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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 7089-7093, August 1993 Genetics Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein JENNIFER K. LODGE*, WOJCIECH K. KANIEWSKI, AND NILGUN E. TUMERtt Monsanto Co., 700 Chesterfield Village Parkway, St. Louis, MO 63198 Communicated by Myron K. Brakke, April 12, 1993 ABSTRACT Exogenous application of pokeweed antiviral show that expression of PAP confers resistance to unrelated protein (PAP), a ribosome-inhibiting protein found in the cell viruses. Virus resistance, previously observed in transgenic walls of Phytolacca americana (pokeweed), protects heterolo- plants expressing coat protein genes or the read-through gous plants from viral infection. A cDNA clone for PAP was component of the tobacco mosaic virus replicase gene, has isolated and introduced into tobacco and potato plants by been specific for the virus from which the genes are derived transformation with Agrobacterium tumefaciens. Transgenic or closely related viruses (8, 9). The results of the experi- plants that expressed either PAP or a double mutant derivative ments described here provide a way of producing transgenic of PAP showed resistance to infection by different viruses. plants that may be resistant to a broad spectrum of plant Resistance was effective against both mechanical and aphid viruses. transmission. Analysis of the vacuum infiltrate of leaves ex- pressing PAP showed that it is enriched in the intercellular MATERIALS AND METHODS fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in Isolation of a PAP cDNA Clone. A probe to identify the infection. Previous methods for creating virus-resistant plants cDNA for the PAP gene was made by using PCR to amplify have been specific for a particular virus or closely related the DNA that encodes the first 30 amino acids of the mature viruses. To protect plants against more than one virus, multiple protein (10). The template DNA in the reaction was cDNA genes must be introduced and expressed in a single transgenic made from poly(A)+ RNA from pokeweed leaves and a 5' lne. Expression of PAP in transgenic plants offers the possi- primer (5'-GGGGTCTAGAATTCGTNAAYACNATHAT- bility of developing resistance to a broad spectrum of plant HTAYAAYGT) that was designed to match the sense strand viruses by expression of a single gene. of putative DNA sequence encoding amino acids 1-8 and a 3' primer (5'-GGRTCYTTYGCYTCRTT) that was designed Ribosome-inhibiting proteins (RIPs), which have been iso- to match the antisense strand of the putative DNA sequence lated from many different plant species, inactivate eukaryotic encoding amino acids 25 through 30, where N = A, G, C, or ribosomes. There are two classes: type I RIPs have a single T; Y = T or C; R = G or A; and H = A, C, or T. A 100-bp polypeptide chain, and type II RIPs have two polypeptides, fragment obtained from PCR was used to isolate the 5' half an A (active) chain and a B chain, which is a galactose- ofthe coding sequence from a AZAP library (Stratagene), and binding lectin (1). RIPs deglycosylate a specific base in the this fragment of the gene was then used to isolate the 28S rRNA and prevent binding of elongation factor 2. Type remainder of the coding sequence from a separate cDNA I RIPs, which do not possess a binding domain, do not bind library. easily to cells and consequently have a relatively low mam- Construction of Plant Transformation Vectors. PCR was malian cytotoxicity (1). used to engineer convenient restriction sites (Bgl II at the 5' RIPs have been of considerable interest recently due to end and Sma I at the 3' end) into the full-length cDNA their therapeutic potential as chimeric toxins, which can be encoding PAP for inserting into plant transformation vectors. targeted to a particular cell type, such as a cancer cell (1). In pMON8443 has the PAP gene expressed from the enhanced addition, type I RIPs and the A chains of type II RIPs have 35S RNA promoter from cauliflower mosaic virus (CaMV antiviral activity. Recently, it has been reported that several E35S) (11) and pMON8484 has the PAP gene expressed from RIPs inhibit the replication ofhuman immunodeficiency virus the 35S RNA promoter from figwort mosaic virus (FMV) 1 in T cells and macrophages in vitro (1). (12). pMON8442 has the variant PAP gene expressed from Three distinct antiviral proteins with similar activities have the 35S RNA promoter from FMV (12). been identified in pokeweed (Phytolacca americana). PAP, Plant Transformations. Agrobacterium tumefaciens con- PAPII, and PAP-S are the forms of pokeweed antiviral taining the plant transformation vectors was used to trans- protein (PAP) that appear in spring leaves, summer leaves, form tobacco (Nicotiana tabacum cv. Samsun and Nicotiana and seeds, respectively (2-4). They are active against plant benthamiana) by the leaf disc method (13) and potato (So- and animal viruses (5, 6). Exogenous application of small lanum tuberosum cv. Russet Burbank) by transformation of amounts of PAP to the surface of plant leaves completely stem sections prevents mechanical transmission of unrelated viruses to (14). several different host plants (5). PAP is stored in the cell wall and can be obtained Abbreviations: RIPs, ribosome-inhibiting proteins; PAP, pokeweed matrix of leaf mesophyll cells readily antiviral protein; NPTII, neomycin phosphotransferase II; GUS, from water extracts of macerated leaf tissue (7). 