Plant Physiol. (1997) 114: 1113-1 121

Expression of Pokeweed Antiviral in Transgenic Plants lnduces Resistance in Grafted Wild-Type Plants lndependently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis’

Sergey Smirnov, Vladimir Shulaev, and Nilgun E. Tumer* Center for Agricultura1 Molecular Biology and Department of Plant Pathology, Rutgers University, P.O. Box 231, New Brunswick, New Jersey 08903-0231

pokeweed (Phytolacca americana) plants. These are Pokeweed antiviral protein (PAP), a 29-kD protein isolated from similar in molecular mass (29, 30, and 29.5 kD, respec- Phytolacca americana, inhibits translation by catalytically removing tively) but are expressed at different developmental stages a specific adenine residue from the large rRNA of the 60s subunit of and in different tissues of pokeweed (Wyatt and Shepherd, eukaryotic ribosomes. Transgenic tobacco (Nicofiana fabacum) 1969; Irvin et al., 1980; Barbieri et al., 1982). PAP depuri- plants expressing PAP or a variant (PAP-V) were shown to be resis- nates ribosomes from pokeweed and other plants (Bonness tant to a broad spectrum of plant . Expression of PAP-v in et al., 19941, as well as mammalian, yeast, and bacterial transgenic plants induces synthesis of pathogenesis-related proteins ribosomes. In addition, PAI’ effectively inhibits infection and a very weak (~2-fold)increase in salicylic acid levels. Using by a number of different plant (Wyatt and Shepherd, 1969; reciproca1 grafting experiments, we demonstrate here that trans- Tomlinson et al., 1974; Chen et al., 1992) and animal viruses genic tobacco rootstocks expressing PAP-v induce resistance to (Tomlinson et al., 1974; Ussery et al., 1977), including hu- tobacco mosaic virus infection in both N. fabacum NN and nn scions. lncreased resistance to potato virus X was also observed in man immunodeficiency virus (Zarling et al., 1990). PAP N. tabacum nn scions grafted on transgenic rootstocks. PAP expres- also inhibits growth of tumor cells (Stirpe et al., 1992). sion was not detected in the wild-type scions or rootstocks that Positive correlations were reported between RIP-catalyzed showed virus resistance, nor was there any increase in salicylic acid depurination of tobacco ribosomes and antiviral activity of levels or pathogenesis-related protein synthesis. Grafting experi- exogenously applied RIPs (Taylor et al., 1994) and between ments with transgenic plants expressing an inactive PAP mutant the extent of inhibition of virus infection by PAP and the demonstrated that an intact active site of PAP is necessary for depurination of host ribosomes (Chen et al., 1992). induction of virus resistance in wild-type scions. These results We reported that transgenic tobacco and potato plants indicate that enzymatic activity of PAP is responsible for generat- expressing PAP show broad-spectrum resistance to infec- ing a signal that renders wild-type scions resistant to virus infec- tion by different viruses (Lodge et al., 1993). In this paper tion in the absence of increased salicylic acid levels and we studied the mechanism of this resistance by grafting pathogenesis-related protein synthesis. wild-type tobacco plants on transgenic tobacco plants ex- pressing PAP. Our results indicate that PAP expression in transgenic rootstocks of grafted plants induces resistance Many plants produce proteins that inactivate ribosomes to vira1 infection in wild-type scions in the absence of by depurinating rRNA in a highly conserved stem-loop detectable levels of PAP, SA accumulation, and PR-protein structure in the 28s RNA (Lord et al., 1991; Stirpe et al., synthesis. In contrast, transgenic plants expressing an 1992). Single-chain RIPs such as PAP and the A chains of active-site mutant PAP are not able to induce resistance in two-chain RIPs such as ricin remove an adenine base by grafted wild-type scions, demonstrating that enzymatic specific cleavage of the N-glycosidic bond at A4324 in rat activity of PAP is required to induce the resistant state in 28s rRNA and at homologous sites on ribosomes from systemic tissues. other organisms. Ribosomes depurinated in this manner are unable to bind the EF-2/GTP complex and protein synthesis is blocked at the translocation step (Montanaro et MATERIALS AND METHODS al., 1975; Osborn et al., 1990). Three different kinds of RIPs, Nontransgenic Nicotiana tabacum cv Samsun nn and NN PAP, PAPII, and PAP-S, have been purified from plants and transgenic N. tabacum cv Samsun nn plants

