Proc. Nadl. Acad. Sci. USAVol. 88, pp. 6721-6725, August 1991Genetics

Expression of an antisense viral gene in transgenic tobacco confersresistance to the DNA virus tomato golden mosaic virusA. G. DAY*t, E. R. BEJARANO*, K. W. BUCK*, M. BURRELL§, AND C. P. LICHTENSTEIN*¶*Centre for Biotechnology and tDepartment of Biology, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AZ, UnitedKingdom; and §Advanced Technologies (Cambridge) Ltd., Cambridge Science Park, Cambridge CB4 4WA, United Kingdom

Communicated by Sydney Brenner, April 18, 1991

ABSTRACT Transgenic tobacco plants carrying a geneticcassette including an antisense DNA sequence of the virallyencoded ALI gene of the geminivirus tomato golden mosaicvirus (TGMV) were constructed; ALI encodes a protein abso-lutely required for TGMV DNA replication. These geneticcassettes also contained, on the same transcription unit, a geneencoding hygromycin resistance, which allowed selection forconcomitant expression of the antisense gene. In transgeniclines, RNA transcripts of the predicted size and strand speci-ficity were detected in antisense plants and sense controls. Afterinfection of plants with TGMV, by agroinoculation, the fre-quency of symptom development was very significantly re-duced in a number of antisense lines and correlated, broadly,with the abundance of antisense RNA transcript and with areduction in viral DNA harvested from infected leaf tissue. Weused an in vitro assay to study viral DNA replication in theabsence of cell-to-cell spread; no replication was seen in five ofthe six antisense lines studied, in contrast to controls.

Antisense RNA, complementary to a target RNA (e.g.,mRNA), has recently been exploited to artificially suppressgene expression with high specificity (1, 2). Both previouslyintroduced and endogenous genes have successfully beentargeted by this technique. The variety of models accountingfor the mechanism include antisense RNA-DNA interactioninterfering with transcription, rapid degradation of the senseRNA-antisense RNA duplex, a block in the transport ofduplex RNA into the cytoplasm, and inhibition oftranslation.Because antisense RNA acts at the transcript level, it iseffective against multiple gene copies and, in addition, acts intrans. It would thus seem to be ideal for the suppression ofviral infection; viruses invade the cell from outside andmultiply to a high copy number.

Antisense RNA has been successfully used to suppressviral infection in bacteria and in animal tissue (1). In plants,cross-protection between RNA viral strains has historicallybeen used as a strategy for achieving virus resistance. Thishas led to engineering resistance by construction of trans-genic plants that overexpress the viral coat protein and plantsthat contain replicating copies of a "satellite RNA" (3). Thereported applications of antisense technology to the suppres-sion of viral infection in plants have, so far, met with onlylimited success (4, 5). This is probably due to the mode ofaction of antisense RNA and the viruses chosen as targets(i.e., RNA viruses that replicate in the cytoplasm).The single-stranded DNA viruses known as geminiviruses

are serious plant pathogens, yet little has been done toengineer plants with resistance. Recently, however, Stanleyet al. (6) have shown that transgenic plants containing adefective geminiviral DNA show some decrease in symptomseverity following infection. Because geminiviruses replicatein the nucleus, they would seem to be ideal targets to apply

antisense technology to engineer resistance. To our knowl-edge, this paper is the first report on the application ofantisense RNA technology, in plants, to achieve resistance toinfection by a geminivirus. We constructed tobacco plantsthat express an antisense viral sequence of tomato goldenmosaic virus (TGMV) transcriptionally fused to a drug re-sistance gene.

