J.
gen. Virol. (1988), 69, 891-896.
Printedin Great Britain
891
Key words: wheat dwarf virus/geminivirus/agroinfection
Agroinfection of Triticum aestivum with Cloned DNA of Wheat Dwarf
Virus
By R. J. H A Y E S , H. M A C D O N A L D , R. H. A. C O U T T S AND K. W. B U C K *
Department of Pure and Applied Biology, Imperial College of Science and Technology,
Prince Consort Road, London S W 7 2BB, U.K,
(Accepted 19 January 1988)
SUMMARY
A head-to-tail dimer of cloned DNA from a Swedish isolate of wheat dwarf virus
(WDV) was integrated between the T-DNA border sequences of a broad host range
binary vector and transferred into cells of wheat seedlings using an Agrobacteriummediated delivery system. Two-thirds of the inoculated plants developed a systemic
infection. Extracts of infected leaves were shown to contain the virus double-stranded
(supercoiled, open circular and linear) and single-stranded DNA forms of unit genome
length and the virus capsid polypeptide. The results demonstrate the infectivity of a
previously sequenced clone of WDV DNA.
Geminiviruses are plant viruses that are characterized by their twinned isometric or
'geminate' particles and genomes of circular ssDNA (for reviews, see Harrison, 1985; Stanley,
1985; Lazarowitz, 1987). Two subgroups of geminiviruses may be distinguished: (i) viruses that
have bipartite genomes, are transmitted by whitefly vectors and have dicotyledonous hosts, e.g.
bean golden mosaic virus (Morinaga et al., 1983), African cassava mosaic virus (synonym
cassava latent virus) (Stanley, 1983) and tomato golden mosaic virus (Hamilton et al., 1983) and
(ii) viruses that have unipartite genomes, are transmitted by leafhopper vectors and have
monocotyledonous hosts, e.g. maize streak virus (MSV) (Grimsley et al., 1987). A third subgroup
may be needed to accommodate beet curly top virus which has a unipartite genome and a
leafhopper vector, but has dicotyledonous hosts and a genome organization similar to that of the
larger DNA component of the whitefly-transmitted geminiviruses (Stanley et al., 1986).
Restriction endonuclease analysis and cloning of the dsDNA form of a Swedish isolate of
wheat dwarf virus (WDV) suggest that, like MSV, the virus genome consists of a single DNA
component (MacDowell et al., 1985). Although WDV and MSV are distinct, serologically
unrelated viruses with different leafhopper vectors (Lindsten et al., 1980), their DNAs show
46% nucleotide sequence homology and their genome organizations have some features in
common, indicating a distant relationship (MacDowell et al., 1985). Neither WDV nor MSV are
mechanically transmissible, but infection of maize was achieved recently by inoculating plants
with Agrobacterium tumefaciens strains carrying Ti plasmid vectors which had head-to-tail
tandem dimers of cloned MSV DNA integrated between the right and left T-DNA border
sequences (Grimsley et al., 1987). We now report the use of this process, termed agroinfection, to
demonstrate the infectivity of a clone of WDV DNA (WD1) the complete nucleotide sequence
of which has been previously determined (MacDowell et al., 1985).
To construct a dimer of WDV DNA, 1 ~tg of HindlII-digested pWD1 (WDV DNA cloned
into the HindlII site of pEMBL9; MacDowell et al., 1985) was incubated with 2 units of T4
DNA ligase (NBL) in 1 ml of 50 mM-Tris-HC1 pH 7-5, 8.5 mM-MgC12, 10 mM-dithiothreitol,
2.5 mM-ATP for 6 h at 14 °C. The ligation products contained circular WDV DNA insert,
circular vector and WDV DNA insert religated to the vector. After digestion with BstEII, which
cuts once in WDV DNA but not in pEMBL9, the DNA was precipitated with ethanol,
resuspended in 10 gl of the ligation buffer described above and incubated with 2 units of T4
0000-8178 © 1988 SGM
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DNA ligase for 3 h at room temperature. The products of ligation were then used to transform
competent Escherichia coli DH5ct (Hanahan, 1985; Jessee, 1986) and the recombinant plasmids,
which were isolated from transformant colonies, were analysed by restriction endonuclease
digestion and agarose gel electrophoresis (Maniatis et al., 1982). One such plasmid, pAil 1,
which contained a head-to-tail dimer of WDV DNA, was partially digested with HindlII to
release the dimeric DNA which was then isolated by electrophoresis in, and extraction from, a
low melting point agarose gel (Maniatis et al., 1982) and ligated into HindIII-digested pBinl9
(Bevan, 1984). Transformation into competent DH5~ cells resulted in a number of colonies
containing pHG l8, a recombinant plasmid containing the dimer of WDV DNA integrated
between the right and left T-DNA border sequences of the binary vector pBinl9.