~~ This work was supported by National Science Foundation Abbreviations: ISR, induced systemic resistance; PAP, grant no. MCB-9419919 and a Johnson & Johnson Discovery grant pokeweed antiviral protein; PAP-v, variant PAP; PGPR, plant to N.E.T. and the New Jersey Commission on Science and Tech- growth-promoting rhizobacteria; PR proteins, pathogenesis- nology. related proteins; PVX, potato virus X; RIP, ribosome-inactivating * Corresponding author; e-mail [email protected]; fax protein; SA, salicylic acid; SAR, systemic acquired resistance; 1-908-932-6535. TMV, tobacco mosaic virus. 1113 1114 Smirnov et ai. Plant Physiol. Vol. 114, 1997 expressing PAP (Lodge et al., 1993) were used in grafting quantified by HPLC. Total SA (the sum of free and Glu- experiments. Homozygous seeds (R, generation) from conjugated SA) was determined as described by Yalpani et transgenic plant lines 33617-1 and 31634 expressing wild- al. (1993). type PAP, lines 26139-19,29491-1, and 29491-7 expressing the PAP-V, and line 144-12 expressing active-site mutant RESULTS PAP (Tumer et al., 1997) were used. PAP-v contains two amino acid changes (L20R and Y49H) (Lodge et al., 1993). Expression of PAP in Grafted Plants Grafts were prepared with 6-week-old scions and root- N. tabacum cv Samsun NN and nn plants were grafted stocks as described by Vernooij et al. (1994). Three weeks with homozygous (R,) progeny from the previously char- after grafting plants were treated with carborundum and acterized N. tabacum cv Samsun nn line 26139-19 express- four leaves of each plant were inoculated with TMV (U1 ing PAP-V, which contains two amino acid changes (L20R strain) in 50 mM phosphate buffer, pH 7.0. Inoculated and Y49H) and shows broad-spectrum resistance to vira1 grafted plants were kept in a growth chamber with a 16-h infection (Lodge et al., 1993). A schematic diagram of these photoperiod. The number and diameters of local lesions grafts is shown in Figure 1. Two different kinds of grafts were measured 7 d after inoculation. Lesion size was were made: the first had transgenic rootstocks and wild- determined for 20 lesions on each inoculated leaf by mea- type scions (T1 and T2); the second had wild-type root- suring the size of 10 lesions localized in a 1- to 2-cm2 area stocks and transgenic scions (T3 and T4). The efficiency of at the center of each half of a leaf. Sizes of 80 lesions per grafting with transgenic plants was approximately the plant were determined and the mean value 2 SD was same as with wild-type tobacco plants in 10 different calculated. Student’s t test was used to determine whether grafted plants of each type. The transgenic tobacco plants the lesion numbers or sizes were significantly different from line 26139-19 usually had symptoms of PAP expres- from the controls. , sion, including chlorotic lesions on the leaves and delayed Grafted plants with wild-type cv Samsun nn scions in- growth (Lodge et al., 1993). No such symptoms appeared oculated with TMV were monitored every 3 d for systemic on the wild-type scions grafted on transgenic rootstocks. mosaic symptom development. Two, three, and four weeks They grew well and flowered without delay. later, three leaf discs were sampled for ELISA from sys- Immunoblot analysis of T1-type grafted plants showed temically infected, upper scion leaves of each inoculated that PAP is expressed at high levels in rootstocks of plant plant. PVX (1 &mL) was inoculated on two bottom nos. 4,5,6,8,9, and 10 (Fig. 2A). PAP expression was very leaves of a wild-type scion or rootstock. Inoculated plants low in rootstocks of plant nos. 20 and 22 but was detectable were kept at 20°C in a growth chamber and samples (0.1 g) by ELISA. The variability in PAP expression among the RI for ELISA were taken from the uninoculated, upper wild- and homozygous R, progeny of line 26139 was previously type leaves. lmmunodetection of Proteins Immunoblot analysis was performed according to the method of Harlow and Lane (1988). Total leaf proteins were extracted with PBS buffer, separated by SDS-PAGE, blotted on a nitrocellulose membrane, and probed with antibodies using a ”Renaissance” chemiluminescence de- tection kit (DuPont). Monoclonal anti-PR-1 antibody, which recognizes the acidic forms of PR-1, was a gift of Dr. Graft c1 c2 c3 D. Klessig (Rutgers University, New Brunswick, NJ). tYPe ELISA detection of PAP, TMV, and PVX was performed ungrafted plants grafted controls according to the methods of Lodge et al. (1993).

RNA Analysis Total RNA was isolated from leaves with TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol. Total RNA was separated on rootstock b a 1.3% agarose gel containing formaldehyde, and RNA gel blots were hybridized with cDNA probes corresponding to PR proteins PR-1, PR-2, and PR-3 of tobacco (gifts of Dr. D. Graft T1 T2 T3 T4 Klessig). tYPe chimeric grafted plants SA Determination Figure 1. Schematic diagram of the grafting experiments. N and n, SA levels in scions and rootstocks of grafted plants were Wild-type N. tabacum cv Samsun NN and nn plants, respectively; T, determined 3 weeks after grafting and before virus inocu- transgenic N. tabacum cv Samsun plants expressing wild-type, vari- lation. SA was extracted from leaf samples (0.3 g) and ant, or active-site mutant PAP. Pokeweed Antiviral Protein-Induced Resistance 1115

synthesis in the transgenic rootstocks but not in the wild- type scion f grafteo s d plants. Detectable amountR P f o s 4 5 « 9 10 20 22 N T PAP proteins are not synthesized in wild-type scions or trans- SRSRSRSRSR SR~ SR SR located from transgenic rootstocks inte wild-typth o e scions.