MATERIALS AND METHODSConstruction of Plasmids. A 1488-base-pair (bp) Nde

I-BamHI fragment of the A DNA molecule of TGMV (TG-MVA) containing the ALI promoter and coding region plusthe 5' regions ofAL2 and AL3 was isolated, rendered bluntended, and subcloned into the Sma I site of pBSM13+(Stratagene). To delete the promoter of ALI, this recombi-nant plasmid was digested with BamHI and Sph I and treatedwith exonuclease III, yielding a plasmid, pBSAL1, in whichthe sequence had been deleted up to position -15 bp of thetranslation start, as confirmed by DNA sequence analysis. A1258-bp fragment of this plasmid, containing the entire ALIcoding region plus 185 bp 3' of this coding region (includingportions of the AL2 and AL3 genes), was released by cuttingwith Kpn I and BamHI and blunt-end cloned into the Sal I siteof the plasmid pJC3 to yield pJC8S and pJC8AS, whichcontained the ALI fragment upstream of the hygromycinresistance gene (hyg) in the sense and antisense orientation,respectively [A.G.D. and C.P.L., unpublished results; pJC3contains a 360-bp cauliflower mosaic virus 35S promoterfragment, p35S (7), the whole coding region of the Esche-richia coli hyg gene, and a 930-bp fragment containing 3'transcriptional regulatory signals of the ocs gene]. This1258-bp fragment was also cloned into the Sma I site of pJC3to yield pJC1OS and pJC1OAS, which contained the ALIfragment downstream of hyg in each orientation. The plas-mids pJC8S and pJC8AS were partially digested with Xba I,completely digested with HindIII, and end-filled; the relevantfragment containing the gene chimeras was cloned into theXba I blunt-ended polylinker site of the transferred DNA(T-DNA) transfer binary vector pDLT201 (J. Tpvar andC.P.L., unpublished data) to yield pP1A and pP5A. Similarly,from plasmids pJC1OS and PJC1OAS, digested with BamHIand HindIll, the relevant fragment was cloned into the XbaI site of pDLT201 to yield pP6A and pP2A. A 260-bpHindIII-EcoRV fragment, containing the enhancer sequenceof the cauliflower mosaic virus 35S promoter, was blunt-endcloned into the Bgl II sites of pPlA, pP2A, pP5A, and pP6Ato finally yield pPlAEN, pP2AEN, pP5AEN, and pP6AEN,respectively. These plasmids were transferred from E. colistrain DH5a to Agrobacterium tumefaciens strain LBA4404

Abbreviations: TGMV, tomato golden mosaic virus; TGMVA andTGMVB, A and B genomes of TGMV, respectively; T-DNA,transferred DNA.tPresent address: Cambridge University Chemical Laboratory,Lensfield Road, Cambridge CB2 lEW, United Kingdom.ITo whom reprint requests should be addressed.

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by triparental mating (8). Analysis of plasmid DNA fromAgrobacterium confirmed that the constructs remained in-tact.

Plant Transformations. Nicotiana tabacum was trans-formed with A. tumefaciens strain LBA4404 containing thebinary plasmids pPlAEN, pP2AEN, pP5AEN, and pP6AENby using the leaf disc method (8). Transformants wereselected for resistance to hygromycin (40 gg/ml). To analyzethe structural integrity of the input DNA, total DNA wasextracted from frozen leaves as described (8); 10-pug DNAsamples were digested, separated on 1% agarose gels, andblotted onto Hybond-N (Amersham) membranes accordingto the manufacturer's instructions. These membranes werehybridized in Church buffer (9) with a radiolabeled ALI DNAprobe (Kpn I-BamHI 1258-bp fragment of pBASL1) at 65TC.The filters were washed in 0.1 x standard saline citrate(SSC)/0.1% SDS at 650C and autoradiographed. The probewas removed from the membranes, and the filters wererehybridized with a hyg DNA probe by using the samehybridization and washing conditions as for the ALI DNAprobe.

Analysis of Expression of Transgenes in Transformed Plants.For Northern blotting, RNA was extracted from leaves asdescribed (10) and fractionated by agarose gel electrophore-sis in the presence of 1.2% formamide. Commercial RNAmolecular size markers were purchased from BoehringerMannheim and treated the same as the RNA samples. RNAwas blotted (according to the manufacturer's instructions)onto Hybond N', and the membrane was prehybridized inChurch buffer and sheared denatured salmon sperm DNA (20mg/ml). The RNA was hybridized in the same buffer to adouble-stranded DNA ALI probe (Kpn I-BamHI 1258-bpfragment of pBASL1) at 65°C. The membrane was washed in0.2x SSC/0.1% SDS at 65°C and autoradiographed.For RNA slot blotting, the total RNA from leaves was

