ABSTRACT
We describe experiments directed towards development of cauliflower mosaic virus
(CaMV) replicons for propagation of functional elements during infection of
plants. Modifications and inserts were introduced into replaceable domains associated with the 35S promoter. The 35S enhancer (-208 to -56) was found to potentiate promoter activity when in reverse
orientation sufficient to establish systemic infection. However, replacement of
the 35S enhancer with that from the
nos
promoter caused loss of infectivity. A 31 bp oligonucleotide containing a
polypurine tract specifying initiation of CaMV plus strand DNA synthesis was
inserted into a 35S enhancer deletion mutant and propagated in plants. Analysis
of progeny DNA showed the presence of an additional discontinuity at its new
location in the 35S enhancer, indicating that the artificial primer had
functioned correctly in an ectopic site. An intron and flanking sequences from
the RNA leader of the
Arabidopsis
phytoene desaturase (
pds
) gene, when inserted into the 35S enhancer in forward orientation was very
efficiently spliced during infection. The CaMV replicon carrying the
pds
gene fragment produced unusual infection characteristics, with plants showing
early symptoms and then recovering. We conclude that infectious CaMV replicons
can be used to carry a variety of elements that target both viral and host
functions.
Plant viruses have a number of features that make them attractive as a means of
delivering foreign nucleic acid sequences to plants (
1
-
3
). Their extrachromosomal genomes replicate to high copy number, they can spread
systemically from the point of infection within days and collectively they have
an extremely large host range. Most plant viruses have RNA genomes, some of
which have been used to deliver foreign RNA to plant cells, including
relatively large marker genes like
GUS
(
4
-
9
). However, RNA virus expression and amplification vectors are cytoplasmic and
they cannot be used to probe nuclear functions. Plant viruses with DNA-based genomes, however, have a nuclear phase in their multiplication
cycle. Of the three groups of plant DNA viruses, the badnaviruses,
caulimoviruses and geminiviruses, only the latter two have so far been
investigated extensively as vectors (
10
,
11
). Vectors based on geminiviruses have been shown to be effective in delivering
foreign DNA to plants, but there are limitations to the size of the insertion (
3
,
12
-
15
). Also, transcription and replication of geminiviruses both occur in the
nucleus, limiting the ability to target cytoplasmic processes.
As pararetroviruses (
16
-
18
), the caulimoviruses, such as cauliflower mosaic virus (CaMV), have separate nuclear transcriptional and cytoplasmic
reverse transcriptional phases. The circular double-stranded 8 kb DNA genome of CaMV also seems to have a strict upper size
limit (
16
), such that insertion of >~250 bp of foreign DNA requires deletion of non-essential viral sequences. Only two of the seven major open reading
frames (ORFs) in the CaMV genome (ORFs VII and II) are dispensable for virus
infectivity. Of these, gene II has been used to insert small genes, including
DHFR (
19
), interferon (
20
) and metallothionine (
21
), which were propagated in infected plants. A heterologous intron from soybean has also been inserted into gene II, but this was spliced from RNA only inefficiently (
22
). However, although gene II has a dispensable function, it contains
cis-
acting elements additional to its protein coding function which could be incompatible with insertion of some constructs. These elements include a plus strand
replication primer (
23
), a putative splice acceptor sequence (
24
) and sequences linking translation with adjacent genes.
A primary early objective in developing viral vectors was to enable them to
carry genes to genetically modify plants, but this has been obviated by the
variety of alternative efficient methods of producing transgenic plants. In
contrast, viral vectors can be exploited by judicious insertion of small
efficacious foreign sequences. For example, addition of foreign oligopeptides
to the surface of virions is being developed for production of animal virus
vaccines in plants (
1
,
9
). Plant viruses are also being exploited to study aspects of viral pathogenesis
(
4
) and other molecular phenomena, such as gene silencing in plants (
25
). We aim to exploit the detailed knowledge of CaMV molecular biology to develop
vectors to carry foreign elements to specific cellular compartments to target
particular processes. Recently (
26
), we discovered that significant portions of the enhancer upstream of the 35S
promoter (Fig.
