Coordinate Regulation of Replication and Virion Sense Gene ...Replication and Gene Expression in WDV 215 inoculation. Figure 2 shows a DNA gel blot analysis (Southern, 1975) of DNA

The Plant Cell, Vol. 4, 213-223, February 1992 O 1992 American Society of Plant Physiologists

Coordinate Regulation of Replication and Virion Sense Gene Expression in Wheat Dwarf Virus

Jul ie M. 1. Hoferya9' Elise L. Dekker,b Helen V. Reynolds,' Crispin J. WoolstonIai2 Brian S. CoxYb and Phil ip M. Mull ineauxa

a John lnnes Institute, John lnnes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, United Kingdom Department of Plant Sciences, University of Oxford, Oxford 0x1 3RA, United Kingdom

We have investigated the relationship between viral DNA replication and virion sense gene expression in wheat dwarf virus (WDV), a member of the geminivirus group, by testing a series of deletion mutants in transfected Triticum monococcum (einkorn) protoplasts. Mutants contained a transcription fusion of the chloramphenicol acetyltransferase coding sequence to the virion sense promoter that replaced the viral coat protein coding sequence. The deletion analysis revealed that WDV replication and virion sense transcription can proceed independently and are controlled in part by nonoverlapping elements in the large intergenic region. These data and those from a C2 open reading frame (ORF) frameshift mutant also showed that the product of the C2 ORF (C1-C2 protein) is independently involved in both DNA replication and activation of the virion sense promoter. The amino acid sequences encoded by C2, which are highly con- sewed in the geminivirus group, show some homology to the DNA binding domain of the myb-related class of plant transcription factors. The possible involvement of the host in controlling the function of the C142 protein and the impli- cation of these data for the development of WDV-based gene vectors are discussed.

INTRODUCTION

The geminiviruses are plant DNA viruses and those that infect monocotyledonous (monocot) hosts are particularly good candidates for the study of interactions between transcription and DNA replication in plants (Accotto et al., 1989; Mullineaux et al., 1990). Wheat dwarf virus (WDV) is a member of the subgroup of monocot-infecting geminiviruses that includes maize streak virus (Howell, 1984; Mullineaux et al., 1984), Digitaria streak virus (DSV; Donson et al., 1987), and chloris striate mosaic virus (Anderson et al., 1988). The genome of WDV is monopartite and consists of a circular 2.7-kb molecule of single-stranded DNA, which replicates in infected tissue as double-stranded DNA (dsDNA) (MacDowell et al., 1985; Woolston et al., 1988). Sequence analysis reveals the presence of four open reading frames (ORFs) (Vl, V2, C1, and C2) common to all monocot-infecting geminiviruses, and mutagenesis studies have been used to determine their functions. V1 may be involved in viral spread and symptom development (Mullineaux et al., 1988; Boulton et al., 1989; Lazarowitz et al., 1989), and V2 is the capsid protein (Morris-Krsinich et al., 1985). Neither of these ORFs is required for dsDNA replication in protoplasts derived from Triti- cum monococcum (einkorn) cell suspension cultures (Woolston et al., 1989; Matzeit et al., 1991); however, both

To whom correspondence should be addressed. Current address: Department of Applied Biology, University of

Hull, Hull HU6 7RX, United Kingdom.

complementary sense ORFs, C1 and C2, are essential (Schalk et al., 1989; Ugaki et al., 1991). The large intergenic region (LIR; Davies et al., 1987) of the viral genome is located between the 5'ends of ORFs V1 and C1. A highly conserved nanonucleotide sequence within the LIR comprises part of the head of a potential stem-loop structure and is thought to be the origin of replication (ofi; MacDowell et al., 1985; Revington et al., 1989). This motif resembles the ori of the single-stranded DNA bacteriophage (pX174 (Shlomai and Kornberg, 1980) and is almost identical to a site required for the initiation of DNA synthesis in the human adenoviruses (Rawlins et al., 1984).

WDV transcription proceeds bidirectionally from within the LIR (Dekker et al., 1991). A single, abundant, virion sense transcript directs the synthesis of the V1 and V2 ORF products. As in DSV, two complementary sense transcripts, one spliced and one unspliced, appear to direct the synthesis of a 41-kD splice-mediated fusion of the C1 and C2 ORFs (C1-C2 protein) and a 30-kD C1 product, respectively (Accotto et al., 1989; Mullineaux et al., 1990; Dekker et al., 1991). Neither product has yet been detected in vivo. Whereas the C1-C2 ORF product is essential for the replication of WDV dsDNA in einkorn protoplasts (Shalk et al., 1989; Matzeit et al., 1991), a role for the putative unspliced complementary sense transcript product, C1, has not yet been defined. The subgroup of geminiviruses that infects dicotyledonous (dicot) plants encodes an AC1 (or AL1) ORF product that is equivalent to the

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214 The Plant Cell

WDV C1-C2 protein (Hanley-Bowdoin et al., 1990; Etessami et al., 1991). All other factors required for geminivirus replication and transcription are probably of cellular origin. This de- pendente on host proteins makes the geminiviruses an excellent model system for investigating replication and its relation to gene expression in plants (Accotto et al., 1989). In addition, the participation of cellular factors in the regulation of gene expression should be addressed if geminiviruses are to be developed further as plant transformation vectors.

