Plant Molecular Biology 11: 35 - 43 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

35

Agroinfection and nucleotide sequence of cloned wheat dwarf virus D N A Crispin J. Woolston l, Richard Barker 2, Helen Gunn l, Margaret I. Boulton 1 and Philip M. Mullineaux 1

IAFRC Institute of Plant Science Research, John Innes Institute, Colney Lane, Norwich, Norfolk, NR4 7UH, UK; 2Sequencing Systems Limited, Unit 184, Cambridge Science Park, Cambridge, CB4 4GN, UK Received 28 October 1987; accepted in revised form 11 March 1988.

Key words: Agrobacterium tumefaciens, Agroinfection, Geminivirus, Wheat Dwarf Virus Abstract

Cloned D N A of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation o f infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component o f 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.

Introduction

Wheat dwarf virus (WDV), a member of the geminivirus group of plant viruses [22], was first observed in certain winter cereals in Czechoslovakia [6], and has a host range which, when determined using the natural leafhopper vector, Psammottetix alienus Dhalb, includes such crop species as Triticure, Arena and Hordeum [37]. A distinct subgroup within the geminiviruses is formed by viruses which infect monocotyledonous plants and which are transmitted by leafhopper vectors. These include WDV and also maize streak virus (MSV; [21]), digitaria streak virus (DSV; [7, 8]) and chloris striate mosaic virus (CSMV; [12]). The geminiviruses have a circular single-stranded DNA (ssDNA) genome which is encapsidated into paired semi-isometric particles (for a review see [34]). In addition to the virion ssDNA, a circular double-stranded DNA (dsDNA) form o f the geminivirus genome is also found in infected plants

[34]. The role of this dsDNA has not yet been elucidated, but it is speculated that it functions as an intermediate in the replication of the virus genome [36]. Extensive cloning and sequence analysis o f either ssDNA extracted from purified virus or viral dsDNA from infected plants has failed to reveal more than a single component to the viral genome for WDV, DSV and MSV [23, 9, 28, 20, 21]. Although this viral DNA is not mechanically infectious and therefore cannot be definitively identified as the complete genome, evidence that a single component is infectious has recently been presented for beet curly top virus (BCTV [35]). This geminivirus has a host range restricted to the dicotyledenous plants, a leafhopper vector and only a single genome component and yet is mechanically transmissible [29, 30, 32]. Cloned viral BCTV DNA is infectious, and therefore the potential for a DNA molecule o f this nature to function as a viral genome has been demonstrated. However, the nucleotide sequence of BCTV indicates a

36 genome organisation very closely related to the bipartite geminiviruses [35], and so these data are not necessarily applicable to the infectivity o f the gramineae-infecting MSV and WDV. Recently the technique of "agroinfection" has become available for the introduction o f cloned viral DNA into plants [15]. This technique is particularly suited to viruses where the normal, insect vector, method of virus transmission, is not available. Naked viral DNA of several monocot-infecting geminiviruses with monopartite genomes is not infectious when rubbed onto abraded leaves or injected into various tissues, suggesting that these methods are inadequate for delivery of the viral DNA to a site from which it can initiate infection [28]. The cloned viral genome is inserted as a tandem dimer between the T-DNA borders of an Agrobacterium tumefaciens Ti-plasmid-based vector. It is presumed that infection is initiated by interaction of the Agrobacterium carrying the viral genome with the ceils of the host plant, possibly followed by the escape of the viral genome from the transferred TDNA by a process o f homologous recombination. No direct evidence for this mechanism has yet been presented although Hille et al. [18] have shown that certain mutations in the vir loci of the Ti-plasmid do affect the subsequent production of viral symptoms when using tandem dimers of cauliflower mosaic virus to "agroinfect" turnips. "Agroinfection" therefore can overcome the barriers to the introduction of viral DNA by other routes, and has previously proved successful for the introduction of cloned MSV DNA into maize plants [16]. We have used the technique to determine that a characterised full-length clone o f the WDV genome derived from the viral dsDNA found in infected wheat tissue is infectious and produces symptoms, viral DNA forms and virus particles typical of naturally occuring WDV infections. The nucleotide sequence of this infectious clone also confirms that WDV has a genome comprised of a single component. Additionally, the results indicate that Agrobacterium tumefaciens can interact, at least in a limited fashion, with the cells of a wheat plant.