1
), together with co-linear elements, were not essential for CaMV infectivity. Moreover, we
found that the 35S enhancer could tolerate insertion of an innocuous 84 bp
bacterial linker sequence which was propagated stably in plants. Since the 35S
promoter region has several co-linear functions active in different cellular compartments, we wished to
test the possibility that this part of the CaMV genome could carry different functional elements to target specific processes.
CaMV was propagated in turnip (
Brassica rapa-rapifera
cv Just Right) or
Arabidopsis thaliana
ecotype Col-0. Turnip plants grown under glasshouse conditions were mechanically inoculated with CaMV or cloned CaMV DNAs as previously described (
27
);
Arabidopsis
was propagated in a growth chamber at 20oC on a 10 h photoperiod. CaMV isolates Cabb B-JI and CM4-184, as infectious clones pCa24 (
28
) and pLW414S (
29
) respectively, were used for construction of chimeric viruses. The following
clones and mutants were as previously described (
26
): pBX2, pSISS, pSISSM676, M676, M9, M10 and M11. CaMV genome co-ordinates of isolate Cabb B-JI are prefixed nt.
Transformation, colony analysis and purification of DNA from bacteria was as
described by Sambrook
et al.
(
30
). A 55 bp deletion in the leader of 35S RNA, starting at +14 (nt 7443) from the
cap site (nt 7429) and ending at +69 (nt 7499), was introduced using the
`Quickchange' mutagenesis procedure (Stratagene). An
Nsi
I site was introduced at the 5'-end using oligonucleotides 1 and 2 (Table
1
) and a
Spe
I site at the 3'-end with oligonucleotides 3 and 4 (Table
1
). The intervening sequence was removed and replaced with a linker (oligonucleotides 5 and 6, Table
1
) to generate M676b1. A fragment (oligonucleotides 7 and 8, Table
1
) based upon the Bluescript polylinker sequence was inserted into the 35S RNA
leader to construct M676b2. The 35S enhancer was reversed in a subclone, pS1SS
(
26
), comprising an
Nco
I-
Dra
II (nt 6906-7877) fragment cloned into pALTER (Promega). This clone contained a
Spe
I sites at the 3'-end (nt 7367) of the enhancer, with a second being introduced at the 5'-border (nt 7223) using the Altered Sites (Promega) mutagenesis method.
This construct was digested with
Spe
I and religated to generate inserts of both orientations, which were subsequently selected by PCR and confirmed by sequencing. The
Nco
I-
Dra
II fragment containing the reversed enhancer replaced the equivalent fragment of the full- length CaMV clone pBX2 to generate mutant M18b. The
Agrobacterium nos
enhancer was isolated from clone pSLJ261 (
31
) after insertion of an
Nhe
I site at -210 from the
nos
cap site by the `Quickchange' method (Stratagene) using oligonucleotides 9 and 10 (Table
1
) to generate SLJ261M1. This was digested with
Nhe
I to isolate the -210 to -66
nos
enhancer fragment, which was inserted into the
Nhe
I site of M9 to generate M9nos.
Synthetic forward and reverse oligonucleotides (oligonucleotides 11 and 12, Table
1
) containing a sequence defining the plus strand discontinuity (G3) located in gene II (Fig.
1
A), with ends complementary to DNA cut with
Nhe
I, were annealed and cloned into the
Nhe
I site in deletion mutant M4 (
26
) in forward and reverse orientations, producing the recombinant viruses M10Gf
and M10Gr respectively.