Leve f SGraften o Ali d Plants Total SA levels were determined in the rootstocks and scion Tl-typf so e grafted plants levelA .S graften si d plants 7 11 15 2 < 10 n/n-1 n/n-3 T from lin. 26139-1eno ng/7 3 9 g ± wer fres1 e32 h weight, S—If S—K~ 5—R" S—K S—R S—R~ 5—IT 5—R compared with 187 ±11 ng/g fresh weight in Cl-type grafted wild-type tobacco plants (Table I). Similarly, as show Tabln i sligha , eI t increas level A detectes S n eswa i d transgenie inth c rootstock f Tl-typo s e grafted plantsn I . contrast, no increase in SA levels was observed in the Figure 2. Analysis of PAP expression in grafted tobacco plants of T1 wild-type scions (Tabl levelA S slightle . esI) ar y elevaten di and T2 types. Protein (10 /xg) was fractionated on 15% SDS- transgenic rootstocks and not in wild-type scions, possibly polyacrylamid d analyzean l ege y immunoblo db t analysi- de s a s because detectabl t synthesizeno ee amountar A S n di f o s scribe "Materialn di Methods.d san , Grafte"A d plant typ1 T ef s o nos. the scion or translocated to the scion from the transgenic , , 8wit102 6 9 , . 2 tabacum, , h N 5 d 20 , 4 an , cv Samsun NN scions rootstock. and line 26139-19 rootstocks. S, Scions; R, rootstocks; N, N. taba- cvm Samsucu n NN;T, transgenic tobacco line 26139-19 PAPd ;an , 10 ng of purified PAP. B, Grafted plants of T2 type nos. 7, 11, 15, 2, Resistance to TMV in Grafted Plants 6, and 10 with N. tabacum cv Samsun nn scions and line 26139-19 To determine whether wild-type plants grafted with rootstocks. n/n-1 and n/n-3, Control grafted plants of C3 type; T, PAP-expressing transgenic plants are resistant to TMV, transgenic tobacco 26139-19. wild-type leaves of the Tl and T3 types of grafted plants were inoculated with 5 /Mg/mL TMV. Corresponding reported (Lodg al.t ee , 1993; Turne al.t re , 1997) source .Th e type2 C graftef d leaveso C e an lth f dso control plants were of this t variatiounknownye s a s ni . Ther s similawa e r also inoculated wit jig/mh5 L TMV shows .A Tabln ni , eII variatio expressioP PA n ni T2-typn ni e grafted plants (Fig. grafte l typdT plante e th showe f o s increasen a d d resis- 2B). Plant nos. 2, 7,10,11, and 15 had high levels and plant tance to TMV in wild-type cv Samsun NN scions. The expressionP verd levelw ha PA ylo 6 f o s. no , whics hwa number of local lesions observed on the wild-type scion detectabl ELISAy eb contrastn I . expressioP t PA ,no s nwa leaves was approximately half the lesion numbers on wild- detected in the wild-type scions of either Tl- or T2-type type scion leaves of Cl-type controls (Table II). Local le- grafted plants (Fig. 2) or in grafted control plants of the C3 sions observe scion o d n leave f Tl-typo s e grafted plants type (Fig. 2B). were also significantly reduce sizn di e (Table II). Grafte typ3 dT eplante demonstrateth f so d resistanco et Expression of Genes Encoding PR Proteins in TM wild-typVn i e rootstocks (Tabl , experimeneII e Th . 2) t Grafted Plants number and size of virus-induced lesions on the leaves of Tl-type grafted plants were analyzed for expression of the cv Samsun NN rootstocks were significantly reduced pathogenesis-related protein PR-la by immunoblot analy- compared wit e numbeth h d siz f lesionan o er rootn o s - sis. As shown in Figure 3, transgenic rootstocks that ex- stocks of C2-type control plants (Table II). These results pressed PAP expressed PR-1 constitutively. The level of demonstrate that wild-type tobacco plants grafted with PR-1 expression in the rootstocks was comparable to that observe leavee . tabacumth N n f i dso Samsuv c plantN nN s Scions Rootstocks TMV 6 d after TMV inoculation (FigN N . 3)T .2 2 In 0 contrast2 5 1 3 1 0 1 , PR- 9 6 1 expresS 2 2 0 -3 2 I1 S 0 1 9 6 5 t detecte wild-type no th sios n ni wa d e scions graften o d transgenic rootstocks t detecteno . s Expressiowa d 1 PR f no rootstockn i (Figs2 2 . froRootstock r 3) .o m 5 1 plan . no ts from plant no. 22 (Fig. 2A) and no. 15 (data not shown) had low levels of PAP expression, which was detectable only by ELISA. Expressio PR-1f no , PR-2 PR-d ,an 3 gene graften si d plant determines swa d using northern-blot analysis. Tran- scripts corresponding to PR-1, PR-2, and PR-3 were de- Figure 3. Analysis of PR1 expression in grafted tobacco plants. Pro- tein (3 /xg) from leaves of grafted plants of T1 type nos. 5, 6, 9, 10, tecte transgenin di c rootstockn i st expressinno t bu P gPA 13, 15, 20, and 22 was fractionated on 15% SDS-polyacrylamide gel wild-type scions. A positive signal was detected with all and analyzed by immunoblot analysis as described in "Materials and three probe TMV-infecten i s d tobaccn i ot plantno t bu s Methods. , TransgeniT " c tobacco line 26139-19 , wild-typN ; . N e uninfected tobacco plants (dat t shown)ano . These results tabacum cv Samsun NN plants inoculated with ( + ) TMV or unin- demonstrate that expression of PAP-v induces PR-protein oculated (-). 1116 Smirnov et al. Plant Physiol. Vol. 114, 1997

Table 1. Analysis of PAP and SA levels in grafted tobacco plants

Crafted Plant Type of Level of PAP Level of SA (Scion/Rootstock) Graft Scion Rootstock Scion Rootstock ng PAP/mg total proteina ng SA/g fresh wt tissueb NNInn‘ c1 O NA^ 21 8.6 t 17.1 187.2 t 11.3 NN/26139-19c T1 O 111.2t 18.1 214.6 t 23.3 313.7 t 35.7

a Three weeks after grafting, 6 to 10 grafted plants of each type were analyzed by ELISA for PAP expression in scions and rootstocks. Mean values t SD are shown. SA levels in scions and rootstocks of grafted plants were determined 3 weeks after grafting. Total SA content was determined as described in ”Materials and Methods” in 6 to 10 grafted plants of each type. Four to 10 samples (0.3 g) were analyzed for each plant. The data presented are the means 2 SD. NN, cv Samsun NN; nn, cv Samsun nn; 26139-19, homozygous progeny of transgenic line 26139 expressing PAP-V. NA, Not analyzed.