incubated with 100 units of RNase-free DNase I (BoehringerMannheim) at 37°C to remove the DNA. After deproteiniza-tion by phenol/chloroform extraction and precipitation withethanol and sodium acetate, the RNA was quantified, andequal amounts were applied to a Hybond-N+ membrane byusing a Schleicher & Schuell Minifold II slot blot apparatus.The conditions for hybridization were the same as for theNorthern blots except that 70°C was used instead of 65°C forhybridization. The strand-specific RNA probes were tran-scribed by T7 RNA polymerase (Boehringer Mannheim) fromthe T7 promoter of pBSM13+ (Stratagene) containing the1258-bp fragment of ALI in the sense and antisense orienta-tion. A double-stranded DNA probe of the N. tabacum geneencoding the small subunit of ribulose bisphosphate carbox-ylase (Rubisco) was obtained by labeling a Sph I-Xmn Ifragment (11). The wash conditions were the same as for theNorthern blot.

Infection of Plants with TGMV by Agroinoculation. Trans-formed and untransformed N. tabacum plantlets were mi-cropropagated in tissue culture for two rounds of subculturewithout antibiotic selection, transferred to soil, and hardenedoff in an insect-free containment growth room at 24°C with a16-hr photoperiod (-10,000 lux with high-pressure sodiumlamps). One week after transfer to soil, the plants were keptin the dark for 24 hr prior to removal of the lower leaves andwere challenged with TGMV by agroinoculation (10 ,u of anovernight culture) (12, 13); here dimers of TGMVA and theB molecule of TGMV (TGMVB) were each cloned into thebinary plasmid vector pGA482 (14) and transferred to A.tumefaciens strain LBA4404. The two resulting strains werecoinoculated as a mixed culture. After agroinoculation theplants were kept in the dark for a further 24 hr (two plantsfrom each line were inoculated with water). The presence orabsence of symptoms, necrotic leaf lesions, was determined14 and 21 days after infection.

Statistical Analysis of Symptom Development. The statisti-cal technique of "empirical logistic transforms" (15) wasused to analyze the data from the infected plants. In thisstatistical analysis, plants were considered to be symptombearing or symptomless; no consideration was taken of theseverity of the symptoms. Each transformed line was com-pared with the control individually, and no attempt was madeto sum together different transformed antisense lines. Whereapplicable, the wild-type and sense control lines were com-pared and found to be statistically identical. They were thensummed together and treated as one block for comparisonwith the antisense lines.

Analysis of Viral DNA Replication in Infected Tissue. Slotblots were carried out using a Schleicher & Schuell MinifoldII slot blot apparatus (according to the manufacturer's in-structions). Two micrograms ofDNA in a final volume of 2001.l was denatured with 0.5 M NaOH/1.5 M NaCl prior toapplication to a Hybond-N membrane. The membrane wasneutralized by immersion in 0.5 M Tris HCI, pH 7.2/1.5 MNaCl and washed in 20x SSC/1% SDS. The blot washybridized in Church buffer (9) to a DNA probe fragmentcontaining either the complete genome of TGMVB or theTGMVA-specific probe used for the Southern blot of leaf discDNA.In Vitro Analysis of TGMV DNA Replication by Leaf Disc

Agroinoculation. Leaf disc assays were carried out as de-scribed by Elmer et al. (16). DNA was extracted from 20 to30 leaf discs by the method of Lichtenstein and Draper (8).The DNA was measured by fluorescence using a TKO 100minifluorometer (Hoefer), and 2.0-pg samples were run in a1.2% agarose gel and transferred to a Hybond-N membrane(according to the manufacturer's instructions). The mem-brane was prehybridized in Church buffer and sheared de-natured salmon sperm DNA (20 mg/ml). The membrane washybridized in the same buffer to a double-stranded TGMVA-specific DNA probe [Sau I-Sau I coat protein gene (AR])fragment]. The membrane was washed in 0.1x SSC/0.1%SDS at 650C and autoradiographed.