We used a protoplast system to investigate the organization of control elements in WDV, and our results revealed that independent sequences in the LIR are required for replication and virion sense promoter activity. We have shown that the Cl-C2 protein is also essential for both processes and can contribute to each independently. We consider the possible involvement of the host cell in regulating transcription and replication and whether the C1-C2 protein might be a transcription factor.

RESULTS

Standard Constructs

We used three standard WDV constructs, shown in Figure l A , to study the relationship between WDV virion sense promoter activity and vira1 dsDNA replication. pWDV2CAT contains the chloramphenicol acetyltransferase (CAT) coding sequence (Alton and Vapnek, 1979) cloned between the BstEll (nucleotide 500) and Mlul (nucleotide 1246) sites of WDV, which replaces the WDV V2 ORF in a transcription fusion to the virion sense promoter (for details of construction, see Meth- ods). Smaller, replicating WDVCAT molecules (a possible replicon form is shown in Figure 16) are generated from pWDV2CAT in transfected protoplasts, probably as a result of replication (Woolston et al., 1989).

pWDV3CAT (Figure 1A) is replication defective due to frameshift mutations in both C2 ORFs, which would result in prematurely terminated translation of the C1-C2 ORF fusion product. In every other respect, pWDV3CAT is identical to pW DV2CAT.

A second replication defective construct, pWDV3.35S- CATNOS (Figure lA), contains the same C2 ORF frameshift mutations; however, the CAT coding sequence is under the control of a cauliflower mosaic virus (CaMV) 35s promoter and a nopaline synthase polyadenylation sequence. The 35SCATNOS fragment was inserted in an antisense orientation with respect to the WDV virion sense promoter to ensure that the CaMV 35s promoter was positioned as far as possible from any regulatory sequences in the LIR.

Time-Course Experiments

pWDV2 7.2kb

Figure 1. pWDVCAT Vectors.

(A) pWDV2 is a 1.4-mer tandem repeat of WDV inserted into pBluescript KS+ (Alting-Mees and Short, 1989). Vira1 ORFs VI, C1, and C2 are marked. A polylinker has been introduced 113 bp 3'to the BstEll site (nucleotide 500) of WDV, and a unique BamHl site has been inserted 7 bp 5' to the BstEll site to facilitate the introduction of reporter genes. UTT is a universal translation termination oligonucleotide (Mullineaux et al., 1988) inserted to prevent translation read through from ORF VI. Deletion mutations removed sequences from ORF V1 and the LIR (indicated by a doubleheaded arrow). The complementary sense TATA box is marked with an open triangle; the virion sense TATA box is marked with afilled triangle. The CAT coding sequence (Alton and Vapnek, 1979) was inserted into the EstEll and Bglll sites of pWDV2 to generate pWDV2CAT. Ndel sites (') were filled in and self-ligated to generate pWDV3CAT, a replication- defective vector containing frameshift mutations in both C2 ORFs. pWDV3.35SCATNOS contains the CaMV 35s promoter, the CAT coding sequence, and the NOS polyadenylation sequence inserted into the Xhol site of the polylinker in an antisense orientation with respect to the WDV virion sense promoter. (B) Predicted form of a WDV2CAT replicon generated from pWDV2CAT DNA in transfected einkorn protoplasts.

Einkorn protoplasts transfected with pWDV2CAT and pWDV3CAT were sampled at 24-hr intervals for 6 days after

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Replication and Gene Expression in WDV 215

inoculation. Figure 2 shows a DNA gel blot analysis (Southern,1975) of DNA extracted from protoplasts inoculated withpWDV2CAT (lanes b to g) that revealed persistent open-circular (oc), supercoiled (sc), and linear forms of plasmidDNA and additional smaller DNA species, which were the scand oc forms of the WDV2CAT replicon (Figure 1B). This as-signment of DNA forms was based on the analysis outlinedbelow and on previous experiments (Woolston et al., 1989).The smaller DNA forms were not detectable in DNA extractedfrom pWDVSCAT-transfected protoplasts (Figure 2, lanes h tom). Digestion of total DNA with BamHI converted plasmidand WDV replicon DNAs to linear forms of the predictedsizes, 7.2 kb and 3.2 kb, respectively, and DNA gel blot analy-sis using a CAT-specific DNA probe showed the reportergene to be present in both DNA species (J.M.I. Hofer and P.M.Mullineaux, unpublished data). Furthermore, the smallerforms of DNA (oc and sc) were susceptible to digestion with

Day Day

1 2 3 4 5 6 1 2 3 4 5 6

InputDNA

oc*

sc'

l!wt oc

wt sc

I wt ss

:.r

Uia b c d e f g h i j k l m n

Figure 2. Replication of WDVCAT Constructs in Einkorn Protoplasts.An autoradiograph is shown of a DNA gel blot of DNA extracted fromtransfected protoplasts that were separated on a 1% agarose gel andprobed using a full-length Ncol monomer of WDV-specific DNA. Lanea contains 2 ng of input pWDV2CAT DNA. Lanes b to g were eachloaded with 5 ng of DNA extracted from pWDV2CAT-transfected pro-toplasts on days 1 to 6 after inoculation. Lanes h to m were eachloaded with 5 ng of DNA extracted from pWDV3CAT-transfected pro-toplasts on days 1 to 6 after inoculation. Mbol-sensitive (*), oc, andsc forms of double-stranded replicon DNA were generated inpWDV2CAT-transfected protoplasts but were absent in pWDVSCAT-transfected protoplasts. Lane n contains DNA extracted from wild-type (wt) WDV-infected wheat seedlings. Single-stranded (ss) viralDNA was not routinely detected in transfected protoplasts.

