documented through studies on the behavior of
foreign proteins or RNA in protoplasts or inoculated leaves (
3,
25,
32). The present results illustrate the potential for
cost-effective, high-throughput screening using TBSV DNA-based
vectors
that are suitable for the direct rub-inoculation of many
different
plant species with miniprep plasmid DNA. The application
of this easy
and rapid alternative expression system may substantially
expedite gene
(or cDNA library) screening schemes or be used to
rapidly evaluate the
biochemical behavior of foreign RNA segments.
This vector system may
also be applied to investigate signaling
in gene silencing by inducing
or suppressing this phenomenon through
the overexpression of particular
genes in inoculated
leaves.
In summary, this report constitutes the first example of an in
vivo-transcribed cDNA of a member of the economically important
and
diverse family
Tombusviridae. Another novel aspect of the
present study is that the presence of the HDVagrz in combination
with a
poly(A) signal improves infectivity upon inoculation of
plants with
untreated plasmid DNA. These features have been linked
to generate a
new versatile and robust TBSV-mediated gene vector
system that permits
the rapid, transient expression of foreign
genes after simple
rub-inoculation of miniprep DNA onto plant
species of different
families.
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ACKNOWLEDGMENTS |
I am grateful to Steve Garcia, Joan Kuecker, and Beth Whitehead for
excellent technical assistance. I thank Karen-Beth G. Scholthof for the
many valuable suggestions during the experimentation and preparation of
the manuscript and, together with Massimo Turina, for the plasmids
pMax9 and p12-Max9. I also thank Bénédicte Desvoyes for
providing purified PMV CP and valuable contributions to the
experimental analyses and Mike Hughes for his assistance in optimizing
the DNA inoculation conditions. I thank George Lomonossoff for pUC-35S
and Andy Ball for plasmid 2.0 and for helpful suggestions regarding in
vitro ribozyme activity assays.
This work was funded by grants from USDA/CSREES-NRI-CGP (95-37303-2289)
and the Texas Higher Education Coordinating Board Advanced Research
Program (999902-056).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Plant Pathology and Microbiology, Texas A&M University, Room 120, L. F. Peterson Building, College Station, TX 77843-2132. Phone:
(409) 862-1495. Fax: (409) 845-6483. E-mail:
herscho{at}acs.tamu.edu.
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Journal of Virology, September 1999, p. 7823-7829, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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