RESULTSTGMV, a geminivirus, has a genome of two small circularsingle-stranded DNA molecules, TGMVA and TGMVB (seeFig. 1A and ref. 18). TGMVA encodes the coat protein andprotein(s) required for virus DNA replication. TGMVB en-codes proteins required for cell-to-cell spread within aninfected plant but is not required for virus replication andencapsidation (19, 20). Because the TGMV coat protein genemay be deleted without abolishing infectivity and in any caseis expressed at high levels (12, 21), it is a poor target forachieving virus resistance. We chose a region encompassingthe ALI gene open reading frame as a target because it isconserved in all geminiviruses, encodes a protein absolutelyrequired for viral DNA replication, and is not abundantlytranscribed (16, 22).Plasmid Construction. To suppress expression of a multi-

copy virus, we felt that it would be necessary to obtain highlevels of expression of the antisense RNA. Thus, to maximizetranscription, we made bifunctional constructs, wherein theantisense sequence was transcriptionally fused to a drugresistance marker gene and driven by a powerful constitutiveplant promoter to allow coselection in plant transformants. Itis difficult to predict the best region to target with antisenseRNA; in this study we chose a region comprising the com-plete coding sequence of ALI and the 5' sequences of twoother open reading frames, AL2 (which overlaps ALI) andAL3. This region was isolated on a 1258-bp fragment fromTGMVA and cloned in either the sense or antisense orien-tation, upstream and downstream, of the hygromycin resis-tance gene (hyg) from E. coli (23) in a plasmid plant trans-

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FIG. 1. (A) Maps of the DNA A and B genomes of TGMV(TGMVA and TGMVB, respectively). Nucleotide positions of thesix open reading frames are marked. Only the coat protein gene,AR], andBR] (transcribed in the rightward direction) are encoded onthe viral plus strand (i.e., by the infecting viral single-strandedDNA). The 200-bp region common to both TGMVA and TGMVB isshown as an open box; within this, a palindromic sequence, yieldinga potential stem-loop structure, and marked in black, is highlyconserved in all geminiviruses and is presumed to be cis-essential forviral DNA replication. (B) Physical map of the chimeric ALI senseand ALI antisense bifunctional hyg gene fusions in plasmidspPlAEN, pP2AEN, pP5AEN, and pP6AEN. RB and LB denote theflanking cis-essential signal sequences required for transfer ofT-DNA from A. tumefaciens to plant cells. In each plasmid, tran-scription of both the hygromycin resistance gene (hyg) and the ALIantisense (or sense) gene is driven by the cauliflower mosaic virus"enhanced" 35S promoter (p35S) (17). The genes have the 3'transcriptional regulatory signals of the octopine synthase gene (ocs)(14). Arrows indicate the sense and antisense orientation of thegenetic units. Enh, enhancer.

formation vector to yield the plasmids pPlAEN, pP5AEN,pP6AEN, and pP2AEN (Fig. 1B).

Plant Transformation. Tobacco (N. tabacum) leaf tissuewas transformed by A. tumefaciens-mediated transforma-tion. The resulting transgenic plants were maintained asindependently transformed lines by micropropagation in tis-sue culture. No transformants were obtained with pPlAEN,despite repeated attempts. In addition it was difficult toobtain transgenic plants from pP5AEN because most trans-formants, while able to grow, failed to root on hygromycinselection medium. However, plant tissue transformed bypPSAEN yielded two antisense lines, P5AEN1 and P5AEN6,that were maintained for further analysis. Similarly, frompP2AEN, five antisense plant lines, P2AEN1-5, were main-tained; and from pP6AEN, two sense lines, P6AENS and

P6AEN6, were maintained. Integration, structure, and copynumber of the constructs in the transformed plants wereconfirmed by Southern blotting of genomic DNA digestedwith EcoRI, EcoRV, and HindIII (data not shown). The datais summarized in Table 1. As P6AEN5 and P6AEN6 ap-peared to be identical, only P6AEN6 was analyzed further.