Mbol, whereas the plasmid DNA was not (J.M.I. Hofer andP.M. Mullineaux, unpublished data). This suggested that theWDV replicon molecules had an altered pattern of DNA meth-ylation, which rendered them sensitive to this enzyme, andwere therefore likely to have been synthesized de novo(Woolston et al., 1989). The replicative DNA forms observedin protoplasts transfected with pWDV2CAT were identical tothose observed in wild-type WDV-transfected protoplastsreported previously (Woolston et al., 1989).

These time course experiments confirmed that an intact C2ORF was required for the generation of replicative WDVdsDNA forms. They also demonstrated that the ability of aninoculated plasmid to give rise to replicating WDV monomersmay be assayed by the presence or absence of the smallMbol-sensitive dsDNA forms identified in Figure 2.

Half of each sample harvested for DNA extraction was re-tained and assayed for CAT activity as shown in Figure 3. Inour expression system, a transcriptionally fused CAT genewas used to measure the activity of the WDV virion sense pro-moter. In protoplasts transfected with pWDV3.35SCATNOS(Figure 3A), maximum CAT activity was observed 24 hr afterinoculation and was maintained throughout the experiment.A similar CaMV 35S promoter activity profile has beendemonstrated in maize protoplasts after uptake of a plasmidcontaining the CAT reporter gene (Maas et al., 1990). Tran-sient gene expression in protoplasts is a well-establishedtechnique (Fromm et al., 1985; Werr and Lorz, 1986; Prols etal., 1988), and our results for construct pWDV3.35SCATNOSwere typical, showing that input plasmid DNA was capableof driving the expression of the CAT reporter gene.

Virion sense promoter activity measured in protoplaststransfected with pWDV2CAT showed a different profile (Fig-ure 3B). Promoter activity was initially low, then increased,concomitant with an increase in the number of molecules ofreplicating dsDNA (Figure 2, lanes b to g). Similar promoteractivity profiles were obtained in protoplasts transfected witha WDV dimer construct, pWDV1-59 (see Methods; data notshown), and a pWDVCAT construct reported previously(Matzeit et al., 1991).

In complete contrast to the results above, no CAT activitywas measured in protoplasts transfected with pWDVSCAT(Figure 3C). This indicated that the virion sense promoter wasnot active on input DNA in pWDVSCAT-transfected pro-toplasts and that either actively replicating DNA or the ex-pression of the C2 ORF was necessary for activation of thevirion sense promoter.

Deletion Mutagenesis of the LIR

Figure 4 shows a series of deletion mutants derived frompWDV2CAT that were constructed to determine the relation-ship between the virion sense promoter and DNA replication.In these experiments, transfected protoplasts were harvestedand assayed 4 days after inoculation when levels of virionsense promoter activity and viral replication were approach-ing a maximum (Figures 2 and 3).

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% CATA activity 70 90 70 90 90 100

3-CM

1-CM

CM

day 1 2 3 4 5 6%CAT

activity 10 30 ?o 70 90 100B

3-CM

1-CM

CM

G

day 1 2 3 4 5 6

CM

day 1 2 3 4 5 6Figure 3. CAT Activity in Einkorn Protoplasts Transfected withWDVCAT Constructs.(A) Protoplasts transfected with pWDV3.35SCATNOS. One hundredpercent is equivalent to 0.25 nmol of chloramphenicol converted to3-CM per 106 protoplasts per hour.(B) Protoplasts transfected with pWDV2CAT. One hundred percent isequivalent to 0.30 nmol of chloramphenicol converted to 3-CM per106 protoplasts per hour.(C) Protoplasts transfected with pWDV3CAT. A1% background of CATactivity was detected.

A preliminary analysis determined that deletion of the V1ORF between nucleotides 299 and 500 affected neitherdsDNA replication nor virion sense gene expression (J.M.I.Hofer, H.V. Reynolds, and P.M. Mullineaux, unpublisheddata), which is consistent with results obtained for WDV V1deletion mutants reported elsewhere (Laufs et al., 1990;Ugaki et al., 1991). Deletions that removed sequences be-tween nucleotides 2746 and 500 (pSSB430 and pABP514;Figure 4A) resulted in a loss of CAT activity and a loss of themutants' ability to replicate. A deletion between nucleotides2683 and 39 (pECA101; Figure 4A) resulted in the samephenotype. Therefore, elements located between nucleotides2683 and 299 were essential for both replication and virionsense gene expression.

A deletion that removed sequences between nucleotides2750 and 39 (pAPA38; Figure 4A) did not affect either virionsense promoter activity or dsDNA replication deleteriously.This small segment of the LIR was therefore not required foreither activity (Figure 48).

Another group of mutants that were deleted betweennucleotides 142 and 500 (pAVB365, pBSB347, and pALB288;Figure 4A) gave rise to WDV replicons, yet CAT activity wasvery low. This indicated that elements between nucleotides142 and 500 in the LIR were dispensable for WDV dsDNAreplication and that dsDNA replication could proceed in theabsence of virion sense promoter activity (Figure 4B).