Materials and methods

Extraction of wheat nucleic acids Wheat tissue infected with WDV was kindly provided by Dr J. Vacke (Ruzyne, Czechoslovakia). We have called this isolate of the virus WDV-CJI. Total nucleic acids were extracted by powdering the infected tissue in liquid nitrogen and allowing it to partially thaw in the presence of an equal amount (w/v) of TLES (50 mM Tris-Cl pH 9.0, 150 mM LiC1, 5 mM EDTA, 5070 SDS). This was extracted with two volumes of phenol/chloroform (1:1, saturated with 0.I M Tris-HCl, p H 7.0), the separated aqueous phase was further extracted three times with equal volumes of phenol/chloroform, and finally with an equal volume of 24:1 chloroform:isoamyl alcohol (saturated with 10 mM Tris-HCl pH 8.0, 1 mM EDTA). RNA was precipitated by addition of an equal volume of 4 M LiCI, and after incubation at - 2 0 ° C for 3 h was pelleted by centrifugation at 12000 g for 15 min. The clear supernatant, which consisted mainly of DNA, was dialysed against 1 ×TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at 4 °C for 16 h, and the nucleic acid recovered by precipitation with 2.5 volumes of ethanol. The DNA was collected by centrifugation at 12000 g for 10 min, resuspended in 1 × TE and further purified by CsC1 gradient centrifugation using standard methods [24].

Cloning o f full-length W D V DNA The total wheat DNA was subjected to agarose gel electrophoresis. DNA species migrating faster than the wheat chromosomal DNA, which might correspond to WDV DNA forms, could be discerned by ethidium bromide staining. A fraction enriched for these DNA forms, obtained by collection of a region from the gel [13], was digested to completion with restriction enzyme Sau 3A. The fragments so obtained were cloned into Barn HI cut M13mpl8 [38]. Di-deoxy sequencing [31] of these clones followed by comparison with the published sequence of WDV [23] yielded one (pSAU 41) which contained an insert that was 100°70 homologous to the published sequence.

37 Total infected wheat DNA, cut with Hind III or Kpn I under standard conditions was inserted into these sites in the polylinker of bacteriophage M13mpl8. Recombinant bacteriophage were screened for the presence o f WDV DNA by plaque hybridisation [24] using the hexadeoxynucleotideprimed a-32p-dCTP-labelled [11] Sau 3A WDV DNA fragment of pSAU 41 as a probe. Clone pWDVH2 is M13mpl8 with a full-length Hind III insert o f the WDV-CJI genome cloned into the Hind III site of the polylinker. Clone pWDVK10 is the equivalent clone constructed using the Kpn I sites present in WDV-CJI and M13mpl8 DNA. DNA hybridisations [33] were carried out as described in Maule et al. [26].

kanamycin selection in Luria Broth (LB) at 25 °C. The subsequent inoculum o f approximately 5 x 109 cells m1-1 was injected as 5 × 5/xl aliquots into the basal 1 cm o f the stem o f wheat seedlings. These seedlings, which were at the two-leaf stage, had been grown in a peat-based compost in glasshouse conditions at 25 °C +_ 5 °C. Natural daylight was supplemented to a 16 h photoperiod with mercury halide lamps. All inoculations of plants with Agrobacterium tumefaciens containing WDV genomes were carried out in a MAFF-approved containment glasshouse. Steps were taken to ensure that irrigation water was not allowed to escape the house untreated, and all plant tissue and growth medium was incinerated after use.