To construct LM11, the
Mlu
I-
Bst
EII fragment from M11 was cloned into pLW414S to generate pLWM1. The
Stu
I fragment from pLWM1 was then ligated to the appropriate
Stu
I fragment from pCa24 to generate LAM2. To allow cloning at the
Nhe
I site, viral DNA was excised from pAT153 by digesting with
Sal
I and cloned into
Sal
I-cut pGEM5, generating LM11. An equivalent recombinant to LM11, called MP12, having the CM4-184 gene II region but the wild-type Cabb B-JI 35S promoter, was constructed with a polylinker site at nt 7222. Two further
constructs, containing fragments of host DNA set in both orientations in the polylinker site of MP12,
were made as follows. First, modification was made to pSISS by inserting an
Nsi
I site at nt 7285 and a
Stu
I site at nt 7222 to generate pSISSj. A polylinker was inserted into the
Stu
I site in both orientations, generating pSISSj1 and pSISSj2. A
Hin
dIII (nt 5846)-
Nco
I (nt 6904) fragment from Cabb B-JI, containing an extra
Dra
II site (nt 6407), was added to the
Nco
I site at nt 6904 of both subclones to give pJSHI and pJSH2 respectively. Phytoene desaturase DNA fragments were obtained by PCR from
Arabidopsis thaliana
total nucleic acid. The primers used (oligonucleotides 13 and 14, Table
1
) were in the 5'-untranslated leader of the gene and included the first ATG in the polypeptide
coding sequence. These primers had 5' extensions introducing
Spe
I-
Sac
I and
Kpn
I-
Sac
I restriction sites at the 5'- and 3'-ends of the PCR fragment respectively. The PCR
fragment was cloned in sense and antisense orientations, as a
Spe
I-
Kpn
I fragment, into JSH1 and JSH2 respectively, producing pASS1 and pASA1. pJSH1
and pJSH2 contained an oligonucleotide polylinker (oligonucleotide 15, Table
1
) inserted in a
Stu
I site introduced at nt 7222 in CaMV isolate Cabb B-JI in a
Nco
I-
Dra
II (nt 5165-7878 in Cabb B-JI) sub-fragment in forward and reverse orientations respectively.
The modified CaMV
Dra
II fragments from pASS1 and pASS1 were ligated to the larger fragment from
Dra
II-cut LM11. The resulting recombinants were called MD1 and MD3, denoting
virus carrying the sense and antisense phytoene desaturase gene (
pds
) fragments respectively. MP12 was derived from these clones by digestion at
Sac
I sites in the polylinker flanking the
pds
inserts.
Table 1
DNA (containing total viral DNA) from infected plants was extracted for PCR
analysis by grinding infected leaf material in extraction buffer (0.5% SDS, 10
mM Tris pH 8.0, 1 mM EDTA) and incubating the sap with proteinase K (final concentration 0.5 mg/ml) and RNase A (final concentration 10 [mu]g/ml) for 30 min at 37oC before spinning down cell debris and precipitating total nucleic acid
from the resulting supernatant with an equal volume of isopropanol. One
fiftieth of the final resuspension of DNA was used per PCR reaction.
Virion DNA used in Southern blots was extracted by a method modified from that
of Hull
et al
. (
32
). A single systemically infected leaf was ground in 800 [mu]l sterile distilled water and Triton X-100 added to a final concentration of 2%. After vortexing thoroughly,
viral inclusion bodies and cell debris were pelleted by centrifugation in a
microfuge. The supernatant was discarded and the pellet washed with water until
all Triton X-100 had been removed. The final pellet was resuspended in DNase buffer (100 mM Tris-HCl, pH 7.4, 2.5 mM MgCl
2
) containing 10 [mu]g DNase I and 10 [mu]g RNase A and incubated at 37oC for 1 h. SDS was then added to 0.5% and the mixture incubated at 65oC for 15 min, when proteinase K was added (to a final
concentration of 10 [mu]g/ml) and incubation continued for a further 30 min at 37oC to disrupt inclusion bodies and release virion DNA. Debris was pelleted by centrifugation and the supernatant phenol/chloroform extracted. Viral DNA was selectively precipitated with PEG (30%
polyethylene glycol 6000, 10 mM MgCl
2
) at room temperature. Discontinuities were mapped by electrophoresis of 10 [mu]g virion DNA in 1% agarose containing 30 mM NaOH, 3 mM EDTA at 1.4 V/cm for 18 h.
In our previous study of the role of the 35S promoter in CaMV pathogenicity (
26
), we identified regions of the 35S enhancer that were non-essential for CaMV infectivity and a 5'-boundary (-208 from the cap site; see Fig.