Protoplasts transfected with a mutant deleted betweennucleotides 2746 and 81 (pAPS84; Figure 4A) yielded a fourthphenotype. A relatively high level of virion sense promoter ac-tivity was maintained, although no dsDNA replicons could bedetected by DNA gel blot analysis (Southern, 1975). Thisdemonstrated that virion sense promoter activity and dsDNAreplication in WDV were independent events, and this mutantdelineated a region between nucleotides 2746 and 81 thatwas necessary for dsDNA replication (Figure 4B). A sum-mary of the domains in the WDV LIR defined by this seriesof deletion mutants shows that although replication and virionsense promoter functions could be separated, some se-quences were also shared (Figure 4B).

We investigated further the activity of the virion sense pro-moter in replication defective WDV constructs. The profileof promoter activity of mutant pAPS84, which increasedwith time in a similar manner to the profile of pWDV2CAT, isshown in Figure 5. Introduction of a frameshift mutation inthe C2 ORF of pAPS84 to generate a double mutant,pAPS84.WDV3CAT (Figure 4A), abolished CAT activity and

Transfected protoplasts were harvested on days 1 to 6 after inocula-tion and assayed for CAT activity. Typical autoradiographs of thedifferent acetylated forms of chloramphenicol (CM), 3-acetyl-chloramphenicol (3-CM), and 1-acetyl-chloramphenicol (1-CM) thatwere separated by thin-layer chromatography are shown. Chloram-phenicol acetylation was quantified by scanning densitometry andexpressed relative to the amount of 3-acetyl-chloramphenicol mea-sured on day 6 (100%).

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t LIR CAT Acthrlty

Relative t o WDV2CAT Repllcatlon

1 CQ

1 I CAT > I CAT >

pWDV3CAT

pAPS84.WDV3CAT

< 2%

< 2%

c 2%

< 2%

< 2%

66% (SE 12%)

45% (SE 11%) < 2%

< 2%

< 2%

J J J X

X

J X

X

X

X

Figure 4. WDV Deletion Mutants.

(A) The WDV V2 ORF was replaced with the CAT coding sequence to generate transcription fusions to the virion sense promoter. Deletion mutations were made in the LIR, a noncoding region of the WDV genome extending between the V I and C1 ORFs. CAT activity was determined for each deletion mutant from an extract prepared from transfected einkorn protoplasts harvested 4 days after inoculation. The values given are averages obtained from at least eight individual transfection experiments. CAT activity measurements were included with the provision that a positive result was obtained from pWDV2CAT-transfected protoplasts in the same experiment. One hundred percent is equivalent to 0.30 nmol of chloramphenicol converted to 3-acetyl-chloramphenicol per 106 protoplasts per hour (SE, standard error). Replication was scored according to detection of WDV replicon DNA (refer to Figure 2). (8) A summary representation of functional regions in the LIR where dotted rectangles delineate the regions required for dsDNA replication and cross-hatched rectangles delineate the regions required for virion sense gene expression defined in this study.

confirmed that the C2 ORF was required for virion sense promoter activity, independent of the ability to produce replicons.

DlSCUSSlON

pWDV2CAT is a WDVvector designed to measure WDV virion sense gene expression from a transcriptional fusion of the virion sense promoter to the CAT reporter gene and was constructed such that tandemly repeated sequences enable the release of replicative dsDNA monomers in einkorn protoplasts. The replication defective construct, pWDV3CAT, showed that an intact C2 ORF, expressed as a splice- mediated fusion to the C1 ORF (Schalk et al., 1989; Dekker et al., 1991), is essential for WDV dsDNA replication in

einkorn protoplasts. Our deletion analysis enabled us to iden- tify two regions within the LIR that are also required for dsDNA replication (Figure 48). The first region, defined by mutants PECA101 and pAPA38 (nucleotides 2689 to 2746), contains both mapped complementary sense transcript start sites and the predicted complementary sense TATA box (nucleotide 2720; Dekker et al., 1991); therefore, failure of mutant PECA101 to replicate was probably due to disruption of the expression of the C1C2 transcription unit. The second essential region, delineated by mutants pAPA38, pAPS84, pSSB430, and pAVB365 (nucleotides 34 to 142; Figure 4B), is probably not directly involved in the expression of complementary sense ORF products because pAPA38 was com- petent in both replication and virion sense transcription, yet pAPS84, with a slightly larger deletion extending away from the complementary sense ORFs, failed to generate replicons.

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218 The Plant Cell

1 O0 90 80

.- a 70 5 60 I- 50 8 40 ' 30

20 10

> cd

.-

1 2 3 4 5 6 Days after inoculation

Figure 5. CAT Activity in Einkorn Protoplasts Transfected with Dele- tion Mutant pAPS84.

Typical time comes of CAT activity are shown for protoplasts transfected with mutant pAPS84 (dashed line) compared to protoplasts transfected with pWDV2CAT (solid lhe) and pAPS84.WDV3CAT (dotted line). One hundred percent represents 0.30 nmol of chloramphenicol converted to 3-acetyl-cloramphenicol per 106 protoplasts per hour.

The second region includes the conserved geminivirus nanonucleotide sequence (nucleotides 77 to 85) and pro- vides further evidence that this motif is part of the geminivirus core ori, either as a DNA nicking site (Rogers et al., 1986; Saunders et al., 1991) or as a recognition site for the binding of a replication complex (Huberman, 1987; Challberg and Kelly, 1989).