Expression of ALl RNA in Transgenic Plants. A singlefull-length transcript of the predicted size (3.5 kilobases) wasobserved in all the transformants analyzed by Northernblotting using a double-stranded DNA probe of ALI (Fig.2A). Thus, it is likely that the transcript comprises the entirehyg coding region and the complete ALl (sense or antisense)sequence. RNA slot blots were performed with strand-specific probes to distinguish between sense and antisensetranscripts and to quantify expression; a probe against anendogenous tobacco transcript was used as an internal stan-dard to calibrate expression (Fig. 2 B and C). We observeda 5- to 6-fold range in the expression ofantisense RNA amongthe different transgenic plant lines. There are promotersinternal to the coding region of ALI that drive transcriptionof AL2 and AL3 that were included in our construct. How-ever from the expression studies described above, we foundno evidence of any transcription from these promoters.

Analysis of Symptom Development and Viral DNA in In-fected Plants. Susceptibility of the transgenic lines to TGMVinfection was analyzed by scoring symptom development andviral DNA replication. Batches of micropropagated untrans-formed and transgenic plant lines were challenged by agroin-oculation with TGMV; agroinoculation is a technique inwhich infection is mediated by Agrobacterium strains con-taining a tandem repeat of the geminivirus DNA withinT-DNA borders. Following infection of the stem, monomersof virion DNA are released, replicate, and spread systemi-cally through the plant, giving rise to symptom developmentin leaves distant from the site of infection. This method ofinfection is much more efficient than mechanical inoculation;in our initial experiments, mechanical infection of untrans-formed plants with whole virus yielded far fewer plantsbearing symptoms than those that were agroinoculated.Symptom development (necrotic leaf lesions) was scored 14days after infection. A second scoring was carried out 7 daysafter this without finding any further increase in symptomdevelopment or severity. A statistical analysis (15), based onthe presence or absence of symptoms and not on symptomseverity or viral replication, was carried out on the datasummarized in Fig. 3. All the transformants with the an-tisense ALl downstream of hyg (P2AEN series) and onecontaining it upstream (P5AEN6) had fewer symptom-bearing plants than the untransformed plants (at a statistically

Table 1. Transformed plant lines

Copies per plantPlant line Construct genome and integrityP2AEN1 pP2AEN 1, intactP2AEN2 pP2AEN 2-4, all intactP2AEN3 pP2AEN 6-10, possible

dimers, some copiesrearranged

P2AEN4 pP2AEN 1, intact; 1, rearrangedP2AEN5 pP2AEN 1, intactP5AEN1 pP5AEN 6-10, all intactP5AEN6 pP5AEN 4, intact; 1, rearrangedP6AEN5 pP6AEN 1, intactP6AEN6 pP6AEN 1, intact

N. tabacum transgenic plants were transformed with the binaryplasmids pP2AEN, pP5AEN, and pP6AEN. Integration, structure,and copy number of the constructs in the transformed plants wereconfirmed by Southern blotting of genomic DNA digested withEcoRI, EcoRV, and HindIII.

A

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FIG. 2. Northern blot (A) and RNA slot blot (B) analysis oftransformed and untransformed plants. The expression of sense andantisense ALI was analyzed using total RNA extracted from leavesof untransformed (C) and transformed plants P2AEN1 (sample 1),P2AEN2 (sample 2), P2AEN3 (sample 3), P2AEN4 (sample 4),P2AENS (sample 5), P5AEN1 (sample 6), and P6AEN6 (sample 8).(A) Northern blot of total RNA (20 ,ug per lane) hybridized with adouble-stranded DNA ALI probe. (B) RNA slot blot. Samples (10 ,ugof total RNA per slot) were hybridized to an antisense-specific ALlRNA probe (I), a sense-specific ALl RNA probe (II), and a double-stranded DNA probe to the ribulose bisphosphate carboxylase(Rubisco) small subunit transcript (III). (C) Histogram representingthe quantification of the RNA slot blot analysis of the transformantexpressing the antisense ALl RNA. The results are expressed as apercentage relative to the P2AEN4 ALl RNA level. kb, Kilobases.

significant level). The sense line showed no difference insusceptibility to the untransformed plants. In the resistantantisense lines, symptom severity was reduced (fewer ne-crotic lesions per leaf and fewer leaves with necrotic lesionsper plant) compared to controls, in those plants that did showsymptoms.