PAP-expressing transgenic plants show resistance to TMV TMV-induced local lesions on wild-type scions, this corre- infection, regardless of the position of the transgenic plants lation was not statistically significant. in the graft. To determine whether N.tabacum cv Samsun nn scions TMV resistance was observed when transgenic plants grafted on transgenic rootstocks were resistant to TMV, from lines 33617 and 31634, expressing wild-type PAP, and scion leaves of T2-type grafted plants and C3-type controls line 29491, expressing PAP-V, were used in T1-type grafts were inoculated with 1.0 and 0.1 pg/mL TMV. After virus (Table 11, experiment 3). Grafted wild-type scions wifh inoculation, plants were incubated in a growth chamber, rootstocks from lines 33617 and 29491 were resistant to viral symptoms on the scion leaves were scored visually, TMV, whereas grafted wild-type scions with rootstocks and virus antigen levels were determined by ELISA. As from line 31634 did not show significant TMV resistance. shown in Figure 4A, mosaic symptoms appeared on wild- This transgenic line showed the lowest level of virus resis- type cv Samsun nn scions with transgenic rootstocks iater tance when tested against PVX and PVY in earlier experi- than the symptoms on wild-type cv Samsun nn scions ments (Lodge et al., 1993). grafted with wild-type cv Samsun nn rootstocks. More- We analyzed SA levels in transgenic rootstocks and wild- over, 4 weeks after virus inoculation with either 0.1 or 1 type scions of T1-type grafted plants to determine whether pg/mL TMV, about 50% of grafted plants with transgenic there is a correlation between viral resistance in wild-type rootstocks had no visible symptoms on their wild-type scions and SA levels in the transgenic rootstocks. Average scion leaves. As shown in Figure 4B, virus accumulation SA levels in the transgenic rootstocks were slightly higher was reduced in wild-type scions with transgenic rootstocks than in the wild-type scions. However, although the data as compared with virus accumulation in wild-type scions shown in Table I11 suggested a negative correlation be- with wild-type rootstocks. Symptom development and tween SA levels in transgenic rootstocks and the size of TMV antigen levels were both reduced in T2-type grafted

. Table II. Susceptibility of grafted N. tabacum cv Samsun NN plants to TMV infection Crafted Plants Craft No. of No. of Size of Lesions (Scion/Rootstock) Type Plants Analyzed Lesions per Leaf”

Experiment 1 NN/nn c1 4 235 2 9 1.31 t 0.12 “1261 39-1 9 T1 8 122 ? 51‘ 0.71 2 0.14d Experiment 2e nn/NN c2 4 710 -+ 187 1.39 t 0.24 261 39-191” T3 8 264? llOd 0.61 2 0.17d Experiment 3 NN/nn c1 207 t 54 1.89 t 0.34 NN/29491-1 T1 61 t 34d 1.30 t 0.14d “129491 -7 T1 75 t 25d 1.28 t 0.31‘ “13361 7-1 T1 65 ir 28d 1.O5 t 0.71 “131634 T1 194 t 8% 1.50 t 0.54

a Four leaves of grafted cv Samsun NN plants were inoculated with 200 pL of 5 pg/mL TMV and the lesions on inoculated leaves were counted 7 d postinoculation. Four to eight plants of each type were inoculated and kept in a growth chamber at 23°C. Sizes of 80 local lesions per plant were measured and then a mean value of the size of local lesions and SD was calculated for each type of grafted plant. An average number of local lesions ? SD per leaf is shown. Significantly different from control at 1% level. Significantly different from control at 0.1% level. e Two rootstock leaves were inoculated. Pokeweed Antiviral Protein-lnduced Resistance 1117

Table 111. Comparison of SA levels and TMV resistance in grafted were analyzed by ELISA (Fig. 5). Only 20% of T2- and plants T4-type grafted plants contained PVX antigen. In contrast, 80% of C3-type control plants contained detectable levels of Leve1 of SAb Size of TMV-lnduced Planta Scion Rootstock Local Lesions PVX antigen by ELISA. These results demonstrate that PAP expression in transgenic rootstocks or scions induces resis- ng SMg fresh wt tissue mm tance to PVX in wild-type cv Samsun nn scions or root- N N/T-4 364 2 87 373 2 40 1.17 -t 0.10 stocks, respectively. NN/T-5 203 2 19 523 2 25 0.95 ? 0.06 NN/T-6 215 2 13 368 2 39 0.98 It 0.08 N N/T-8 1762 18 324? 1 0.79 t 0.06 NN/T-9 192 26 29024 1.O7 2 0.1O NN/T-13 307 2 84 348 2 105 1 .O8 t 0.08 NN/T-15 133t7 181 21 1.20 +- 0.10 NNn-20 1932 18 211 29 1.10 5 0.10 NN/T-22 201 224 NA 1.O4 +- 0.06 A Average 220 2 71 327 2 106' 1.O4 2 0.1 2d NNInn-2 NA NA 1.34 2 0.1 2 NN/nn-3 236 2 19 198 2 36 1.54 2 0.20 1251 NN/nn-11 NA NA 1.36 2 0.14 NN/nn-15 219 +- 17 187 2 11 1.35 2 0.20 Average 227212 193 28 1.40 2 0.1 O