1
B) upstream of which deletions abolished virus infectivity. To complete this
analysis and determine whether sequences downstream of the cap site could
tolerate modification, we deleted a 55 bp fragment of the 35S RNA leader (+14
to +69). Insertion of two new restriction sites at the boundary of the deletion
site (construct M676b; Fig.
2
) did not affect CaMV infectivity but deletion of the fragment (in M676b1) or
replacement (in M676b2) with a linker we have previously shown to be innocuous
to CaMV infectivity (
26
) abolished infectivity (Fig.
2
). Thus, possible modifications in the 35S promoter region are limited to the
enhancer.
To determine whether different CaMV elements could function ectopically from
within the 35S promoter domain, we constructed an artificial plus strand
priming sequence based upon the polypurine tract located in gene II. The sequence (oligonucleotide 11, Table
1
) consisted of 31 bp upstream of the priming site flanked by
Nhe
I sites. This was cloned into the
Nhe
I site of the 35S promoter deletion mutant M10 (Fig.
2
). Since the oligonucleotide fragment could insert in either orientation into the restriction site, we
selected clones by PCR analysis with an insert in both the forward (M4Gf) and
reverse (M4Gr) orientations. The inserts were confirmed by sequencing and the
constructs inoculated into plants. The CaMV replicons carrying artificial plus
strand priming sequences set in both orientations in the 35S enhancer were
found to be infectious (Fig.
2
).
To test ectopic functionality of the polypurine tract, we analysed progeny
virion DNA for the presence of an extra discontinuity. Wild-type CaMV virion DNA contains three discontinuities, one in the minus
strand and two in the plus strand, adjacent to the replication priming sequences (
33
). On denaturation, wild-type virion DNA produces three major single-stranded molecules of 8.0, 5.4 and 2.6 kb, denoted the [alpha], [beta] and [gamma] strands respectively, together with various
minor components (Fig.
3
). A mutant virus (MP12) with most of gene II deleted and lacking one of the two
plus strand discontinuities generates only single strands of 8.0 kb as the
major component (Fig.
3
). However, denaturation of virion DNA of the mutant carrying the artificial
polypurine tract in the 35S enhancer had single-stranded DNAs of 8.0 ([alpha]), 3.1 ([beta]*), 2.6 ([gamma]), and 2.3 ([beta]**) kb, consistent with the presence of an extra
discontinuity splitting the [beta] strand into two fragments at the site of insertion. This indicates that
the artificial plus strand priming sequence functioned quite normally in its
abnormal location. Sequencing of progeny virus showed that the insert was
unchanged (data not shown). Moreover, although the insert in the opposite
orientation was propagated by the vector (M10Gr), a discontinuity was not
generated (Fig.
3
), consistent with the functional polarity of this
cis
-acting element. The symptoms produced by both M10Gf and M10Gr were not
significantly different from those of the unmodified M10 vector (Fig.
2
).
We wished next to test the ability of the 35S enhancer to accommodate
heterologous functional sequences that were not essential to the infection
process. We have already shown that an 85 bp innocuous bacterial sequence can
be stably propagated in the 35S enhancer (
26
). In an attempt to extend the amount of heterologous DNA which could be
propagated, a chimeric virus (LM11) was constructed between CaMV isolate CM4-184, lacking most of gene II (
34
), and M11, a mutant with a 110 bp deletion in the 35S enhancer. The chimera had
a combined deletion of 531 bp. However, upon infection of plants, LM11 displayed symptoms several weeks later than wild-type infections, limiting its usefulness (Fig.
2
). A further chimera was constructed without a deletion in the 35S enhancer but
with a short polylinker sequence in the position of the left border defined by
deletion mutant M11, producing the same truncation in gene VI (Fig.
2
). This construct (MP12), with an overall loss of 421 bp, produced symptoms
similar to those of wild-type CM4-184 but was slightly delayed compared with isolate Cabb B-JI, from which the major portion of the hybrid virus had been
derived.