Total DNA was extracted from leaves of some plants thathad developed symptoms and from leaves of all the plantswithout symptoms. DNA slot blot analysis using TGMVBDNA as probe demonstrated that all the symptom-bearingplants contained high levels of viral DNA (data not shown).Analysis of DNA from symptomless untransformed plantsindicated that 70% had viral DNA present at an average of58% of the level of that found in symptom-bearing plants.Viral DNA was present in a similar percentage of symptom-less plants from the P2AEN lines (antisense region down-stream of hyg); however, the amount of viral DNA wassignificantly less than that of the untransformed plants (see

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FIG. 3. Percentage of untransformed (bar C; 75 plants) andtransformed P2AEN1 (bar 1; 56 plants), P2AEN2 (bar 2; 44 plants),P2AEN3 (bar 3; 61 plants), P2AEN4 (bar 4; 57 plants), P2AEN5 (bar5; 54 plants), P5AEN1 (bar 6; 32 plants), P5AEN6 (bar 7; 40 plants),and P6AEN6 (bar 8; 25 plants) plants that have developed symptomsafter agroinoculation with TGMVA and TGMVB. The presence orabsence of symptoms was determined 14 and 21 days after infection.Percentages were calculated by adding the results oftwo independentexperiments; no statistical differences were found between theexperiments. The statistical technique of empirical logistic trans-forms (15) was used to analyze the data from the infected plants. Inthis statistical analysis, plants were considered to be symptombearing or symptomless; no consideration was taken of the severityof the symptoms or viral DNA replication in symptomless plants.Each transformed line was compared with the control individually,and no attempt was made to sum together different transformedantisense lines. Lines were considered not resistant when the con-fidence limit was below 95% (P5AEN1 and P6AEN6). The confi-dence limits for the resistant lines were >99.9% (P2AENI), >99%o(P2AEN2), >99.99% (P2AEN3), >99.99% (P2AEN4), >99%(P2AENS), and >99.99o (P5AEN6).

Table 2). Interestingly, in the P5AEN lines (antisense regionupstream of hyg), the suppression of viral DNA replicationwas found to be almost complete in symptomless plants.In Vitro Analysi of TGMV DNA Repicatlon. Symptom

development involves both cell-to-cell spread and viral DNAreplication. To examine the effect on DNA replication in theabsence of cell-to-cell spread, we performed in vitro leaf discagroinoculation assays (16) with TGMVA alone. TGMVADNA can only replicate in the agroinoculated cell but cannotspread. Analysis of untransformed leaf disc tissue gave three

Table 2. Percentage of symptomless plants containing viral DNAand amount of viral DNA present in their leaves

% symptomless % viral DNA in symptomlessplants containing plants relative to

Plant line viral DNA symptom-bearing plantsUntransformed 70 58 + 8P2AEN1 69 28 ± 8P2AEN2 58 23 + 8P2AEN3 69 32 + 5P2AEN4 86 37 ± 6P2AEN5 53 19 ± 8P5AEN1 0P5AEN6 7 2 1Leaves from symptomless plants (from one of the two viral

challenge experiments described in Fig. 3) were harvested 21 daysafter agroinoculation with TGMV, and total DNA was extracted andexamined for the presence of viral DNA by slot blot analysis. Thevalues indicate the percentage of symptomless plants that containany viral DNA and, ofthose that do, the amount ofviralDNA presentin the leaves relative to that found in leaves showing symptoms. Thevalue is the average ± SE obtained from analysis ofDNA extractedfrom 10 untransformed plants, 16 plants oftransformant P2AEN1, 12plants of P2AEN2, 29 plants of P2AEN3, 35 plants of P2AEN4, 15plants of P2AENS, 6 plants of PSAEN1, and 14 plants of PSAEN6.The same results were obtained when either TGMVA or TGMVBDNA probes were used.

