Proc. Natl. Acad. Sci. USAVol. 92, pp. 6117-6121, June 1995Plant Biology

Broad resistance to plant viruses in transgenic plants conferredby antisense inhibition of a host gene essential inS-adenosylmethionine-dependent transmethylation reactionsCHIKARA MASUTA, HIDEo TANAKA, KYOUKO UEHARA, SHIGERU KUWATA, AKIRA KoIWAI, AND MASANA NOMALife Science Research Laboratory, Japan Tobacco, Inc., 6-2 Umegaoka, Aoba-ku, Yokohama 227, Japan

Communicated by Theodor 0. Diener, University of Maryland, College Park, MD, March 21, 1995 (received for review November 30, 1994)

ABSTRACT S-Adenosylhomocysteine hydrolase (SAHH)is a key enzyme in transmethylation reactions that use S-adenosylmethionine as the methyl donor. Because of theimportance of SAHH in a number of S-adenosylmethionine-dependent transmethylation reactions, particularly the 5'capping of mRNA during viral replication, SAHH has beenconsidered as a target of potential antiviral agents againstanimal viruses. To test the possibility of engineering a broadtype of resistance to plant viruses, we expressed the antisenseRNA for tobacco SAHH in transgenic tobacco plants. Asexpected, transgenic plants constitutively expressing an anti-sense SAHH gene showed resistance to infection by variousplant viruses. Among those plants, about half exhibited somelevel of morphological change (typically stunting). Analysis ofthe physiological change in those plants showed that theycontained excess levels of cytokinin. Because cytokinin hasbeen found to induce acquired resistance, there is also a strongpossibility that the observed resistance was induced by cyto-kinin.

A number of strategies have been used to control plant virusdisease with the expression of different viral sequences, in-cluding viral coat-protein genes (1, 2), virus-specific antisenseRNAs (3, 4), satellite RNAs (5, 6), nonstructural genes (7, 8),and even nontranslatable sequences (9). The resistance con-ferred by these methods is generally against one specific virusor closely related viruses. Our working hypothesis was that theinhibition of one of the nonessential host-cell enzymes re-quired for viral replication might lead to protection against abroad spectrum of viruses.

S-Adenosylhomocysteine hydrolase (SAHH) controls bio-logical methylation reactions by mediating the intracellularS-adenosylhomocysteine (SAH)/S-adenosylmethionine (SAM)ratio. DNA and protein methylation and demethylation play animportant role in signal transduction in a variety of organisms.In addition, SAHH probably controls 5'-cap mRNA methyl-ation by regulating the intracellular SAH/SAM ratio (10-12),and undermethylation at the 5' terminus of viral mRNAinduced by inhibition of cellular SAHH has been correlatedwith inhibition of viral replication (13). In fact, SAHH inhib-itors are known to be effective antiviral agents for animalviruses (11, 14). These inhibitors also suppress plant viruses, asshown by leaf disk assays (15, 16). To determine whetherSAHH antisense RNA could inhibit viral infection in thetobacco plant, we constructed transgenic plants that expressantisense RNA of a tobacco SAHH gene (TobSAHH). Theresults show that the expression of SAHH was reduced inplanta through inhibition by antisense RNA and that thesubsequent impairment of cellular SAHH function(s) resultedin the inhibition of viral replication.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

MATERIALS AND METHODSIdentification of the TobSAHH Gene. By differential screen-

ing of a cDNA library obtained from flower-bud regeneratingexplants, we have isolated 12 cDNA clones representing genesthat are preferentially expressed during in vitro flower forma-tion (unpublished work). Nucleotide sequence analysis of oneof the cDNAs showed that the putative protein encoded by thegene has the same amino acid sequence as that encoded by theSAHH cDNA later isolated by Mitsui et aL (17).