a NN/T, Crafted plants of T1 type with transgenic rootstocks from c111 the homozygous plant line 261 39-19. NN/nn, Control grafted plants P -a of C1 type. The numbers of individual plants correspond to those shown in Figures 2A and 3. SA levels in scions and rootstocks of grafted plants were determined as described in "Materials and Methods" 3 weeks before virus inoculation. Two samples were an- alyzed for each plant. The results represented are the means 2 SE. Four leaves of wild-type scions were inoculated with 200 p,L of 1 5 10 15 20 25 30 &nLTMV. Plants were incubated in a growth chamber at 23°C for 7 d and sizes of local lesions were measured. Mean values & SD are B shown. Analysis of correlation between size of lesions and SA levels in scions and rootstocks of grafted transgenic plants show the Pear- son value for scions, r = 0.247 (k = 9-2 = 7), and for rootstocks, r = -0.478 (k = 8-2 = 6). Null hypothesis cannot be eliminated for both cases at level 5%. Significantly different from control at 1% level. Significantly different from control at 0.1% level. plants compared with C3-type controls. Analysis of protein extracts by SDS-PAGE showed that wild-type scions that did not show symptoms or contain TMV by ELISA did not contain TMV coat protein (data not shown). Mosaic symp- toms on wild-type cv Samsun nn scions with transgenic rootstocks were weaker than on control plants with wild- type cv Samsun nn rootstocks. These results demonstrate that PAP expression in transgenic rootstocks induces TMV Days after inoculation resistance in cv Samsun nn scions. TMV resistance is in- duced in both hypersensitive (cv Samsun NN) and sys- temic (cv Samsun nn) hosts of TMV, suggesting that the U nd26139, 0.1 pglml TMV induced resistance is not due to the hypersensitive re- ...... 0...... nd26139, 1.0 pg/mI TMV sponse to TMV infection observed in cv Samsun NN plants. .--.o..__ nn/nn, 0.1 pg/d TMV Resistance to PVX in Crafted Plants ----A--- ndnn, l.Opg/d TMV Resistance to PVX has been analyzed in the T2 and T4 Figure 4. Qsistance to TMV infection in grafted tobacco plants with types of grafted plants with scions or rootstocks from the cv Samsun nn scions. Three bottom leaves of Samsun nn scions were transgenic line 26139-19. C3-type grafted plants were used inoculated with 1 .O or 0.1 pg/mL TMV. Ten plants with transgenic 261 39 rootstock and O) and 4 plants with Samsun nn rootstock as controls. Three weeks after grafting, wild-type cv Sam- (O (O and A) were used in each experiment. Plants were grown in a sun nn scions or rootstocks were inoculated with 1 pg/mL growth chamber at 20°C. A, Symptom development on cv Samsun nn PVX on two bottom leaves and kept in a growth chamber. scions; B, TMV accumulation in systemically infected cv Samsun nn Three weeks postinoculation, samples from systemically scions. Samples (0.1 g) were taken from systemically infected leaves infected scion leaves (T2 and C3) or rootstock leaves (T4) and TMV levels were determined by ELISA. 1118 Smirnov et al. Plant Physiol. Vol. 114, 1997

site mutant PAP were 3 times higher than the level of PAP-v expressed in transgenic rootstocks of line 26139-19 (Table IV). SA levels in ungrafted plants from line 144-12 were not significantly different from the SA levels in wild- type control plants (data not shown). As shown in Table IV, wild-type scions grafted on transgenic rootstocks express- ing the active-site mutant PAP demonstrated no resistance to TMV. In contrast, wild-type scions with transgenic root- stocks expressing PAP-v had fewer TMV lesions and the size of the TMV lesions on these plants was significantly reduced compared with the lesions on control plants (Table IV). These results demonstrate that an intact active site of PAP is necessary for induction of virus resistance in wild- type scions grafted on transgenic rootstocks.

dm nnlt6139 M139b I SCUSSl O N Figure 5. Susceptibility of grafted tobacco plants to PVX.'Two bot- D tom scion leaves of grafted nn/26139 plants were inoculated with We have previously shown that transgenic plants ex- 100 pL of PVX (1 &mL). Three weeks after inoculation 0.1-g pressing PAP are resistant to broad-spectrum virus infec- samples were taken from systemically infected scion (nn) leaves and tion (Lodge et al., 1993). Using reciproca1 grafting experi- analyzed for PVX by ELISA. Two bottom rootstock leaves of grafted ments, we demonstrate here that expression of PAP in 261 39/nn were inoculated with PVX, and 3 weeks after inoculation systemically infected rootstock (nn) leaves were analyzed by ELISA. transgenic plants induces resistance to TMV infection in Ten plants from each type of graft were used in the experiment. wild-type scions or rootstocks. PAP-induced TMV resis- nn/nn, Samsun nn scionfamsun nn rootstock; nn/26139, Samsun nn tance was observed in both N. tabacum NN and N. tabacum scion/transgenic line 261 39-1 9 rootstock; and 261 39/nn, transgenic nn scions grafted on transgenic rootstocks. Increased resis- line 261 39-1 9 scionlsamsun nn rootstock. tance to PVX was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type parts of grafted plants, nor was Resistance to TMV in Wild-Type Plants Crafted with there any increase in SA levels and PR-protein synthesis. Transgenic Plants Expressing an Active-Site Mutant PAP Grafting experiments with transgenic plants expressing an Recently, a number of different nontoxic PAP mutants inactive PAP mutant demonstrated that an intact active site were isolated and characterized by our group (Hur et al., of PAI' is necessary for induction of virus resistance in 1995). One of these mutants had a mutation at its active site grafted wild-type plants. (E176V) and had lost ribosome depurination activity (Hur Irvin (1995) suggested that a possible biological role of et al., 1995). This mutant was not antiviral when exog- RIPs in plants is to prevent natural grafting. Inhibition of enously applied to tobacco leaves, and transgenic tobacco grafting was not observed when plants expressing PAP plants expressing the active-site mutant were not resistant were grafted with wild-type plants. The percentage of suc- to vira1 infection (Tumer et al., 1997). To determine cessful grafts was similar for grafts between transgenic and whether enzymatic activity of PAP is required for the wild-type plants compared with grafts between wild-type resistance observed in grafted plants, T1-type grafts were plants (data not shown). However, the level of PAP expres- made with transgenic rootstocks from line 144-12, which sion in transgenic tobacco is much lower than in pokeweed, expressed high levels of the active-site mutant (Tumer et in which it may interfere with grafting. al., 1997). C1-type grafted plants were used as controls. As previously reported, transgenic tobacco plants from PAP levels in transgenic rootstocks expressing the active- line 26139 expressing PAP-v are stunted and show chlo-