Next, we isolated a 300 bp sequence from the 5'-untranslated leader of the
Arabidopsis
pds
gene by PCR amplification of cell total DNA. By analogy with the tomato
pds
gene (
35
), it was predicted that the appropriate fragment should contain an intron.
Following PCR using primers with
Spe
-
Sac
5'- and
Sac
-
Kpn
3'-ends which were homologous to sequences 100 bp apart on the
Arabidopsis
pds
cDNA (
36
), a 300 bp fragment was obtained consistent with the presence of a 200 bp
intron (data not shown). On sequencing, the intron was found to be 198 bp long
with a similar, but not identical, structure to that in tomato. The 300 bp
fragment was cloned into the polylinker site of a CaMV construct based upon
MP12 in both sense and antisense orientations, producing constructs MD1 and MD3
respectively (Fig.
2
). Following inoculation into turnip plants, both constructs were found to be
infectious. Analysis of virion DNA by PCR from 9/10 plants infected with MD1
containing the
pds
insert in sense orientation indicated that the insert had lost 200 bp,
consistent with removal of the intron sequence. No product was detected in the
tenth plant. To confirm that the truncation was due to splicing, the
appropriate region of recovered virion DNA was amplified by PCR, cloned and
sequenced for comparison with the original insert (Fig.
4
). This showed that the truncated DNA had a sequence identical to that of the
pds
cDNA, confirming that correct splicing of the 198 bp intron had occurred from
the vector (Fig.
4
). We observed no unspliced molecules in any infections of vectors carrying
pds
in sense orientation. Analysis of virion DNA from plants infected with MD3
containing the
pds
insert in antisense orientation showed that only part of the insert had been
retained. Sequencing showed that 269 bp of the original 345 bp insert
(including the polylinker) had been deleted. Deletion had occurred between two
GAA motifs: one in the 5' polylinker, the second towards the 3'-end of the reversed
pds
insert (Fig.
3
).
We have defined elements associated with the CaMV 35S promoter that are
essential and non-essential to viral infectivity in plants. This was to enable development
of replicons to carry inserted elements that could function in different
cellular compartments. Essential elements have now been delimited both upstream
of the cap site at -208 (
26
) and, in this study, downstream between +14 and +69. This downstream region comprises part of the 35S RNA terminal repeat containing
cis
-acting elements important in translation (
17
), replication (
18
) and, possibly, transcription (
37
). Definition of the 35S enhancer region upstream of the cap site as an
orientation-independent enhancer has come from experiments with excised sequences
expressed in non-host transgenic plants driving a non-essential reporter gene (
38
,
39
). We have now shown that the reversed enhancer potentiates activity of the 35S promoter under conditions where the functionality threshold of infectivity is applied, rather than the
quantitative assay afforded by a reporter gene. To our knowledge, this is the
first use of a reversed enhancer to potentiate viral infectivity in a whole
organism. Moreover, the ability of the enhancer to function in reverse
orientation without disrupting co-linear viral functions re-affirms our previous conclusion (
26
) that the complete enhancer contains replaceable elements, the minimum
requirement for infectivity being the presence of one of the two interchangeable components (see Fig.
1
). However, we were not able to re-establish infectivity of CaMV by replacing the 35S enhancer with the
nos
enhancer. Activity of the
nos
enhancer is generally much lower than that of the 35S enhancer in heterologous
contexts (
40
) and is presumably not sufficient to potentiate transcription above the infectivity threshold.
Recognition of the artificial plus strand replication primer (polypurine tract),
functional in the cytoplasm, was indicated by the presence of an extra
discontinuity in virion DNA within the 35S enhancer. This observation shows
that the additional
cis
-acting element in an already complex region of the viral genome did not
disrupt any co-linear or distal functions. This is the first report of ectopic expression
of such a priming sequence from a pararetrovirus and shows that a maximum of 31
bp is required to specify plus strand priming in CaMV. We also confirmed
functional polarity of the sequence, as has been shown previously (
23
).