Expression ofTobSAHH in Escherichia coli. The TobSAHHcDNA was inserted between the EcoRI and Xba I sites inpMAL-cRi (New England Biolabs), which placed it in thesame translational reading frame as the vector's malE gene.This resulted in a maltose-binding protein (MBP)-TobSAHHfusion protein. Expression of the fusion protein in E. coli JM83was performed essentially as described in the instructions forthe pMAL vectors. After purification of the fusion protein, theTobSAHH domain was separated from MBP by factor Xacleavage carried out at a factor Xa/fusion protein weight ratioof 0.1% in 20 mM Tris Cl, pH 8.0/100mM NaCl/2mM CaCl2.The reaction mixture was incubated at 4°C overnight.Assay for SAHH Activity. The enzyme assay was performed

as described by Poulton and Butt (18), with some modifica-tions. SAHH catalyzes the hydrolysis of SAH in a reversiblereaction (SAH + H20 adenosine + homocysteine). For theforward reaction, 100 ,ul of incubation mixture (pH 7.2)contained 5 mM SAH, 1 mM dithiothreitol, 50 mM potassiumphosphate, 0.2 unit of adenosine deaminase, and E. coli-produced SAHH. Adenosine deaminase was included to con-vert adenosine to inosine, because the production of adenosineinhibits this reaction. For the backward reaction, 100 ,ul ofincubation mixture (pH 7.2) contained 5 mM homocysteine, 5mM adenosine, 1 mM dithiothreitol, 20 mM potassium phos-phate, and E. coli-produced SAHH. The reaction mixtureswere incubated at 25°C for 30 min; an aliquot (20 ,ul) of thereaction mixture was applied to a Polygram CEL 300 UV 254sheet; and the chromatogram was developed with isopropylalcohol/ethyl acetate/ammonia/water (23:27:4:4 by volume).

Construction of an Antisense SAHH Transgene. The frag-ment containing the entire cDNA (1.8 kb) was inserted into thepolylinker of a plant expression vector, pBI121 (Clontech), inthe antisense orientation relative to the cauliflower mosaicvirus 35S promoter. The recombinant plant expression vectorwas introduced into Agrobacterium tumefaciens LBA 4404 andwas used to transform tobacco (Nicotiana tabacum cv. BY-4 orN. tabacum cv. Xanthi nc) by the leaf disk method (19).

Plant Virus-Resistance Assays. Cucumber mosaic virus(CuMV) strains 0 and N had been maintained in our labo-ratory (19, 20). Tobacco mosaic virus strain OM (TMV-OM),potato virus X (PVX), and an ordinary strain of potato virus

Abbreviations: SAH, S-adenosylhomocysteine; SAHH, SAH hydro-lase; SAM, S-adenosylmethionine; CuMV, cucumber mosaic virus;TMV, tobacco mosaic virus; PVX, potato virus X; PVY, potato virusY; MBP, maltose-binding protein.

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Y (PVY-O) were obtained from the National Institute ofAgrobiological Resources (Tsukuba, Japan). Plants weredusted with carborundum, rub-inoculated with viruses, andkept in a greenhouse at 25°C under natural light conditions.

Preparation and Inoculation of Protoplasts. One gram oftobacco leaf tissue was cut transversely into slices 1-2 mm wideand the sections were soaked in 10 ml of an enzyme solutioncontaining 2% Cellulase R-10 (Yakult Pharmaceutical, To-kyo) and 0.5% Macerozyme (Yakult Pharmaceutical) in 0.5 Mmannitol (pH 5.8). After a 4-hr incubation at 25°C, thedigested tissue was filtered through a nylon mesh (100 ,um).The protoplasts were then collected by centrifugation at 100 xg for 3 min and washed twice with 0.5 M mannitol. Viral RNAwas introduced into protoplasts in the presence of polyethyleneglycol (PEG) and CaCl2 (21). Protoplasts were incubatedunder continuous fluorescent light (-1000 lx) and virus ac-cumulation was determined by conventional ELISA.ELISA. Viruses were extracted from leaf tissues or proto-

plasts by grinding in phosphate-buffered saline (PBS) contain-ing 0.05% Tween 20, and a series of dilutions were madeaccordingly. The levels of CuMV and PVY were quantitatedas described (22). The levels of TMV and PVX were deter-mined with ELISA kits (Sigma). The values obtained wereconverted to micrograms of virus per gram of tissue or topicograms of virus per protoplast for the protoplast experi-ment. The t test was used to determine whether there was anysignificant difference in the virus levels of transgenic andcontrol plants.Northern Blot Analysis. Total RNA was isolated from leaves

by the guanidinium isothiocyanate extraction followed bycentrifugation with a CsCl cushion (23). Poly(A)+ RNA wasprepared by using Oligotex-dT30 (Takara Shuzo, Kyoto). Onemicrogram of poly(A)+ RNA per lane was separated in adenaturing formaldehyde/agarose gel and blotted onto aGeneScreenPlus membrane (NEN). Northern blots were hy-bridized with 32P-labeled RNA probes transcribed from pBlue-script II (Stratagene) vectors having the TobSAHH cDNA.Hybridization was performed as described by the manufac-turer of the membrane.Western Blot Analysis. Leaf tissue (1 g) was homogenized in

1 ml of PBS/Tween. After centrifugation at 10,000 x g for 20min, an aliquot (50 ,tg of protein) of the supernatant wassubjected to electrophoresis in an SDS/10% polyacrylamidegel, and the proteins were transferred to an Immobilon-Pmembrane (Millipore) by conventional electroblotting andprobed with anti-SAHH antibodies by using a kit purchasedfrom Vectastain.

