Plant Physiol.

(1997) 114: 11 13-1 121

Expression of Pokeweed Antiviral Protein in Transgenic Plants

lnduces Virus 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 proteins 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 t o be resis- nates ribosomes from pokeweed and other plants (Bonness
tant t o a broad spectrum of plant viruses. 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 - f o l d )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 t o
tobacco mosaic virus infection in both N. fabacum N N and nn
(Tomlinson et al., 1974; Ussery et al., 1977), including hu-
scions. lncreased resistance t o 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 i n 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 i s necessary for depurination of host ribosomes (Chen et al., 1992).
induction of virus resistance i n wild-type scions. These results We reported that transgenic tobacco and potato plants
indicate that enzymatic activity of PAP i s responsible for generat- expressing PAP show broad-spectrum resistance to infec-
ing a signal that renders wild-type scions resistant t o 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
MATERIALS A N D METHODS
synthesis is blocked at the translocation step (Montanaro et
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 tumer@aesop.rutgers.edu; 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
ungrafted plants grafted controls
ELISA detection of PAP, TMV, and PVX was performed
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 n n 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 scions of grafted plants. Detectable amounts of PR
4 5 « 9 10 20 22 N T PAP
proteins are not synthesized in wild-type scions or trans-
S R S R S R S R S R SR~ S R S R located from transgenic rootstocks into the wild-type
scions.

Level of SA in Grafted Plants

Total SA levels were determined in the rootstocks and

scions of Tl-type grafted plants. SA levels in grafted plants
7 11 15 2 < 10 n/n-1 n/n-3 T from line no. 26139-19 were 321 ± 37 ng/g fresh weight,
S—If S—K~ 5—R" S—K S—R S—R~ 5—IT 5—R compared with 187 ± 1 1 ng/g fresh weight in Cl-type
grafted wild-type tobacco plants (Table I). Similarly, as
shown in Table I, a slight increase in SA levels was detected
in the transgenic rootstocks of Tl-type grafted plants. In
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 (Table I). SA levels are slightly elevated in
and T2 types. Protein (10 /xg) was fractionated on 15% SDS- transgenic rootstocks and not in wild-type scions, possibly
polyacrylamide gel and analyzed by immunoblot analysis as de- because detectable amounts of SA are not synthesized in
scribed in "Materials and Methods." A, Grafted plants of T1 type nos. the scion or translocated to the scion from the transgenic
4, 5, 6, 8, 9, 10, 20, and 22 with N. tabacum cv Samsun NN scions
rootstock.
and line 26139-19 rootstocks. S, Scions; R, rootstocks; N, N. taba-
cum cv Samsun NN;T, transgenic tobacco line 26139-19; and PAP,
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
rootstocks. n/n-1 and n/n-3, Control grafted plants of C3 type; T,
To determine whether wild-type plants grafted with
transgenic tobacco 26139-19.
PAP-expressing transgenic plants are resistant to TMV,
wild-type leaves of the Tl and T3 types of grafted plants
were inoculated with 5 /Mg/mL TMV. Corresponding
reported (Lodge et al., 1993; Turner et al., 1997). The source leaves of the Cl and C2 types of grafted control plants were
of this variation is as yet unknown. There was similar also inoculated with 5 jig/mL TMV. As shown in Table II,
variation in PAP expression in T2-type grafted plants (Fig. grafted plants of the Tl type showed an increased 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
no. 6 had very low levels of PAP expression, which was number of local lesions observed on the wild-type scion
detectable by ELISA. In contrast, PAP expression was not 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 observed on scion leaves of Tl-type grafted plants
type (Fig. 2B). were also significantly reduced in size (Table II).
Grafted plants of the T3 type demonstrated resistance to
Expression of Genes Encoding PR Proteins in TMV in wild-type rootstocks (Table II, experiment 2). The
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 with the number and size of lesions on root-
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
observed in the leaves of N. tabacum cv Samsun NN plants Scions Rootstocks TMV
6 d after TMV inoculation (Fig. 3). In contrast, PR-1 expres- 5 6 9 10 13 IS 20 22 S 6 9 10 13 15 20 22 T N N
sion was not detected in the wild-type scions grafted on
transgenic rootstocks. Expression of PR1 was not detected
in rootstocks from plant no. 15 or 22 (Fig. 3). Rootstocks
from plant no. 22 (Fig. 2A) and no. 15 (data not shown) had
low levels of PAP expression, which was detectable only by
ELISA. Expression of PR-1, PR-2, and PR-3 genes in grafted
plants was determined using northern-blot analysis. Tran-
Figure 3. Analysis of PR1 expression in grafted tobacco plants. Pro-
scripts corresponding to PR-1, PR-2, and PR-3 were de-
tein (3 /xg) from leaves of grafted plants of T1 type nos. 5, 6, 9, 10,
tected in transgenic rootstocks expressing PAP but not in 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 probes in TMV-infected tobacco plants but not in Methods." T, Transgenic tobacco line 26139-19; N, wild-type N.
uninfected tobacco plants (data not shown). 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 a n d 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 1 1 1 . 2 t 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; n n , cv Samsun n n ; 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 c v 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? l l O d 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 N N plants were inoculated with 200 p L 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 o f 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,
Leve1 of SAb 80% of C3-type control plants contained detectable levels of
Size of TMV-lnduced
Planta PVX antigen by ELISA. These results demonstrate that PAP
Scion Rootstock Local Lesions
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
NN/T-8 1 7 6 2 18 324? 1 0.79 t 0.06
NN/T-9 192 2 6 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 2 1 1.20 +- 0.10
NNn-20 1 9 3 2 18 211 2 9 1.10 5 0.10
NN/T-22 201 2 2 4 NA 1.O4 +- 0.06 A
Average 220 2 71 327 2 106' 1.O4 2 0.1 2d
NNInn-2
NN/nn-3
NN/nn-11
NA
236 2 19
NA
NA
198 2 36
NA
1.34 2 0.1 2
1.54 2 0.20
1.36 2 0.14
12
NN/nn-15 219 +- 17 187 2 11 1.35 2 0.20
Average 227212 193 2 8 1.40 2 0.1 O