Table IV. Susceptibility of grafted plants expressing the active-site mutant PAP to TMV Crafted Plants No. of Lesions PAP Levela Size of Lesionsc (Scion/Rootstock) per Leafb ns/ms mm cv Samsun NN/Samsun nn 0 400 2 83 1.88 2 0.27 cv Samsun NN/26139-19 NA^ 189 -+ 77e 1.26 2 0.1 5' cv Samsun NN/144-12 307.9 2 125.7 439 2 151 1.75 2 0.16

a PAP levels in rootstocks of 10 different grafted plants of each type were analyzed by ELISA. Mean values 2 SD are shown. Line 144-12 expresses the active-site mutant PAP and line 26139-19 expresses PAP-V. Four leaves of cv Samsun NN scions were inoculated with TMV as previously described. Eight plants of each type were inoculated. Average nos. of local lesions per leaf ? SD are shown. Sizes of 80 local lesions per plant were measured and the mean value & SD for each type of graft was calculated. The level of PAP in T1 -type grafted plants (cv Samsun NN/26139-19) is 11 1.2 5 18.1 as shown in Table I. e Significantly different from control at 0.1% level. Pokeweed Antiviral Protein-lnduced Resistance 1119 rotic lesions (Lodge et al., 1993). These symptoms were not (Yalpani et al., 1991). Our results show that grafted trans- detected on wild-type plants grafted on transgenic plants. genic plants expressing PAP constitutively synthesize PR-1 The absence of PAP in wild-type scions was confirmed by at similar levels to TMV-inoculated N. tabacum cv Samsun immunoblot and ELISA analyses. Although PAP expres- NN leaves (Fig. 3). Constitutive expression of other PR sion was not detected in wild-type scions, they showed proteins was observed by northern-blot analysis. However, fewer TMV lesions and smaller lesions compared with the transgenic rootstocks expressing PAP did not induce PR- lesions on wild-type scions grafted on wild-type root- protein synthesis in wild-type scions. These results suggest stocks. The induced resistance was observed with severa1 that PAP induces PR-protein expression locally in cells different independently transformed plant lines. Resistance containing the PAP gene rather than systemically. to TMV was observed with both Samsun NN and nn plants SA levels in wild-type scions grafted on transgenic root- grafted on PAP-expressing transgenic plants, suggesting stocks were not significantly different from the SA levels in that it does not depend on the hypersensitive response. wild-type scions grafted on wild-type rootstocks. Analysis This resistance was not restricted to TMV but was also of virus resistance in grafted transgenic and wild-type effective against PVX, which belongs to a different virus plants did not show a strong correlation between TMV group. These results suggest that PAP expression in resistance and SA levels. SA levels in ungrafted plants from grafted transgenic plants generates a signal that can trans- line 33617, expressing PAP, were not significantly different locate across the graft union and induce nonspecific viral from wild-type tobacco plants (O. Zoubenko and N. Tu- resistance in grafted wild-type tissues. mer, unpublished data). Yet wild-type scions grafted with In many plants, including tobacco, the primary infection rootstocks from line 33617 showed resistance to TMV (Ta- with necrotizing pathogen or treatment with certain chem- ble II), indicating that PAP-induced resistance observed in icals can trigger enhanced systemic resistance of the plant wild-type scions is not due to an increase in SA levels. to subsequent infection by a variety of pathogens. This Similarly, we did not observe any correlation between nonspecific resistance is known as SAR. SA is the most PAP-induced virus resistance and PR-protein expression. probable signal molecule for SAR in plants (Malamy et al., Some plants did not produce detectable amounts of PR-1 in 1990; Ward et al., 1991). There is evidence that SA is a their transgenic rootstocks but still showed virus resistance long-distance, phloem-transmissible signal that moves in their wild-type scions (Fig. 3; Table 111). These results from the site of initial pathogen infection throughout the suggest that the slight increase in SA levels observed in plant (Yalpani et al., 1991). Transgenic tobacco plants ex- transgenic rootstocks and the constitutive synthesis of PR pressing a salicylate hydroxylase gene (nahG) from Pseudo- proteins have no effect on PAP-induced virus resistance in monas putida do not manifest a SAR response (Gaffney et wild-type scions. Experiments with grafted plants in which al., 1993). Recent results from studies of in vivo-labeled SA SAR was activated with TMV pretreatment showed that show that most of the SA detected in the healthy tissue PAP expression induces virus resistance that is indepen- after TMV inoculation is synthesized in the inoculated dent of the SAR response (S. Smirnov and N. Tumer, leaves and transported to the dista1 parts of the plants unpublished data). These results suggest that PAP expres- (Shulaev et al., 1995). sion induces a pathway different from the classic SAR and In certain cases, SAR can be activated in the absence of a that this pathway leads to systemic resistance indepen- pathogen. These include "disease lesion-mimic mutants" of dently of SA accumulation and PR-protein expression. maize, barley, tomato, and Arabidopsis, and transgenic We recently showed that transgenic tobacco plants ex- plants expressing certain transgenes. Transgenic plants ex- pressing an active-site mutant PAP are not resistant to viral pressing the bacteriopsin gene from Halobacterium kalobium infection, indicating that an intact active site of PAP is contain high levels of SA, constitutively express PR pro- required for the antiviral activity of PAP (Tumer et al., teins, and show resistance to TMV (Mittler et al., 1995). 1997). The results presented here demonstrate that an Transgenic tobacco plants expressing cholera toxin accu- active-site mutant PAP does not induce virus resistance in mulate SA and PR proteins and show nonspecific viral wild-type scions. These results suggest that enzymatic ac- resistance (Beffa et al., 1995). Expression of yeast invertase tivity of PAP is responsible for generating a signal that increases the SA levels and induces SAR in transgenic moves across the graft union to the wild-type plants, where tobacco (Herbers et al., 1996). These observations sug- it induces virus resistance. This signal can be translocated gested that induction of resistance in PAP-expressing to the upper as well as to the lower parts of the grafted transgenic plants may also be mediated by the SAR re- plants and is not linked to SA accumulation or induction of sponse. Surprisingly, SA levels in PAP-v-expressing trans- PRl synthesis. genic plants were very low. Only a 1.7-fold increase in SA ISR has been observed in plants growing in contact with levels was observed in transgenic plants from line 26139-19 selected nonpathogenic, root-colonizing biocontrol rhi- compared with the control plants. zobacteria (Alstrom, 1991; van Peer et al., 1991; Wei et al., In contrast, TMV inoculation of NN plants causes 15- to 1991). The PGPR-mediated systemic resistance is indepen- 40- and 5- to 10-fold increases of SA levels in inoculated dent of SA accumulation and PR-gene expression as well and systemic leaves, respectively (Malamy et al., 1990). (Pieterse et al., 1996). PGPR-mediated ISR against tobacco Five- to 10- fold increases in SA levels are necessary for necrosis virus has been demonstrated for tobacco plants efficient induction of PR-protein synthesis. Smaller in- (Maurhofer et al., 1994). Extracellular rhizobacterial lipo- creases in SA levels induce very weak PR-protein synthesis polysaccharides are sufficient to elicit ISR in plants (van 1120 Smirnov et al. Plant Physiol. Vol. 114, 1997