The intron from the leader sequence of the
Arabidopsis
pds
gene was isolated from plants, inserted into the 35S enhancer and was
efficiently removed by splicing. Since the splicing occurred independently of
any essential viral function, this raises the possibility of using CaMV vectors
for studying splicing sequence specificity
in vivo
, since constructs can be rapidly introduced into plants by mechanical
inoculation. The intron had been inserted towards the 3'-ends of two co-linear viral RNAs (see Fig.
1
). The most likely route for loss of the intron was from the pregenomic 35S RNA,
with the spliced form being amplified by reverse transcription. The efficiency
of splicing of the
Arabidopsis
intron in our vector was much higher than that previously reported for a
soybean leghaemoglobin intron inserted into CaMV gene II (
22
). Possible explanations for the differences in reported processing efficiency
are, first, that soybean is not a CaMV host and, second, splicing from within
gene II is most likely complicated by the fact that the foreign intron was
inserted into an existing CaMV intron (
24
).
Loss of all but 76 bases of the
pds
fragment inserted in antisense orientation indicates low stability during
infection. A similar lack of stability was observed for the reversed soybean
intron inserted into gene II (
22
). Although this could be a feature of such sequences, a further possibility is
that the 35S enhancer is unable to tolerate large inserts, which might
compromise spacing of important transcriptional elements. Splicing of the sense
pds
intron could thus have been favoured if a more efficient enhancer had resulted.
However, a shortened form of the antisense
pds
insert could not be generated by splicing, so a piece was removed by an alternative process. Since the deletion had occurred between two GAA motifs, this suggests deletion occurred by strand switching, as has been observed before with CaMV deletions (
41
).
Unexpectedly, turnip plants containing the sense
pds
construct showed recovery by becoming asymptomatic. This contrasts with the use
of a plant RNA viral vector carrying an antisense
pds
sequence in which expression of the host
pds
gene, involved in carotenoid biosynthesis, appeared to be silenced, causing symptoms of carotenoid deficiency (
42
). The difference may be due to the fact that CaMV DNA enters the nucleus and
evokes a different type of response from the host.
We gratefully acknowledge the BBSRC for a Graduate Studentship to R.J.N. and for
additional funding. We thank R.Hull, J.W.Davies and G.P.Lomonossoff for helpful
comments. We are grateful to J.Jones for clone pSLJ261 and S.Howell for clone
pLW414S. Experiments were performed under MAFF license no. PHF 1491/982/34.
*To whom correspondence should be addressed. Tel: +44 1603 452 571; Fax: +44
1603 456 844; Email: covey@bbsrc.ac.uk
Oligonucleotide
Sequence
1
5'-GTAGAGAGAGACTGATGCATTTCAGCGTGTCC-3'
2
5'-GGACACGCTGAAATGCATCAGTCTCTCTCTAC-3'
3
5'-CGGGAAACTAGTCACACATTA-3'
4
5'-TAATGTGTGACTAGTTTCCCG-3'
5
5'-TCGTTGACCAGGCTAGCGGT-3'
6
5'-CTAGACGCGTAGCCTGGTCAACGATGCA-3'
7
5'-CTAGTCAGGTTGACGGTATCCATAAGCTTGCTATTGAATTCCTGCAGCCCGGACTGCCACTAGTTCT-3'
8
5'-CTAGAGAACTAGTGGCAGTCCGCGCTGCAGGAATTCAATAGCAAGCTTATGGATACCGACTACCTGA-3'
9
5'-GATGACGCGGGGCTAGCCGTTTTAC-3'
10
5'-GTAAAACGGCTAGCCCCGCGTCATC-3'
11
5'-CTAGCAAAAACCATTTTTAAGAGTGGGGGGGTTG -3'
12
5'-CTAGCAACCCCCCCACTCTTAAAAATGGTTTTTG -3'
13
5'-GGACTAGTGAGCTCGAGCTACTTCCACTAGCCTC-3'
14
5'-GGGGTACCGAGCTCTAAAGCTATGTCCCATTAG-3'
15
5'-CTAGAACTAGTGAGCTCGGTACCGAATTCCTCGAGTCTAG-3'
REFERENCES
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