Agrobacterium conjugation

Virus purification

WDV-CJI DNA released from either pWDVH2 or pWDVKI0 at the cloning sites was ligated in a molar excess into either Hind III or Kpn I cut pBin 19 [1]. Tandem dimers o f WDV were identified by restriction analysis. Clone pJIT 31 comprises pBinl9 with a tandem dimer o f the Hind III-linearised W D V C J I genome from pWDVH2 inserted at the polylinker Hind III site. Clone pJIT 33 comprises pBin 19 with a tandem dimeric insert o f the Kpn I linearised WDV-CJI DNA from pWDVK10 inserted at the Kpn I site in the polylinker. All pBin 19-based plasmids were maintained in E. coli JM83 recA (P. M. Mullineaux, unpublished data). Both these constructs were conjugated [5] into Agrobacterium tumefaciens strain C58 nal R [17]. Exconjugants were selected on kanamycin (50 mg 1-1) and naladixic acid (60 mg 1-l) and were screened for the presence of both authentic pJIT 31 or pJIT 33 and the presence of the wild-type C58 Tiplasmid.

Virus purified from infected plant tissue [3] was coated onto grids, negatively stained with 2% uranyl acetate and examined with a Joel 100B electron microscope. Single-stranded viral DNA was purified from this virus preparation by phenol extraction [3].

Plant inoculation The methods employed were those described previously [16] with the following modifications. Bacterial cultures for inoculation were grown under

Nucleotide sequencing of an infectious clone of WDV-CJI DNA The WDV-CJI DNA from clone pWDVK10 was released by digestion with Kpn I and transferred into the Kpn I site of vector plC 20R [25]. The resultant monomeric clones, pJIT 34 and pJIT 43 (depending on orientation relative to the polylinker), were sequenced by chemical cleavage methods [27]. The sense o f the viral ssDNA was confirmed by hybridisation of ssDNA prepared from purified virus which had been dot-blotted onto nitrocellulose filters [26], with strand-specific WDV DNA probes prepared by cDNA synthesis on templates of ssDNA of clones pWDVS20 and pWDVS30, Sst I subclones o f WDV-CJI in M13mpl8 [4]. The variations between the published WDV sequence [23] and the sequence of WDV-CJI are presented in the same sense as the viral DNA in Table 1.

38 Results

Table 1.

Nucleotide position

Nucleotide in WDV-S

Nucleotide in W D V - C J I

A m i n o acid changes caused

3 15 26 30 51 160 174 200 364 426 533 536 540 542 548 551 600 602 821 1040 1073 1079 1082 1172 1196 1205 1256 1351 1370 1470

A T A T C T G G T T G G A G A T G G G G T A T T T T T T G

T G T G T C T C C A A T C C G A A T A A G C C C C C C T C A

None None None None None None None None O R F 5 Gin to Leu O R F 5 Lys to Ile O R F 5 Leu to Phe O R F 5 Pro to Thr ORF5HistoGlu ORF2MettoLeu O R F 5 Phe to Leu O R F 5 Thr to Ser ORF5ArgtoSer ORF2AlatoThr None None None None None None None None None None None None

1503 1505 1535 1575 1844 1850 1943 1944

C T T C A A G A

T A C T G G A G

1989 2250 2297 2360 2362 2363 2530 2656 2694

C A G T A G C C A

G G A A G A G G T

(WDV-S)

) J

( ~

~ J

~ (

Southern blot analysis o f total wheat D N A infected with WDV-CJI demonstrated the presence of D N A forms typical of a geminivirus infection (Fig. 1, lane 3). Restriction enzyme analysis of this D N A confirmed that enzymes Hind III or Kpn I cut only once in the viral d s D N A (data not shown) and so these sites were used to obtain genomic clones of WDV-CJI. Several apparently full-length clones

~ O R F 4 T h r to Set

)

O R F 4 T h r to Ala None O R F 4 Cys to Arg ORF 4 Tyr to His ") J O R F 3 lie to Thr O R F 3 Ser to Thr O R F 3 Val to Ala None None None None O R F 3 Glu to Gln None None

Fig. 1. Southern blot probes for sequences specific to WDV. Lane 1, clone pWDVkl0 cut with Kpn I; Lane 2, total D N A extracted from uninoculated plants; Lane 3, total D N A extracted from plants naturally infected with WDV; Lane 4, total D N A from plants agroinfected with WDV-CJI. Letters A to E identify viral D N A forms. A, open circular dsDNA; B, linear dsDNA; C, covalently closed circular dsDNA, identified by restriction digestion. Forms D and E are viral ssDNA, identified by strand-specific probes (data not shown).