Peer and Shippers, 1992; Leeman et al., 1995). We demon- Leeman M, van Pelt JA, den Ouden FM, Heinsbroek M, Bakker strate here that PAP-induced virus resistance, like PGPR- PAHM, Schippers B (1995) Induction of systemic resistance against fusarium wilt of radish by lipopolysaccharides of mediated ISR, does not depend on SA levels and PR-gene Pseudomonas fluorescens. Phytopathology 85: 1021-1027 expression. However, PAP-induced virus resistance and Lodge JK, Kaniewski WK, Tumer NE (1993) Broad-spectrum PGPR-mediated ISR differ significantly, at least in the first virus resistance in transgenic plants expressing pokeweed anti- step of resistance induction. PGPR-dependent ISR is acti- viral protein. Proc Natl Acad Sci USA 90: 7089-7093 vated by rhizobacteria via lipopolysaccharides and PAP- Lord JM, Hartley MR, Roberts LM (1991) Ribosome inactivating proteins of plants. Semin Biol 2: 15-22 induced resistance is mediated by the enzymatic activity of Malamy J, Carr JP, Klessig DF, Raskin I (1990) Salicylic acid: a PAP. These results suggest that resistance induced by an likely endogenous signal in the resistance response of tobacco to SA-independent signaling pathway, different from that viral infection. Science 250 1002-1004 controlling SAR, can be triggered by at least two different Maurhofer M, Hase C, Meuwly P, Metraux J-I', Defago G (1994) inducers. Further analysis of PAP-mediated resistance will Induction of systemic resistance of tobacco to tobacco necrosis virus by the root-colonizing Pseudomonas fluorescens strain provide important information concerning the regulation CHAO: influence of the gacA gene and pyoverdine production. of this pathway. Phytopathology 84: 139-146 Mittler R, Shulaev V, Lam E (1995) Coordinated activation of programmed cell death and defense mechanisms in transgenic ACKNOWLEDCMENTS tobacco plants expressing a bacterial proton pump. Plant Cell7: 29-42 We would like to thank Drs. Peter Day, Ilan Chet, Ilya Raskin, Montanaro L, Sperti S, Mattioli A, Testoni G, Stirpe F (1975) and Michael Lawton for critica1 reading of the manuscript and Dr. Inhibition by ricin of protein synthesis in vitro. Biochem J 146: Dan Klessig for PRI monoclonal antibody and cDNA probes 127-131 Osborn RW, Hartley MR (1990) Dual effects of the ricin A chain against PRl, PR2, and PR3. on protein synthesis in rabbit reticulocyte lysate. Eur J Biochem 193: 401-407 Received December 23,1996; accepted April 15,1997. Pieterse CM, van Weels SCM, Hoffland E, van Pelt JA, van Loon Copyright Clearance Center: 0032-0889/97/ 114/1113/09. LC (1996) Systemic resistance in Arabidopsis induced by biocon- trol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression. Plant Cell 8 1225-1237 LITERATURE ClTED Shulaev V, Leon J, Raskin I (1995) 1s salicylic acid a translocated signal of systemic acquired resistance in tobacco? Plant Cell 7: Alstrom S (1991) Induction of disease resistance in common bean 1691-1701 susceptible to halo blight bacterial pathogen after seed bacteri- Stirpe F, Barbieri L, Battelli MG, Soris M, Lappi DA (1992) sation with rhizosphere pseudomonas. J Gen Appl Microbiol37: Ribosome-inactivating proteins from plants. Biotechnology 10: 495-501 405412 Barbieri L, Aron GM, Irvin JD, Stirpe F (1982) Purification and Stirpe F, Hugnes RC (1989) Specificity of ribosome inactivating partia1 characterization of another form of the antiviral protein proteins with N-glycosidase activity. Biochem J 262 1001-1002 from seeds of Phytolacca americana L (pokeweed). Biochem J 203: Taylor S, Massíah A, Lomonossoff G, Roberts LM, Lord JM, 55-59 Hartley M (1994) Correlation between the activities of tobacco Beffa R, Szell M, Meuwly P, Pay A, Vogeli-Lange R, Metraux J-P, ribosome-inactivating proteins in depurination of tobacco ribo- Neuhaus G, Meins F, Nagy F (1995) Cholera-toxin elevates somes and inhibition of tobacco mosaic virus infection. Plant J 5 pathogen resistance and induces