Separation and Analysis of SAH and SAM. SAH and SAMwere analyzed by HPLC (24). Leaf tissue (1 g) from four plantswas homogenized with 4 ml of ice-cold 5% (wt/vol) sulfosal-icylic acid. After centrifugation at 10,000 x g for 5 min, thesupernatant was collected and filtered through a GelmanHPLC Acro-LC 13 filter. Fifty microliters of the sample wasanalyzed on a Partisil 10 SCX column (Whatman). The elutiongradient was arranged with two buffers [buffer A, 1 mMammonium formate adjusted to pH 4 with formic acid; bufferB, 0.2 M ammonium formate (pH 4) plus 0.8 M ammoniumsulfate]. The flow rate was 1 ml/min.

Cytokinin Assay. Root exudate was used to estimate theendogenous level of cytokinin (25, 26). Cytokinin activity wasassayed by a method employing tobacco callus essentially asdescribed by Torigoe et at (27). Root exudate was collected asthe source of cytokinin and diluted 3- and 5-fold with water.The anticytokinin 4-chloropentylamino-2-methylpyrrolo[2,3-d]pyrimidine, which is known to be a specific competitor ofcytokinin for the putative receptor (28), was used to ensurethat the callus formation was really stimulated by the cytokininin the sap. The concentration of the anticytokinin was adjustedto that of the root exudate dilution. The cytokinin activity wasdetermined by placing tobacco callus on a medium containing

the root exudates, which had been filter-sterilized after theaddition of Murashige-Skoog salts, vitamins, sucrose, andauxin. After about 1 month of incubation at 26°C in the dark,the fresh weight of the callus was measured, and the mean

value for 10 calluses was compared with that of the kinetincontrols.

RESULTS AND DISCUSSIONNucleotide sequence analysis revealed that the cDNA clonecontained 1818 nt (GenBank accession no. D45204). A com-parison with the nucleotide sequence of another tobaccoSAHH cDNA clone, TCB1 (2120 nt), reported by Mitsui et at(17), showed that they had 95% identity. The proteins encodedby the two genes have the same amino acid sequences (485residues). The isolated E. coli-produced TobSAHH had theexpected size (53 kDa) after factor Xa digestion (Fig. 1A, lane4). As the amount of SAHH increased, more and morereaction products (inosine in the forward reaction and SAH inthe backward reaction) were detected (Fig. 1 B and C),showing that the cDNA clone encodes a functional proteinwith SAHH enzymatic activity.Two cultivars were used for transformations: BY-4, a sys-

temic host for TMV, and Xanthi nc, a local-lesion host forTMV. We obtained 120 BY-4 (SHB) plants and 110 Xanthi nc(SHX) plants. All the Ro plants were first tested for TMVresistance. To determine the effect of the antisense inhibitionof SAHH on initial infection and local viral replication, weinoculated leaves of SHX plants (Ro) with TMV. Comparedwith the control plants, the local lesions on the leaves of theSHX plants were smaller and fewer. Inoculated SHB plantsshowed delayed systemic symptom development, and some ofthe plants yielded less TMV. To determine whether theapparent viral resistance of Ro transformants was heritable,several of the most resistant Ro plants were selfed and theirseeds were collected. Seeds from these plants were germinatedin the presence of kanamycin (50 ,ug/ml) and resistant plant-lets were selected. We initially selected 10 SHX and 10 SHBplant lines at random from the Ro population. Ten indepen-dent kanamycin-resistant progeny SHB plants and 5 indepen-dent kanamycin-resistant progeny SHX plants were used toassay TMV resistance. Two SHX lines developed about asmany local lesions on the inoculated leaves as the control

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FIG. 1. (A) Production and isolation of E. coli-produced SAHH.Lane 1, marker proteins of 45, 66, 97, 116, and 200 kDa; lane 2, totalproteins of E. coli extract; lane 3, 3 ,ug of the fusion protein; lane 4, 3