6724 Genetics: Day et al.D

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FIG. 4. Southern blot analysis of DNA extracted from leaf discs10 days after agroinoculation with TGMVA ofuntransformed (C) andtransformed plants P2AEN1 (lane 1), P2AEN2 (lane 2), P2AEN3(lane 3), P2AEN4 (lane 4), P2AEN5 (lane 5), and P5AEN1 (lane 6).The membrane was hybridized to a TGMVA-specific DNA probe[Sau I-Sau I coat protein gene (ARJ) fragment]. The experiment was

repeated under the same conditions (data not shown), and the resultwas similar except that no replication was observed in the trans-formant P2AEN5. Molecular size markers are from A DNA cut withHindIll and electrophoretically separated in the same gel. Thepositions of single-stranded (ss) DNA, supercoiled (sc), and open

circular (oc) double-stranded DNA forms ofTGMVA are indicated.kb, Kilobases.

DNA species offree TGMVADNA corresponding to double-stranded open circular, supercoiled (postulated replicativeintermediates), and single-stranded circular DNAs. In trans-genic tissue of lines P2AEN1-4, no TGMVA DNA was

detected; in P2AEN5, noTGMVADNA was observed in oneassay and very low levels were observed in a duplicate assay

(Fig. 4). In P5AEN1 tissue a slight reduction was observed.

DISCUSSIONWe have shown that transgenic plant lines expressing an

antisense AL1 transcript show a statistically significant re-

duction in symptom development following TGMV agroin-oculation. In the most resistant line, P2AEN4, only 5 out of57 plants challenged displayed any symptoms at all, and inthese 5 plants the symptoms were reduced.Our rationale in placing the antisense region upstream of

the drug marker was that this should reduce the translationalefficiency of the marker gene, and hence drug resistanceshould require correspondingly higher levels ofRNA expres-

sion. Though fewer transformants were obtained than whenthe ALI gene was downstream of hyg, our sample size oftransgenic lines studied is too small to determine whichconfiguration of antisense AL1 relative to hyg gives thehighest levels of antisense RNA expression. Thus it isdifficult to assess the significance of the unexpected obser-vation that the two lines expressing the most antisense RNAactually have the antisense AL1 downstream of hyg.We observed a broad correlation between antisense RNA

levels and resistance (symptom development); those twolines expressing the most antisense RNA, P2AEN3 andP2AEN4, showed greatest resistance (Figs. 2C and 3). Weobserved no correlation between copy number of the inte-grated DNA and either degree of resistance or level ofantisense RNA expression. Variation in gene expressionbetween lines transformed by the same construct is well-documented in plant transformation experiments (23); al-though there is no clear explanation, it is perhaps related tothe transcriptional activity of the region surrounding theintegration site of the input DNA.

Presumably, a threshold level of virus must accumulate ininfected tissue to give symptoms. Below that threshold, nosymptoms may be evident, despite the presence of virus. Thereduction in symptom development that we observed in theantisense lines is reflected in a reduction in the amount ofviral DNA accumulating in the leaf tissue.Though ALI is required for DNA replication, the antisense

constructs might confer resistance by prevention of cell-to-cell spread. The leaf disc agroinoculation experiments, usingonly the TGMVA genome, preclude cell-to-cell spread andallow examination of viral DNA replication only in theinfected cells; these experiments show clearly that the an-tisense ALl RNA blocks replication. Moreover, the degreeof such blocking correlates with the degree of symptomdevelopment measured in whole plants.Our results are very encouraging and suggest that the use

of antisense RNA technology may find application in thecontrol of economically important geminivirus diseases.

This work represents an equal contribution from the first twoauthors. We thank Dr. Robert J. Hayes for advice on agroinoculationprocedures. We gratefully acknowledge the support of the Scienceand Engineering Research Council (A.G.D.) and the EuropeanCommunity and European Molecular Biology Organization for se-nior fellowships (E.R.B.).

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Jongedijk, E., Hoekema, A. & Cornelissen, B. J. C. (1989)Plant Mol. Biol. 13, 337-346.

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6. Stanley, J., Frischmuth, T. & Ellwood, S. (1990) Proc. Nati.Acad. Sci. USA 87, 6291-6295.

7. Odell, J. T., Nagy, F. & Chua, N. H. (1985) Nature (London)313, 810-812.

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Genetics: Day et al.

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