Two regions of the WDV LIR are required for virion sense promoter activity (Figure 46). The first region was delineated by mutants PECA101 and pAPA38 and includes the complementary sense TATA box (Dekker et al., 1991). As previously described, the virion sense promoter is activated by the C1-C2 protein and, therefore, is indirectly dependent on an active, complementary sense promoter. The second domain (nucleotides 81 to 299; Figure 46) contains the virion sense transcription start sites and sequences 105 bp upstream, in- cluding the putative virion sense TATA box at nucleotide 225 (Dekker et al., 1991). Mutants pAPS84, pAV6365, and our preliminary V1 ORF deletions defined this region. Other parts of the LIR may also contribute to virion sense promoter function in the form of enhancer or activator sequences. The LIR of

maize streak virus contains GC-rich elements, homologous to the Spl binding site of mammalian promoters, which activate virion sense transcription in maize protoplasts (Fenoll et al., 1988,1990). Similar GC boxes may be present in the WDV LIR. The 84-bp deletion of mutant pAPS84 removed two GC-rich stretches 5'to the geminivirus nanonucleotide motif, and their absence may account for a reduction in virion sense promoter activity observed. Other enhancer elements also may be located upstream of the virion sense TATA box. Partic- ularly striking are repeated polypyrimidine tracts (CTTTT) that may affect DNA curvature (Travers, 1989) and CCAAT box motifs (Jones et al., 1985, 1987; Cohen et al., 1986) at nucleotides 2699 and 2708, which are present as imperfect palindromes in a number of other geminiviruses.

A correlation between replication and virion sense promoter activity was indicated by concomitant increases in CAT activity and replicon dsDNA forms in protoplasts transfected with pWDV2CAT. Constructs pWDV3CAT and PECA101 revealed that the C1-C2 protein was necessary for virion sense promoter activity, whereas mutant pAPS84 indicated that actively replicating DNA monomers were not required. Our results suggest that the relationship between replication and virion sense transcription in WDV is mediated by the C1-C2 ORF fusion product, which activates the virion sense promoter in conjunction with, but separate from, its role in replication (Schalk et al., 1989). The AC2 (AL2) ORF of a bipartite, dicot-infecting geminivirus, tomato golden mosaic virus, has been shown to trans-activate the virion sense promoter of genome component A and may also trans-activate the virion sense promoter of genome component 6 (Sunter and Bisaro, 1991). None of the monocot-infecting geminiviruses sequenced to date contains an ORF equivalent to AC2. In WDV, the C1-C2 ORF product therefore appears to be a multifunctional protein, equivalent to both the AC1 and AC2 proteins of dicot-infecting geminiviruses. In many ways, the activity of the C1-C2 protein is analogous to the large T-antigen of sim- ian virus 40, which is another multifunctional protein involved in the initiation of DNA replication and activation of late promoter activity (Prives, 1990).

It is clear from our data that all virion sense fusions in WDV require an activating component. In previous studies in which WDV-based replicons have been used for the development of plant vectors (Matzeit et al., 1991; Ugaki et al., 1991), the contribution of replication to the level of reporter gene expression has been estimated by comparison with replication defective mutants that have disrupted expression of their complementary sense ORFs. This comparison may have given an overestimate of the contribution of vector replication to the level of reporter gene expression because in all cases the reporter gene was fused to the virion sense promoter. We note that deletion mutant pAPS84, which has part of the putative geminivirus ori deleted but retains an intact complementary sense transcription unit, gave levels of CAT activity approximately half that of pWDV2CAT (Figures 4A and 5). Therefore, under our experimental conditions, the contribution of replication to the total level of CAT activity was no more than twofold.

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Our data do not reveal whether activation of the virion sense promoter is a direct or an indirect interaction between the C1-C2 protein and viral sequences. Other host factors may be involved in a multicomponent complex, or C1-C2 may regulate host genes, which, in turn, influence viral replication and virion sense transcription. Figure 6 shows a region of the C1-C2 ORF, identified as an NTP-binding domain (Gorbalenya and Koonin, 1989), which contains amino acid residues that are also conserved in the N-terminal DNA binding domain of the avian myeloblastosis (myb) oncogene- homologous regulatory genes C7 (Paz-Ares et al., 1987) and P7 (Grotewold et al., 1991) of maize and Hv7 (Marocco et al., 1989) of barley. Apart from this region of homology, the C1-C2 polypeptide does not show features characteristic of myb-like gene products in plants and animals, for example, repeat domains (Klempnauer and Sippel, 1987) and evenly spaced

Repeat 2 <----------------------------

DDI..PFKFT DDI..PFKFC DDI..PFKFC DDI..PFKFV DDVDPTYLKR DDVTPQYLKL DDISPNYLKL DDVDPHYLKH

PNWKCFVGAQ PCWKQLIGCQ PCWKQLVGCQ PCWKGLVGSQ KHWKHLIGAQ KHWKELIGAQ KHWKELIGAQ ..FKEFNGSQ

Figure 6. Amino Acid Sequence Comparison.