39 were obtained using a D N A fraction from infected plants enriched for WDV DNA. Two of these, pWDVH2 and pWDVK10, were selected for infectivity tests. However, the only size criterion which could be applied was comparative migration o f the cloned viral insert relative to the source D N A on agarose gel electrophoresis (Fig. 1, lane 1) and so the possibility that the inserts were sub-genomic could not be dismissed. A numer of clones which were obviously shorter than genome length were also obtained but these have not yet been further characterised. Seedling wheat plants were inoculated with Agrobacterium tumefaciens carrying tandem dimeric inserts of the WDV inserts o f pWDVH2 or pWDVK10 in pBin 19 (pJIT 31 or pJIT 33). Within

3 weeks of inoculation the first symptoms characteristic of WDV infection could be discerned, i.e. stunting of the plants and a mottling on the leaves (Fig. 2A and B). The severity o f the symptoms varied with the cultivar of wheat used. "Maris Huntsman" showed the most severe dwarfing and leaf mottie, whearas "Sappo" plants infected with WDV-CJI showed a very mild leaf mottling, with significant dwarfing. Routinely 30% of the plants inoculated became infected. Plants inoculated in the same fashion with either E. coli JM83 RecAor Agrobacterium tumefaciens strain A6 carrying pJIT31 or pJIT33 failed to develop any symptoms. Similarly plants inoculated with Agrobacterium tumefaciens C58 which was not carrying any viral D N A did not develop symptoms.

Fig. 2. (A: left) Photograph of wheat "Sappo" plants. The plant on tne ~eft is uninoculated whilst the plant on the right is infected with WDV-CJI. (B: top right) Wheat "Maris Huntsman" leaves. The leaf on the left is from an uninoculated plant whilst the leaf on the right is from a plant infected with WDV-CJI. (C: bottom right) Electron micrograph of negatively stained geminate virus panicles purified from agroinfected wheat tissue. The bar represents 50 nm.

40 seen in the n a t u r a l l y infected tissue o b t a i n e d f r o m C z e c h o s l o v a k i a (Fig. 1, lanes 3 a n d 4). N o h y b r i d i s a t i o n o f the p r o b e was o b s e r v e d in n o n - i n o c u l a t e d p l a n t s (Fig. 1, l a n e 2) o r in p l a n t s i n o c u l a t e d with either C58 c a r r y i n g n o viral D N A , o r with E. coli o r Agrobacterium tumefaciens A 6 c o n t a i n i n g the dim e r i c c o n s t r u c t s o f W D V in pBin 19 ( d a t a n o t shown). G e m i n a t e virus particles c o u l d be extracted f r o m t h e W D V infected tissue (Fig. 2C), a n d h y b r i d i s a t i o n analysis o f t h e s s D N A extracted f r o m these p u rified virions c o n f i r m e d the viral ( + ) sense to be the s a m e as p r e v i o u s l y r e p o r t e d [23] (Fig. 3). We have c o m p a r e d t h e n u c l e o t i d e sequence o f o u r infectious clone o f the W D V - C J I genome, as d e t e r m i n e d f r o m p J I T 34 a n d p J I T 43, with the sequence o f the Swedish isolate o f W D V (WDV-S [23]), a n d the differences b e t w e e n the two sequences are s u m m a r i s e d in Table 1 a n d illustrated in Fig. 4. I n all there are 47 b a s e c h a n g e s b e t w e e n t h e isolates, with W D V - C J I

Fig. 3. Dot-blot of DNA extracted from virus purified out of agroinfected plants. The bound DNA was hybridised with singlestranded probes complementary to the published (+) sense (Spot 4) and to the ( - ) sense (Spot 2) WDV DNA. Spots 1 and 3 were control spots of WDV dsDNA. Row B is a replicate of row A.