,ug of the products after factor Xa cleavage. S-M, SAHH-MBP; S,SAHH; M, MBP. (B and C) Detection of the enzymatic activity of E.coli-produced SAHH. A mixture of SAHH, MBP, factor Xa, and thefusion protein was used for this assay because MBP and factor Xa donot have any SAHH activity. The protein concentrations were spec-trophotometrically estimated. For the forward reaction of SAHH (B)lanes 1-4 show products obtained with 0, 1, 5, and 10 Ag of SAHH,respectively. Hypoxanthine (Hyp) was detected in lanes 2-4 becausethe preparation of adenosine deaminase included in the reactionmixture was contaminated with an enzyme that converts inosine (I) toHyp. For the backward reaction of SAHH (C) lanes 1-4 show productsobtained with 0, 1, 5, and 10 ,ug of SAHH, respectively. Lane 5contained 50 nmol of SAH and 50 nmol of adenosine (A).

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Table 1. Susceptibility of SHX R1 plants to infection by TMV andCuMV

Plant line

SHX2SHX4SHX5SHX6SHX9SHX13SHX23SHX26SHX32SHX35Control (Xanthi nc)

No. of lesions per leaf(mean ± SD)

TMV-OM CuMV-N

2 ± 220 ± 428 ± 588 ± 9*7 ± 2

33 ± 869 ± 540 ± 628 ± 5102 ± 8*91 ± 5

2± 117 ± 532 ± 465 ± 5*19 ± 432 ± 357 ± 551 ± 736 ± 574 ± 5*70 ± 3

judged from the mobility of the control band (lane C), theupper of the two discrete bands appears to be the target senseRNA. As two different tobacco SAHH genes (80% nucleotidesequence identity) differing in size by about 200 bp havealready been identified (17), we assume that the lower bandrepresents the transcript of another SAHH gene that is ex-pressed in leaf tissue. The levels of SAHH mRNA in thetransgenic plants, except for one of the susceptible plant lines(SHX6), were considerably lower than those observed in thenontransgenic plants (Fig. 4A). Although the levels of anti-sense SAHH RNA in the transgenic plants were very low,antisense RNA species were detected in SHB3, SHX2, andSHX4 (Fig. 4A). As expected, the antisense RNA was notdetected in lanes B, X, and C. The SAHH protein was notdetected in leaves of any of the transgenic lines, whereasSAHH was present in leaves of the control tobacco plants (Fig.4B). Consequently, the decrease in SAHH mRNA and in

Five plants from each line (two leaves per plant) were inoculatedwith TMV (0.5 jig/ml) or with a 50-fold diluted sap from CuMV-N-infected Vinca rosea leaves. Four days after inoculation, lesions werecounted.*Judged to be susceptible plant lines.

plants, whereas the rest exhibited fewer lesions (Table 1).Three SHB lines expressed symptoms in all 10 plants of thatline by day 8, whereas the rest showed delayed symptoms(Table 2). Among the plant lines exhibiting some resistance,two from each tobacco variety showing the highest TMVresistance (SHX2 and SHX3) and two showing intermediateresistance (SHX4 and SHB5) were selected for further anal-ysis. All four transformants used for further study were foundto contain a single copy of the gene at Ro (data not shown).SHX R1 progeny showed a highly reduced incidence of infec-tion byTMV-OM and by CuMV-N, yielding fewer and smallerlesions than the control plants (Table 1). The lesions on thosetransformants developed very slowly. Inoculated SHB R1progeny showed lower accumulations of TMV-OM andCuMV-O (Fig. 2 A and B), and there was a marked delay inthe appearance of systemic symptoms in virus-infected trans-genic plants compared with nontransgenic plants (Fig. 3A andB). Most of the transformants inoculated with PVX (10 ,tg/ml)did not develop any visible symptoms even 2 months afterinoculation (Fig. 3C), and virus concentrations in the symp-tomless leaves were <1% of the level in nontransgenic controls(Fig. 2C).