The deduced amino acid sequences from the N-terminal region of myb gene homologs in maize (ZmC7, Paz-Ares et al., 1987; ZmP1, Grotewold et al., 1991) and barley (Hv7, Marocco et al., 1989) were compared to a highly conserved domain in the C1-C2 ORF of four monocot-infecting geminiviruses, WDV, DSV, maize streak virus (MSV), and chloris striate mosaic virus (CSMV) and an equivalent domain in the C1 ORF of genome component A of four dicot-infecting geminiviruses, beet curly top virus (BCTV Stanley et al., 1986), tomato golden mosaic virus (TGMV Hamilton et al., 1984), bean golden mosaic virus (BGMV; Howarth et al., 1985), and African cassava mosaic virus (ACMV; Stanley and Gay, 1983). Conserved amino acids and conservative changes with a threshold pair value of >0.2 (calculated according to the scoring system of Gribskov and Burgess, 1986) are boxed. Consensus phosphorylation sites are marked ('). The two convergent ends of repeat sequences characteristic of plant myb-like genes (Marocco et al., 1989) but not present in the geminivirus sequences are shown at the top of the alignment.

tryptophans (Saikumar et al., 1990). It is interesting to note that proteins encoded by the maize P7 gene arise as a result of alternative splicing at their 3'ends (Grotewold et al., 1991), a process similar to the intron skipping observed in the transcription of the complementary sense ORFs of WDV and DSV (Accotto et al., 1989; Schalk et al., 1989; Mullineaux et al., 1990; Dekker et al., 1991).

The host cell is thought to play a role in the regulation of geminivirus replication (Townsend et al., 1986). Despite the fact that the complementary sense promoter of WDV must be active on input DNA in einkorn protoplasts (to generate viral replicons), the onset of virion sense promoter activity ap- peared to be delayed compared with the CaMV 35s promoter (Figures 3A and 3B), and WDV2CAT replicon DNA was not detected immediately after transfection (Figure 2A). We have shown that the C1-C2 protein plays a pivotal role in viral replication and gene expression; it is, therefore, a likely target for temporal regulation by host cell factors. These factors could regulate complementary sense transcription and the splicing of complementary sense transcripts (Mullineaux et al., 1990; Dekker et al., 1991), and could modify the activity of their translated proteins. The ratios of C l and C1-C2 proteins would be significant, for example, if cooperative binding to viral or host genomes was necessary, such as the heteromeric binding of the fos and jun protooncogene-encoded transcription factors (Kouzarides and Ziff, 1989) or if the proteins func- tioned antagonistically, as do the products of alternatively spliced fosB transcripts (Murnberg et al., 1991).

At least two potential phosphorylation sites are located in the putative NTP-binding domain (Gorbalenya and Koonin, 1989) of the C1-C2 protein and are conserved between all the geminiviruses sequenced to date (Figure 6). RXX$/T is a consensus site for calmodulin-dependent protein kinases (Sommer et al., 1990), RIGK was identified using the computer program Scrutineer (Sibbald and Argos, 1990) and is a consensus site for phosphorylation by protein kinase C. These and other adjacent Ser and Thr residues may be tar- gets for post-translational modification by the host cell. The DNA binding activity of the c-myb-encoded protein is regulated by phosphorylation and may be linked to severa1 host cell signal transduction pathways (Lüscher et al., 1990). Sim- ian virus 40 DNA replication has also been shown to be linked to the host cell cycle by the activity of cellular phosphatases and kinases that regulate the phosphorylation status of the viral T-antigen (McVey et al., 1989; Virshup et al., 1989) and by cyclin (Prelich et al., 1987), a nuclear protein that is encoded by monocot and dicot plant genomes (Suzuka et al., 1989). WDV may interact with the host cell in a similar manner.

METHODS

Molecular Techniques

Plasmids were constructed using standard recombinant DNA proce- dures (Sambrook et al., 1989). Restriction endonucleases and DNA

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220 The Plant Cell

modifying enzymes were obtained from Bethesda Research Labora- tories; radioisotope-labeled chemicals were obtained from Du Pont- New England Nuclear.

Construction of WDVCAT Transcription Fusions

pJIT34 (Woolston et al., 1988) is a 2750-bp monomer of a Czecho- slovakian isolate of WDV inserted into the Kpnl site of pUC18 (Yanisch-Perron et al., 1985). pJIT34 was used as the source of vira1 DNA for pWDV1-59, a WDVCAT dimer. Construction was as follows: (1) The V2 ORF ATG was removed by digesting pJIT34 with Cfrl (nucleotide 486) and BstEll (nucleotide 500), rendering the ends blunt with T4 DNA polymerase, and subsequently ligating the ends in the presence of 100 M excess of BamHl linker (5’CGGGATCCCG 33. The resulting plasmid, pJIT34A4, contained Saml, BamHI, and BstEll sites at the deletion junction. This manipulation changed two predicted amino acid residues of the V1 ORF. (2) The 772-bp Taql fragment containing the Tn9 CAT coding sequence (Alton and Vapnek, 1979) from pBR328 was inserted into the Sphl and Kpnl sites of pUC19 after the ends of the DNA fragments were made flush. This plasmid was called “pJIT24.” (3) The CAT coding sequence was released from pJIT24 as a Hindlll-Sacl fragment, the ends were made flush, and the fragment was inserted into two Alul sites (nucleotides 539 and 630) of pJIT34A4 in a three-step ligation. (4) The resulting plasmid, pJIT34A4CAT, was digested with Kpnl to release a WDVCAT monomer, which was self-ligated and inserted as a tandem dimer into the Kpnl site of pUC18 to generate pWDV1-59.