H y b r i d i s a t i o n analysis o f s o u t h e r n - b l o t t e d t o t a l D N A extracted f r o m tissue showing W D V s y m p t o m s c o n f i r m e d t h e presence o f W D V D N A f o r m s in these p l a n t s w h i c h c o m i g r a t e d exactly with, a n d were in t h e s a m e s t o i c h i o m e t r i c p r o p o r t i o n as t h o s e

T GT

CT C

G A

c A

A G ~?

"

~/

T

WDV-CJZ 2750 bp

% A

C +

Fig. 4. Physical map of the genome organisation of WDV-CJI showing the positions at which the nucleotide sequence varies with that of WDV-S. Potential protein coding regions which could give a product with a M r greater that 10000 are shown for both (+) and ( - ) strands of the DNA.

41 being one nucleotide longer than WDV-S. This gives an overall nucleotide sequence homology o f 98.3 %. The WDV-S open reading frames (ORFs) [23] are conserved in WDV-CJI (Fig. 4). In the majority of cases the nucleotide changes which occur in the viral ORFs cause little or no change to the potential amino-acid sequence (Table 1). An exception comes in the case of viral ORF 5. A cluster o f base changes in the middle of this ORF cause a large number o f amino acid changes in the potential product. The stable hairpin structures reported for WDV-S [23], and which includes the major form c o m m o n to all the gemini-viruses for which sequence data are available [34] are conserved in WDV-CJI. The overlapping ( - ) sense ORFs, a feature common to the monoparite gemini-viruses are also found in WDV-CJI (Fig. 4), and the second of these ORFs (ORF 4) does not have an AUG codon at its N-terminus.

Discussion

The use o f Agrobacterium tumefaciens mediated delivery of cloned viral DNA to plant cells has allowed us to determine the infectivity o f a sequenced clone o f the WDV genome. "Agroinfected" plants develop symptoms typical o f WDV infection, and subsequent analysis clearly demonstrates the presence of dsDNA and ssDNA forms identical to those found in naturally infected tissue. Additionally, we can detect geminate virus particles in "agroinfected" plants. Thus we are confident in stating that the cloned WDV DNA used for these infectivity tests represents the full WDV genome. The only aspect o f the viral infection not tested is that o f insect transmission from "agroinfected" plants. There remains a possibility that a virus-coded function influencing insect transmission of the virus resides on another, uncharacterised, DNA component, in which case we would not have defined the full genome. However, we are prohibited from attempting such transmission tests and so cannot resolve this question. Due to the lack of evidence for such a DNA component despite exhaustive sequence analysis o f the DNA forms found in infected tissue [23, 9, 28, 20, 21] we consider this possibility to be very slight.

Previous work has demonstrated the infectivity of cloned MSV DNA by agroinfection [16]. However some clarification is needed as to the exact derivation o f the viral DNA used in these experiments. The nucleotide sequence o f MSV-N [28] represents a compilation of sequence data derived both from subcloned fragments o f the viral dsDNA, and directly from dsDNA synthesised in vitro from a viral ssDNA template. As such the sequence presented was that of a population of molecules, and the resultant heterogeneity was clearly indicated [28]. The MSV clone used for the agroinfection experiments pMSV12 [16] was a fortuitous dimer o f MSV derived directly from viral dsDNA [J. W. Davies, personal communication]. The authenticity of pMSV12 was determined by restriction mapping, but the complete nucleotide sequence has not been determined. Therefore, the relationship between the published "consensus" sequence and this infectious clone cannot be stated definitively. Little is yet known about the role of the T-DNA transfer process in the initiation o f viral infection, although in some cases the involvement of the Vir region of the Ti-plasmid can be demonstrated [18]. Our observation that 30% o f the inoculated plants subsequently become infected possibly reflects the efficiency with which Agrobacterium interacts with the plant cells. It is apparent that several Agrobacterium tumefaciens strains are capable o f at least a limited interaction with monocotyledonous ceils [19, 14, 15, 16]. The "agroinfection" of WDV now provides a sensitive marker for the studying the interaction of Agrobacterium tumefaciens and wheat cells, due to the amplification of the viral DNA and the production of symptoms and virus particles. It remains to be determined whether, as in the case o f cauliflower mosaic virus, the usual host range o f the virus can be extended by agroinfection [15]. The site o f inoculation may also influence the efficiency o f initiation of infection. Intercellular spread o f the leaf hopper-transmitted geminiviruses may be an inefficient process for the establishment of a systemic viral infection, with the important event being the infection o f actively dividing cells in the region o f the meristem. Our choice of the basal 1 cm of the seedling is based on the observation that the apical meristem is located in this region.