pathogenesis-related gene ex- 827-835 pression in tobacco. EMBO J 145753-5761 Tomlinson JA, Walker VM, Flewett TH, Barclay GR (1974) The Bonness MS, Ready MP, Irvin JD, Mabry TJ (1994) Pokeweed inhibition of infection by cucumber mosaic virus and antiviral protein inactivates pokeweed ribosomes; implications virus by extracts from Phytolacca americana. J Gen Viro1 22 for the antiviral mechanism. Plant J 5 173-183 225-232 Chen ZC, Antoniw JF, White RF, Lin Q (1992) Effect of pokeweed Tumer NE, Hwang DJ, Bonness M (1997) A nontoxic C-terminal antiviral protein (PAI') on the infection of plant viruses. Plant deletion mutant of pokeweed antiviral protein inhibits viral Pathol 40 612-620 infection. Proc Natl Acad Sci USA 94 3866-3871 Gaffney T, Friedrick L, Vernooij B, Negrotto D, Nye G, Uknes S, Ussery MA, Irvin JD, Hardesty B (1977) Inhibition of poliovirus Ward E, Kessmann H, Ryals J (1993) Requirement of salicylic replication by a plant antiviral peptide. Ann NY Acad Sci 284: acid for the induction of systemic acquired resistance. Science 431-440 261: 754-756 van Peer R, Niemann GJ, Schippers B (1991) Induced resistance Harlow E, Lane D (1988) Immunoblotting. In Antibodies. A Lab- and phytoalexin accumulation in biological control of fusarium oratory Manual. Cold Spring Harbor Laboratory, Cold Spring wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopa- Harbor, NY, pp 471-510 thology 81: 728-734 Herbers K, Meuwly P, Frommer WB, Metraux J-P, Sonnewald U van Peer R, Shippers B (1992) Lipopolysaccharides of plant (1996) Systemic acquired resistance mediated by the ectopic growth promoting Pseudomonas sp strain WCS417R induce re- expression of invertase: possible hexose sensing in the secretory sistance in carnation to fusarium wilt. Neth J Plant Pathol 98: pathway. Plant Cell 8: 793-803 129-139 Hur Y, Hwang D-J, Zoubenko O, Coetzer C, Uckun, F, Tumer NE Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, (1995) Isolation and characterization of pokeweed antiviral pro- Ward E, Uknes S, Kessmann H, Ryals J (1994) Salicylic acid is tein mutations in Saccharomyces cerevisiae: identification of resi- not the translocated signal responsible for inducing systemic dues important for toxicity. Proc Natl Acad Sci USA 92 8448-8452 acquired resistance but is required in signal transduction. Plant Irvin JD (1995) Antiviral proteins from Phytolacca. In M Chessin, Cell 6: 959-965 D DeBorde, A Zipf, eds, Antiviral Proteins in Higher Plants. Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, CRC, Boca Raton, FL, pp 65-94 Alexander DC, Ahl-Goy P, Metraux J-I', Ryals JA (1991) Coor- Irvin JD, Kelly T, Robertus JD (1980) Purification and properties dinate gene activity in response to agents that induce systemic of a second antiviral protein from Phytolacca americana which acquired resistance. Plant Cell 3: 1085-1094 inactivates eukaryotic ribosomes. Arch Biochem Biophys 200 Wei G, Kloepper JW, Tuzun S (1991) Induction of systemic resis- 418-425 tance of cucumber to Colletofrichum orbiculnre by select strains of Pokeweed Antiviral Protein-lnduced Resistance 1121

plant growth-promoting rhizobacteria. Phytopathology 81: Yalpani N, Silverman P, Wilson TMA, Kleier DA, Raskin I (1991) 1508-1512 Salicylic acid is a systemic signal and an inducer of Wyatt SD, Shepherd RJ (1969) Isolation and characterization of a pathogenesis-related proteins in virus-infected tobacco. Plant virus inhibitor from Phytolacca americana. Phytopathology 59: Cell3: 809-818 1787-1 793 Zarling JM, Maran PA, Haffar D, Sias J, Richman DD, Spina CA, Yalpani N, Shulaev V, Raskin I (1993) Endogenous salicylic acid Myers DA, Kuebelbeck V, Ledbetter JA, Uckun FM (1990) In- levels correlate with accumulation of pathogenesis-related pro- hibition of HIV replication by pokeweed antiviral protein tar- teins and virus resistance in tobacco. Phytopathology 83: 202-208 geted CD4+ cells by monoclonal antibodies. Nature 347: 92-95