3-glucuronidase; BMV, brome mosaic virus; PVX and PVY, potato The fact that PAP is a general inhibitor of virus infection viruses X and Y; PLRV, potato leafroll virus; CMV, cucumber makes it an ideal candidate for developing virus-resistant mosaic virus. plants. We cloned the cDNA encoding PAP from pokeweed *Present address: Department of Molecular Biology and Pharma- leaves and used it to transform tobacco and potato plants. We cology, Washington University Medical School, St. Louis, MO 63110. tPresent address: Agricultural Biotechnology Center, Rutgers Uni- The publication costs of this article were defrayed in part by page charge versity, Cook College, P.O. Box 231, New Brunswick, NJ 08903- payment. This article must therefore be hereby marked "advertisement" 0231. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be sent at the present address. 7089 Downloaded by guest on September 23, 2021 7090 Genetics: Lodge et al. Proc. Natl. Acad. Sci. USA 90 (1993) Virus Resistance Analysis. Leaf samples from transgenic Table 1. Protection against virus infection with exogenously plants were analyzed for expression of PAP by ELISA. One applied PAP disc (15 mm in diameter) was sampled from each plant and % infected homogenized in 500 ,ul of phosphate-buffered saline (PBS), 0.05% Tween-20, and 0.2% ovalbumin (PBSTO). Plates were Without With coated with 1:1000 dilution of a 1 mg/ml solution of anti-PAP Virus Group Host PAP PAP IgG. The plant extract (250 ,ul) was added and the plates were Mechanically transmitted* incubated overnight at 4°C. The bound protein was detected PVX Potexvirus N. tabacum 100 0 with alkaline phosphatase-conjugated anti-PAP IgG (1:5000). PVX Potexvirus S. tuberosum 100 0 To estimate virus levels in plants, two discs were sampled PVY Potyvirus N. tabacum 100 0 from the second systemically infected leaf at various times PVY Potyvirus S. tuberosum 30 0 after inoculation and antigen levels were determined by Aphid transmittedt ELISA as previously described (15). PVY Potyvirus S. tuberosum 80 60 RIP Activity of PAP and Variant PAP. Leaf disks from N. PLRV Luteovirus S. tuberosum 80 85 tabacum plants expressing PAP or variant PAP were ground *PAP from seeds was obtained from Calbiochem. Six to 20 plants in PBS and spun for 5 min in a microcentrifuge. Several were inoculated on two leaves per plant with PVY at 20 gg/ml or dilutions of the extracts were made and the amount of PAP PVX at 5 pg/ml, in the absence of PAP or in its presence at 1 Mg in each dilution was measured by ELISA and by Western per leaf. Two weeks after inoculation, each plant was analyzed by blotting. Two and one-halfmicroliters ofeach extract dilution ELISA (16). was added to a modified rabbit reticulocyte lysate. Titrations tFive aphids, which were fed for 1-2 min on a PVY-infected plant, were done to determine lysate conditions which were satu- were transferred to each potato plant after the leaves had been rating for amino acids and RNA but were limiting for ribo- coated with PAP (15 ug per leaf). For PLRV experiments, 10 somes. 20 ul of lysate viruliferous aphids were transferred to each potato plant after the The modified lysate contained leaves had been coated with PAP (15 Mg per leaf). Aphids were killed (Promega), 15 ,ul of the S-100 fraction of the same lysate, 5.7 5 days later and plants were analyzed by ELISA 4 weeks after ,ul of H20, 1 Al of a mixture containing each amino acid inoculation. (minus methionine) at 1 mM, 0.8 ul of 35S-labeled methionine (Amersham), and 4.0 p/ of brome mosaic virus (BMV) RNA position 20 ofthe mature protein was changed to arginine and at 0.25 mg/ml (added after the PAP containing extract). After tyrosine at position 49 was changed to histidine. Fig. 1 shows 30 min at 30°C, 5-Al aliquots were spotted onto filter paper the maps of the transformation vectors and the two amino and washed in 5% trichloroacetic acid, ethanol, and ether, acid changes of the variant PAP. Tobacco leaf disks and and their radioactivities were measured. potato stem sections were transformed by cocultivation with Vacuum Infiltration. Two leaves were excised from each A.

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