Sense and antisense SAHH transcripts from transformedplants were detected by Northern blot analysis (Fig. 4A). As

Table 2. Susceptibility of SHB R1 plants to infection by TMV,CuMV, PVX, and PVY

No. of infected plants

Plant line TMV-OM CuMV-O PVX* PWX-OSHB3 3/10 1/10 0/10 (0/10) 6/10SHB5 6/10 7/10 0/10 (1/10) 7/10SHB12 6/10 8/10 0/10 (1/10) 10/10tSHB21 10/10t 10/10t 2/10 (9/10) 10/lotSHB28 5/10 4/10 0/10 (3/10) 9/10SIHB31 10/10t 10/lOt 3/10 (10/10)t 10/10tSHB52 10/10t 10/lOt 2/10 (10/10)t 10/lOtSHB59 4/10 5/10 1/10 (4/10) 7/10SHB68 9/10 10/10t 2/10 (8/10) 10/lotSHB72 8/10 6/10 2/10(8/10) 9/10Control (BY) 10/10 10/10 2/10 (10/10) 10/10Ten plants from each line were inoculated with TMV-O (0.5 ,ug/ml),

CuMV-O (2 ,ug/ml), PVX (10 ,ug/ml), or PVY-O (2 ,gg/ml). Plantsthat did not develop systemic symptoms by 8 days after inoculationwere considered to be resistant.*In parentheses, values 14 days after inoculation.tJudged to be susceptible plant lines.

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FIG. 2. Accumulation of virus in control (solid bars) and transgenic(hatched bars) plants inoculated with TMV, CuMV, PVX, or PVY.The SHB3 (R1) and control nontransgenic plants were inoculated withTMV-OM (0.5 jig/ml) (A), CuMV-O (2 ,ug/ml) (B), PVX (10 Ag/ml)(C), or PVY-O (2 ,ug/ml) (D). Fifty plants were inoculated and fourindependent symptomatic plants were chosen. Since the plants inoc-ulated with PVX did not show any visible symptoms, we harvestednonsymptomatic leaves or leaves showing possible chlorotic spots.Eight days after inoculation, inoculated and second systemic leaveswere collected and two leaf punches (1 cm in diameter) were taken fromeach of the harvested leaves. Virus levels were determined by ELISA.

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SAHH protein in all the transgenic lines and the detection ofantisense RNA production in SHB3, SHX2, and SHX4 seemto be correlated with the milder symptoms in those transgenicplants.To determine whether the antisense SAHH plants would

also resist infection by a virus that does not require a capstructure for its replication, we chose PVY-O. When this viruswas inoculated onto SHB plants, a low level of resistance, withreduced virus accumulation and delayed systemic symptoms,was observed (Figs. 2D and 3D).To verify the effects of antisense inhibition on viral infec-

tion, we inoculated protoplasts with either PVX or CuMV, towhich the transformants showed a relatively high resistanceamong the viruses tested. Protoplasts expressing an antisenseSAHH gene accumulated lower amounts of viral coat proteins(Fig. 5). These results suggest that much but not all of theresistance observed in our transgenic plants occurred at thelevel of viral replication.

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Time after inoculation, days

FIG. 3. Development of systemic symptoms in control and trans-genic plants inoculated with TMV, CuMV, PVX, or PVY. The SHB3(R1), SHB5 (R1), and control nontransgenic plants (10 each) were

inoculated with TMV-OM (0.5 ,ug/ml) (A), CuMV-O (2 ,ug/ml) (B),PVX (10 jig/ml) (C), or PVY-O (2 jig/ml) (D), and symptomdevelopment was monitored.

FIG. 4. (A) Northern blot analysis of sense and antisense expres-sion of the TobSAHH gene in leaves of SHB and SHX R1 transfor-mants. Lane C (control) contains poly(A)+ RNA (1 jig) from flowerbuds of BY-4, which is rich in SAHH mRNA; lane B, RNA fromnontransgenic BY-4; lanes SHB3 and SHB5, RNA from transgenicBY-4; lane X, RNA from nontransgenic Xanthi nc; lanes SHX6,SHX2, and SHX4, RNA from transgenic Xanthi nc. Sense andantisense RNAs are indicated by arrows. (B) Western blot analysis ofSAHH in leaves ofSHB and SHX R1 transformants. Total proteins (50,ug) from SHB3, SHB5, SHX2, SHX4, BY-4 (B), and Xanthi nc (X)were separated in an SDS/10% polyacrylamide gel, and SAHH wasdetected with anti-SAHH antibodies.