The source of WDV DNA for constructs pWDV2 and pWDV2CAT (Figure 1) was a WDV dimer equivalent of pJIT34A4, called “pJIT34A4D.” pWDV2 was generated as follows: (1) A Hindll monomer was released from pJIT34A4D and inserted into the Hindlll site of pBluescript KS+ (Alting-Mees and Short, 1989) to create pJIT34BS1, A 1.4-kb Mlul (end-filled, nucleotide 1246)-Ncol (nucleotide 2591) fragment was released from pJIT34A4D and inserted into pJIT34BSl at the Ncol (nucleotide 2591) and EcoRV (polylinker) sites to create pJIT34BS1.4. (2) The V2 ORF was deleted by digesting with Mlul (nucleotide 1246) and BstEll (nucleotide 500), filling the sites, and recircularizing the plasmid (BstEll site restored, pJIT34BS1.4AV2). (3) Restriction sites in the pBluescript KS+ polylinker were removed in two steps by digestion with Asp718 and Clal, making the ends flush, and self-ligating (Kpnl restored), then repeating the procedure using the Pstl and Xbal sites to create pJIT34BS1.4AV2Amcs. (4) A 619-bp BamHI-Rsal fragment from pJIT34A4 was ligated to a 21-mer oligonucleotide containing Bglll, Xhol, EcoRV, and Sphl sites. The resulting 640-bp fragment was inserted into the BamHl and EcoRV sites of pBluescript SK+ to create pBS2. (5) pBS2 was digested with Sall, end filled, and subsequently digested with BamHl to release a 663-bp fragment that was inserted into the BamHl and BstEll (filled- in) sites of pJIT34BS1.4AV2Amcs to create pWDVl. (6) To prevent in- terference with translation of inserted sequences fused to the V1 ORF, an oligonucleotide with TAA non-sense codons in all three ORFs (Mullineaux et al., 1988) was inserted at the Smal site. Transla- tion of the VI ORF would be truncated by nine codons. This plasmid was called “pWDV2.” pWDV2CAT was generated by recovering a 1.0-kb BstEll-BamHI fragment containing the CAT coding sequence from pWDV1-59 and inserting it into the BstEll and Bglll sites of pWDV2 (Figure 1).

pWDV3CAT contains a frameshift mutation in both C2 ORFs obtained by destroying two Ndel sites in the following way: (1) A 2.1-kb Sacll-EcoRVfragment was released from pWDV2CAT and ligated into

pBluescript SK+, and digested with the same restriction enzymes. (2) The resulting plasmid, containing only one copy of the C2 ORF, was digested with Ndel, blunt ended, and recircularized. (3) The mociified Sacll-EcoRV fragment was reinserted into the same sites of pWDVPCAT, replacing one of the wild-type C2 ORFs. (4) The remaining Ndel site was removed by linearizing the plasmid with Ndel, making the ends flush and recircularizing to obtain pWDV3CAT.

pWDV3.35SCATNOS was constructed by releasing a 2.3-kb Sall- Xhol fragment from p35SCATNOS and inserting it into the Xhol site in the polylinker of pWDV3CAT in an antisense orientation with respect to the WDV virion sense promoter. p35SCATNOS is a pUC- based vector containing three fragments: 4.0 kb of the NOS 3’polyadenylation sequence from the Sphl site to the Hindlll site (Depicker et al., 1982); the CAT coding sequence, inserted as a Stul-Sacl fragment from pJIT25 (equivalent to pJIT24, with the CAT coding sequence in the opposite orientation); and the CaMV 35s promoter, released as a BamHl fragment from pJIT112. pJIT112 is derived from pJIT58 (Guerineau et al., 1990) and contains the 355 promoter sequence from CaMV (strain Cabb B-JI), corresponding to coordinates 7040 to 7432 of the CaMV Strasbourg strain (Franck et al., 1980).

Deletion Mutants

Deletion mutants were named according to the first two letters of the 5‘restriction site, followed by the first letter of the 3’restriction site and the size of the deletion in base pairs. pJIT36 is an M13mp18 vector, containing a BstEll site, which was inserted as an oligonucleotide into the M13 polylinker. pJIT36 was used as a cloning intermediate for the construction of deletion mutants pBSB347, pAVB365, and pAPB514. An 807-bp Sacl-BstEll fragment was released from pJIT34 and inserted into the corresponding sites of pJIT36. Following digestion with BamHl and the appropriate second enzyme (BstXI, Avall, and Apal, respectively), the DNA ends were made flush and self-ligated. The modified segments were transferred to pWDV2 as Ncol (nucleotide 2591)-BstEll (nucleotide 500) fragments into the corresponding unique sites in pWDV2. pAPA38 was created by digesting pWDV1-59 with Apal, self-ligating, and transferring the modified segment into pWDV2 as an NcoCBstEll fragment, as described above. For the construction of mutants pALB288 and pSSB430, Ncol (nucleotide 2591)-AIul (nucleotide 242) and Ncol-Sspl (nucleotide 80) fragments were released from pJIT34 and inserted into the Ncol and BamHl (filled) sites of pWDV2. pAPS84 was constructed by sequentially ligating an 80-bp BamHI-Sspl (nucleotide 426) fragment and a 346-bp Sspl (nucleotides 80 to 426) fragment from pJIT34 into the Sspl and BamHl sites of pBluescript SK+. A 426-bp partially digested BamHI- Sspl fragment was released and inserted into the Apal (nucleotide 2746) and BamHl sites of mutant pAPA38, to create pAPS84. PECA101 was constructed by releasing an EcoRl (nucleotide 2679, filled)-Ncol (nucleotide 2591) fragment from pJIT34 and inserting it into the Ncol and Apl (nucleotide 2746) sites of pWDV2. The CAT coding sequence was introduced into all the deletion mutants as a BstEll-BamHI fragment, released from pWDV1-59 and inserted into the BstEll and Bglll sites of each mutant.