42 Evidence is emerging that the second D N A component o f CLV codes for factors involved in cell-tocell spread of this virus [36] and thus it might be argued that the mechanical infectivity o f this virus is consistent with the presence of a second genome component. Likewise the lack of mechanical infectivity o f WDV and MSV may be attributable to a lack of a second D N A component. The strength of this argument is reduced by consideration o f BCTV which is mechanically transmissible [32, 29] and yet has only a single D N A c o m p o n e n t [35]. However, successful mechanical inoculation o f BCTV requires needle puncture o f the D N A to the meristematic region of the plant, suggesting that cell-to-cell spread may be compromised in this virus [29, 35]. The nucleotide sequence o f a clone of WDV has previously been presented [23]. However, in the absence o f any infectivity tests, it could not been shown that the clone o f WDV-S D N A was a complete, infectious copy o f the viral genome. The nucleotide sequence of WDV-CJI, when compared to that o f WDV-S, shows a very high degree o f similarity (98.3070) despite their geographical separation. Sequence conservation may be a feature c o m m o n to other geminiviruses. The two isolates of MSV thus far sequenced, MSV-N [28] and MSV-K [20, 21] show a homology o f 98°70, whereas DSV which has a host range which can include maize [10] shows only 64070 homology with either o f the MSV sequences and thus is considered a distinct virus rather than another isolate o f MSV [9]. In c o m m o n with WDV-S, a translation of the nucleotide sequence of WDV-CJI shows the existence o f two potential open reading frames in the ( + ) sense (MR 10146 and MR 29407) and three in the ( - ) sense ( M R 30156, M R 17292, M R 14556). It should be noted that the M R 17292 open reading frame does not have an AUG codon at its 5' end but has been considered as a viral O R F by analogy with MSV in which there is a O R F of similar size and position on the genome which has an AUG codon. Interestingly, the equivalent O R F in DSV also does not have an AUG codon and therefore it is speculated that either a frameshift or splicing mechanism would have to operate if this viral O R F is to be expressed in either WDV or DSV. The presence of an AUG codon does not, however, preclude a similar mechanism in MSV. Analysis o f the alterations to the potential amino-

acid sequence of the viral ORFs by the nucleotide changes which occur between WDV-CJI and WDV-S leads us to speculate either that the product of O R F 5 (Fig. 4 and Table 1) is very tolerant o f alteration or that this O R F arises fortuitously and does not represent a viral gene. It is also apparent that the nucleotide changes between the two isolates tend to be clustered in two areas. One such cluster occurs between nucleotides 3 and 51, within the large intergenic region, and another occurs between nucleotides 533 and 551. This area lies at the junction o f ORFs 1 and 2. The significance, if any, o f these clusters remains to be determined. In the absence of a side-byside comparison o f the two isolates we cannot comment on the possible phenotypic effects o f any of the changes within the viral ORFs. However, the ability to produce infection with cloned D N A provides a route for the study of mutant viral genomes produced in vitro and the study of marker genes in whole plants, allowing progress towards both the understanding of geminivirus molecular biology and the use of cereal geminiviruses as plant transformation vectors.

Acknowledgements The m a j o r part of this work was funded by the DTI consortium " P l a n t Gene Tool K i t " project. M.I.B. is funded by Agrigenetics Research Associates. Experiments were carred out under M A F F licence number P H F 49/152 and 49A/41. The authors would like to acknowledge the help of their colleagues at the John Innes Institute, particularly Marion Pinner for the electron microscopy of purified virus, and Jonathan Donson for critical discussion o f the manuscript, and also Dr Vacke (Ruzyne, Czechoslovakia) for providing WDV-infecting tissue.

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Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA.

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular doubl...
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