The rifampicin-resistant A. tumefaciens strain PC2669 (LBA958) harbouring the nopaline Ti
plasmid pTiC58 (Hooykaas et al., 1980) was grown in yeast extract broth (YEB) (Lichtenstein &
Draper, 1985) at 30 °C, and the E. coli strains DH5ct (priG18) and MC1022 (pRK2013)
(Figurski & Helinski, 1979) were grown at 37 °C in L.-broth (Maniatis et al., 1982) supplemented
with 50 ~tg/ml of kanamycin. These cultures were then used to mobilize priG18 into the
Agrobacterium strain by triparental mating (Lichtenstein & Draper, 1985). Exconjugants
containing pTiC58 and priG18 were selected by plating on YEB containing rifampicin
(100 ~tg/ml) and kanamycin (50 ~tg/ml). The constructs in Agrobacterium were verified by
restriction endonuclease digestions and Southern blotting.
A. tumefaciens (pTiC58 :priG 18) was grown for 48 h at 30 °C in YEB containing rifampicin
and kanamycin. The cells were pelleted in a bench top centrifuge and resuspended, at 1/20 of the
original volume, in Murashige & Skoog medium (Gibco-Bethesda Research Laboratories).
Four-week-old wheat seedlings (Triticum aestivum cv. Flanders), grown in a Fisons Fi-totron
growth cabinet at 22 °C with a 16 h day length, were inoculated by injecting a total of 20 ~tlof the
resuspended cells into four sites in the stem base in the region from 0.5 cm below to 0.5 cm above
the soil. After 4 weeks, total DNA was extracted from 0.1 g of freeze-dried leaf material as
described by Lichtenstein & Draper (1985) and resuspended in 100 p.1 of TE (10 mM-Tris-HC1
pH 8.0, 1 mM-EDTA). Five ~tl portions of the DNA were electrophoresed through a 1.2~
agarose gel containing 0.5 ~tg/ml ethidium bromide in TBE buffer (Maniatis et al., 1982), and the
DNA was then depurinated by soaking the gel in 0-25 M-HC1 for 15 min and transferred to a
GeneScreen Plus membrane (New England Nuclear) under denaturing conditions (Hayes et al.,
1988). The GeneScreen was then neutralized, prehybridized and hybridized to 32p-labelled,
nick-translated pWD1 (Rigby et al., 1977) according to the manufacturer's instructions.
DNA from 22 of the 34 plants inoculated with A. tumefaciens carrying both pTiC58 and
priG 18 hybridized to pWD1. No hybridization was detected between pWD1 and DNA from
mock-inoculated plants, or with DNA from plants inoculated with A. tumefaciens harbouring
only pTiC58.
DNA from the leaves of agroinfected wheat plants is compared with DNA from the leaves of
wheat plants (kindly supplied by K. Lindsten) infected with WDV by the leafhopper vector
Psammotettix alienus in the Southern blot shown in Fig. 1 (a). Bands corresponding to the ds
(supercoiled, open circular and linear) forms and ss forms of WDV DNA were detected in both
types of infected material (lanes 2, 4 and 5). When the DNA was digested with HindlII (single
site in WDV DNA) the dsDNA forms were converted to the linear form of genome length
(approx. 2.7 kb) (lane 7), whereas digestion with EcoRI (two sites in WDV DNA) produced two
bands of approx. 2-2 kb and 0.5 kb (lane 6) as predicted from the nucleotide sequence of WDV
DNA (MacDowell et al., 1985).
Blotting of the DNA samples under non-denaturing (neutral) conditions in which only the
ssDNA species are transferred (Stachel et al., 1986; Hayes et al., 1988) confirmed the nature and
presence of the WDV ssDNA species (Fig. 1b). The two closely separated bands detected in all
the preparations probably represent the linear and circular forms of the virion ssDNA.
Separation of the linear and circular forms of the virion DNA of a number of geminiviruses has
previously been observed after electrophoresis in polyacrylamide gels containing urea (Harrison
et al., 1977; Hamilton et al., 1981) or after electrophoresis of glyoxalated DNA in agarose gels
(Stanley, 1983).
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(a)
(b)
1
2
3
4
5
6
7
8
- -
4072'-3084~
10
OC
--lin
2036"
SC
1635
S
S
~
1018
516
Fig. 1. Autoradiograph of WDV DNA forms produced during infection. Total DNA was
electrophoresed in a 1-2~ agarose gel, transferred to GeneScreen Plus with alkali (Fig. 1a) or 10 x SSC
(Fig. 1b) and hybridized with 3zp-labelled, nick-translated pWDI. Lane 1, HindlII-digested pWD1;
lanes 2 and 8, DNA from wheat infected with WDV by P. alienus; lanes 3 and 9, WDV virion DNA;
lanes 4 and 10, DNA from an agroinfected plant; lane 5, DNA from a second agroinfected plant; lanes
6 and 7, DN A shown in lanes 4 and 10 digested with EcoRI and HindllI respectively, oc represents open
circle DNA, sc represents supercoiled DNA, lin represents linear DNA, ss represents single-stranded
DNA. Positions of relevant molecular size markers (Bethesda Research Laboratories kilobase ladder)
are shown.
