Expression of Pokeweed Antiviral Protein in Transgenic Plants
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Plant Physiol. (1997) 114: 1113-1 121 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 to be resis- nates ribosomes from pokeweed and other plants (Bonness tant to 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-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.