-P
a NN/T, Crafted plants of T1 type with transgenic rootstocks from c
111

the homozygous plant line 261 39-19. NN/nn, Control grafted plants
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
& n L TMV. 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.
..__nn/nn,
.--.o 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
inoculated with 1 .O or 0.1 pg/mL TMV. Ten plants with transgenic
transgenic line 26139-19. C3-type grafted plants were used
261 39 rootstock (Oand O ) and 4 plants with Samsun nn rootstock
as controls. Three weeks after grafting, wild-type cv Sam- (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.
d m nnlt6139 M139b
Figure 5 . Susceptibility of grafted tobacco plants to PVX.'Two bot-
D I SCUSSlO N
tom scion leaves of grafted nn/26139 plants were inoculated with We have previously shown that transgenic plants ex-
100 p L 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
261 39/nn were inoculated with PVX, and 3 weeks after inoculation
ments, we demonstrate here that expression of PAP in
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 g r a f t e d 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 1 5 1 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 N N 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)
i s 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 e t al. Plant Physiol. Vol. 114, 1997

Peer and Shippers, 1992; Leeman e t al., 1995). W e 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 a n d 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 t h e first virus resistance in transgenic plants expressing pokeweed anti-
s t e p of resistance induction. PGPR-dependent ISR is acti- viral protein. Proc Natl Acad Sci USA 90: 7089-7093
vated by rhizobacteria via lipopolysaccharides a n d PAP- Lord JM, Hartley MR, Roberts LM (1991) Ribosome inactivating
proteins of plants. Semin Cell Biol 2: 15-22
i n d u c e d resistance is mediated by t h e enzymatic activity of Malamy J, Carr JP, Klessig DF, Raskin I (1990) Salicylic acid: a
PAP. These results suggest that resistance i n d u c e d by a n 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, c a n 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 t h e 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 1 4 5753-5761 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 influenza
antiviral protein inactivates pokeweed ribosomes; implications virus by extracts from Phytolacca americana. J Gen Viro1 2 2
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 4 0 612-620 infection. Proc Natl Acad Sci USA 9 4 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 9 2 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, p p 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