It is clear from the results of both the alkaline, denaturing, blot (Fig. 1 a) and neutral, nondenaturing, blot (Fig. 1 b) that the W D V D N A species obtained either from agroinfected wheat
plants or from wheat plants infected with W D V by the leafhopper vector are indistinguishable.
Further evidence for W D V infection was obtained by probing of polypeptides from a partially
purified virus preparation with an antiserum raised against purified W D V virions (kindly
supplied by K. Lindsten). Leaf material (5 g fresh weight) from agroinfected or mock-infected
wheat seedlings was homogenized in l0 ml of 10 mM-sodium phosphate, 10 mM-EDTA, 0.1 ~o
thioglycollic acid, pH 4.0 in a Waring blender. After stirring for 30 min at 4 °C the homogenate
was centrifuged for 10 min at 22000g. The supernatant fluid was then centrifuged for 2 h at
50000 r.p.m, in a Beckman Ti70 rotor at 4 °C. The pellets were resuspended in 10 mM-sodium
phosphate pH 7.0 and centrifuged through a 1 ml cushion of 20~o (w/v) sucrose in the same
buffer at 35000 r.p.m, for 4 h at 4 °C in a Beckman SW50.1 rotor. The final pellets, containing
partially purified WDV, or the equivalent material from healthy plants, were resuspended in
100 gl of TE plus 1 ~ SDS and 1 ~ 2-mercaptoethanol, heated at 100 °C for 3 min, and 10 gl
portions were electrophoresed in a 12 ~o SDS-polyacry!amide gel alongside marker proteins. The
proteins were then transferred to nitrocellulose (Towbin et al., 1979) which was cut into two
portions and stained with amido black (Fig. 2a) or probed with the W D V antiserum. Bound
antibodies were detected using a Protein A-peroxidase conjugate together with 4-chloro-1naphthol and hydrogen peroxide as described by Sherwood (1987) (Fig. 2b). The partially
purified virus preparation from the agroinfected plants (lane 3) contained a polypeptide of Mr
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(a)
(b)
1
2
3
4
5
6
a.
b.
C.
d
Fig. 2. Blot of preparations of partially purified virus from agroinfected wheat leaves (lanes 3 and 5) or
equivalent material from healthy plants (lanes 2 and 6). Lanes 1 and 4 contain human gamma globulin
(bands a and c, Mr 5500 and 23500 respectively), lactate dehydrogenase (band b, Mr 36000) and
myoglobin (band d, Mr 17000). Lanes 1 to 4 (a) were stained with amido black. Lanes 5 and 6 (b) were
probed with antiserum to WDV.
29000 (29K), the same size as that detected previously in virus preparations from wheat plants
infected with WDV by P. alienus (MacDowell et al., 1985). A polypeptide of this size was not
detected in comparable material from healthy plants (lane 2). Only the Mr 29K polypeptide
reacted with WDV antiserum, confirming that the WDV coat protein is produced during
agroinfection.
None of the agroinfected wheat seedlings or the seedlings infected with WDV by P. alienus,
supplied by K. Lindsten, showed any obvious symptoms. The characteristic symptoms of WDVinfected plants in the field in Sweden include dwarfing and increased tillering with suppressed
heading and are manifested at a later stage in the development of the plant (Lindsten et al.,
1980).
Agroinfection of maize with dimers of cloned MSV D N A previously established that viral
genome length dsDNA was formed (Grimsley et al., 1987). Here we have shown that
agroinfection of wheat with dimers of cloned WDV D N A gives rise to a systemic infection in
which WDV dsDNA, WDV ssDNA and WDV coat protein are produced and are
indistinguishable from those found in plants infected with WDV by the leafhopper vector. The
results also establish the infectivity of the cloned WDV D N A the complete nucleotide sequence
of which is known (MacDowell et al., 1985). It should be possible to use this system to determine
which of the open reading frames in the WDV genome (MacDowell et al., 1985) are essential for
virus infectivity.
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895
There are now a number of examples of infection of plants with dimers of cloned geminivirus
D N A , either by mechanical inoculation with a recombinant plasmid containing the dimeric
D N A (Hayes et al., 1988) or by transfer of the D N A into plant cells mediated by an
Agrobacterium/Ti plasmid vector system (Rogers et al., 1986; Grimsley et al., 1987; this paper).
In all cases the viral D N A detected was of unit genome size. It will be interesting to determine
whether release of unit-length D N A from the head-to-tail dimers occurs by intramolecular
recombination or by a replicative mechanism.
We thank Dr K. Lindsten for supplying wheat seedlings infected with WDV and for the W D V antiserum.
Professor R. Schilperoort for A. tumefaciem PC2669 (pTiC58) and the Science and Engineering Research Council
for the award of a research grant to K.W.B. and R.H.A.C. Agroinfection experiments with W D V were carried out
with a licence [No. PHF29B/116 (107)] issued under the Plant Pests (Great Britain) Order 1980 by the Ministry of
Agriculture, Fisheries and Food.
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