Based on the idea that the SAH/SAM ratio controls bio-logical transmethylation reactions, including 5' methylation inmRNA capping, we analyzed the levels of SAH and SAM inthe transgenic plants expressing the SAHH antisense RNA. Inthe transgenic plants, significantly (- 10 times) more SAH was

detected than in the control (Fig. 6), indicating that theSAH/SAM ratio was affected by the antisense inhibition ofSAHH. This observation regarding alterations of the methyl-ation reactions in transgenic plants, together with establishedresults from research using animal viruses (11), suggests the

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FIG. 5. Susceptibility of protoplasts from transgenic tobacco plants(SHB3) to infection with CuMV RNA or PVX RNA. Protoplasts (105)from control (solid bars) or transgenic (hatched bars) were inoculatedwith 2 jitg of viral RNA in the presence of PEG and CaCl2.The extentof infection was assayed after incubation for 24 hr, and the virusaccumulation was determined by ELISA. The percentage of infectedprotoplasts was determined by fluorescent antibody staining (controland SHB infected with CuMV, 63% and 41%; control and SHBinfected with PVX, 72% and 31%), and the number of survivingprotoplasts was found to be almost the same within the experimentalgroups. The difference in the proportion of infected protoplasts oftransgenic and control plants does not seem to be very large (at most1.5-2 times), whereas we obtained a much greater difference betweenthe ELISA values representing total levels of viral accumulation (5-10times), suggesting that viral replication rather than viral infection wasinhibited.

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2-40 60 0 20 40 60

Time, min

FIG. 6. HPLC analysis of SAH and SAM. The samples wereprepared from a control BY plant (A) and from the transgenic plantSHB3 (R1) (B). Note the difference (about 10-fold) in the SAH levelsofA and B. The SAH levels of the other transgenic lines (SHB5, SHX2,and SHX4) were also greater than that of the control, by a factor of2-3 (data not shown).

possibility that the viral resistance observed was mediatedthrough hypomethylation of the 5' cap of viral mRNA.Although quite a number of the transgenic plants appeared

normal, about half of them showed growth alterations, andeven the SHB and SHX plants used for this study were slightlystunted. These phenotypes are reminiscent of abnormalitiesresulting from changes in the endogenous levels of phytohor-mone(s) such as cytokinins. Hence, we investigated the endo-genous level of cytokinins in the stunted transgenic plants andfound that these plants contained about 3 times more cytoki-nin than nontransgenic plants (Fig. 7). Tobacco SAHH hasbeen reported to be a cytokinin-binding protein (17) and thusis a bifunctional protein. We assume that antisense inhibitionresults in a decrease in the amount of SAHH and a consequentrelative increase in the number of free active cytokinin mol-ecules. It has been suggested that an increase in endogenouscytokinin levels triggers the induction of systemic acquiredresistance to plant viruses (29, 30). In fact, we detectedpathogenesis-related proteins expressed in the transgenicplants, indicating that the system for acquired resistance wasactivated (data not shown). Research on animal viruses sug-gests that viral suppression through SAHH inhibition resultsfrom undermethylation of the viral mRNA cap structure (11).Since most plant viruses require a methylated cap structure atthe 5' terminus of their mRNA for viral replication, it seemslikely that the plant virus resistance mediated by antisenseinhibition of SAHH operates by the same mechanism as thatdemonstrated for animal viruses. However, we believe that an

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FIG. 7. Endogenous level of cytokinin estimated from cytokinin inthe root exudate. Cytokinin concentrations are expressed in kinetin

equivalents. Forty transgenic plants were sacrificed to collect the rootexudate. Bars A and B, root exudate from SHX transgenic and

nontransgenic plants, respectively; bars C and D, root exudate fromSHX plants and control plants, respectively, to which 1 ,uM anticyto-kinin (4-chloropentylamino-2-methylpyrrolo[2,3-d]pyrimidine) wasadded before the bioassays.

acquired resistance induced by cytokinins could also play animportant role in the plant virus resistance observed in ourtransgenic plants. This cytokinin-induced acquired resistancecould explain the partial resistance of the SHB plants to PVY.

In conclusion, we may have serendipitously activated twodifferent mechanisms for plant virus resistance by the anti-sense inhibition of a single gene of the plant. Regardless of theprecise mechanism(s), our strategy permits production oftransgenic plants with resistance to a broad spectrum ofviruses.

We thank Dr. A. 0. Jackson (Plant Biology Department, Universityof California, Berkeley) for advice and critical reading of the manu-script and Dr. H. Iwamura (Faculty of Agriculture, Kyoto University)for providing us with anticytokinins used for the cytokinin assay.

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