Protoplast Transfection

The einkorn (Tn’ticum monococcum) suspension culture was obtained originally from Dr. H. Lorz, Max Planck lnstitute (Cologne, Ger- many). Liquid cultures (40 mL) were shaken continuously at 120 rpm,

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at 25OC in P10 medium (Kao, 1977). A 5-mL packed volume of cells was transferred to fresh medium every 7 days. Prior to transfection, a 15-mL packed volume of cells was digested overnight in 100 mL of filter-sterilized CPW solution (10 mM CaCI2, 10 mM MgS04, 3 mM 2[N-morpholino]ethanesulfonic acid, 1 mM KN03, 0.2 mM KH2P04, 0.5 M mannitol, 0.1% [w/v] potassium dextran sulphate, pH 5.6), containing 1% (w/v) cellulase RS and 0.Wo (w/v) pectolyase Y-23. Pro- toplasts were filtered through 50-pm meshes and washed in successive 100" volumes of CPW. lntact protoplasts were purified on a 6-mL 0.5 M sucrose cushion in a 0.5-mL volume of CPW after centrifugation at 1089 in a MSE Mistral 2000 swing out rotor (MSE Scientific Instruments, Loughborough, United Kingdom). Fifty micro- grams of CsCl gradient-purified plasmid DNA was added to aliquots of 2 to 5 x 106 protoplasts and gently resuspended in 0.35 mL of SM buffer (0.4 M sucrose, 15 mM MgCI2, O.l*/o [wlv] 2[N-morpholino]ethanesulfonic acid, pH 5.6). A 0.65-mL polyethylene glycol solution was added (40% polyethylene glycol 3350, 0.4 M sucrose, 0.1 M CaN03, pH 9), and the protoplasts were left to incubate at room tem- perature for 15 min. Five successive 2-mL volumes of 0.2 M CaCIZ were added at 5-min intervals. Protoplasts were washed twice in 10 mL of CPW and incubated in the dark at 25OC in 4 mL of Murashige and Skoog (1962) medium (product number 9-300-40, Imperial Labs., Andover, United Kingdom), pH 5.8, supplemented with 0.25 M glu- cose, 0.25 M sorbitol, 90 mM sucrose, 10% (vlv) coconut milk (Sigma), 0.01% (w/v) myo-inositol, 0.05% (w/v) casein enzymatic hydrolysate, 2.5 pM 2,4-dichlorophenoxyacetic acid, 2.8 pM indole acetic acid, 0.9 pM benzylaminopurine, and 0.5 pM zeatin.

DNA Gel Blot Analysis

Transfected protoplasts were ground with fine sand in TLES medium (Woolston et al., 1988) and extracted twice with Tris-saturated phe- nollchloroform (1:l). RNA was precipitated from total nucleic acids after incubation on ice with an equal volume of 4 M LiCI. Purified DNA was extracted with phenol/chloroform, and any remaining RNA was digested with DNase-free RNase at 3PC for 1 hr. DNA was precipitated in ethanol, and 5 pg was loaded in each lane of a 1% agarose gel in TAE buffer (Sambrook et al., 1989). Following electrophoresis at 90 mA for 6 hr, DNA was transferred to a nitrocellulose filter. A full- length WDV monomer, released as a Ncol fragment from pWDVKlOD (Woolston et al., 1989), was used to probe the filter.

CAT Assay

Protoplasts were ground with fine sand in extraction buffer (50 mM Tris-HCI, 50 mM potassium acetate, 1 mM EDTA, 2.5% [v/v] glycerol, 0.5 mM phenylmethyl-sulfonyl fluoride, 3 mM P-mercaptoethanol). Extracts were incubated at 65OC for 15 min prior to the CAT assay (Gorman et al., 1982). Acetyl coenzyme A (3.8 mM) and 14C-chloramphenicol (7.4 KBq [specific activity, 2.2 GBqlmmol]) were used in each reaction, and the products were separated by thin-layer chromatography.

ACKNOWLEDGMENTS

The authors are grateful to Baldeep Kular for skilled technical support, to Dr. Dong Fang Chen for advice on protoplast transfection, and

to @rs. David Baulcombe and Gary Creissen for critical reading of the manuscript. This work was supported in part by a grant-in-aid to the John lnnes lnstitute from the Agriculture and Food Research Council and by the Agriculture and Food Research Council Plant Molecular Biology Programme. J.M.I.H. gratefully acknowledges the support of a John lnnes Foundation Studentship. E.L.D. was supported by a Eu- ropean Molecular Biology Organization (Heidelberg, Germany) fel- lowship, and C.J.W. was supported by the Department of Trade and lndustry Plant Gene To01 Kit Consortium (Laboratory of the Govern- ment Chemist, Middlesex, United Kingdom).

Received October 11, 1991; accepted December 9, 1991.

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