Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis

Abstract

Pokeweed antiviral protein (PAP), a 29-kD protein isolated fromPhytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60s subunit of eukaryotic ribosomes. Transgenic tobacco (Nicofiana fabacum) plants expressing PAP or a variant (PAP-V) were shown to be resistant t o a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (~2 -f o l d ) increase in salicylic acid levels. Using reciproca1 grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. fabacum N N and nn scions. lncreased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected i n the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance i n wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis.Many plants produce proteins that inactivate ribosomes by depurinating rRNA in a highly conserved stem-loop structure in the 28s RNA (Lord et al., 1991; Stirpe et al., 1992). Single-chain RIPs such as PAP and the A chains of two-chain RIPs such as ricin remove an adenine base by specific cleavage of the N-glycosidic bond at A4324 in rat 28s rRNA and at homologous sites on ribosomes from 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 al., 1975; Osborn et al., 1990). Three different kinds of RIPs, PAP, PAPII, and PAP-S, have been purified from pokeweed (Phytolacca americana) plants. These proteins are similar in molecular mass (29, 30, and 29.5 kD, respectively) but are expressed at different developmental stages and in different tissues of pokeweed (Wyatt and Shepherd, 1969;Irvin et al., 1980;Barbieri et al., 1982). PAP depurinates ribosomes from pokeweed and other plants (Bonness et al., 19941, as well as mammalian, yeast, and bacterial ribosomes. In addition, PAI' effectively inhibits infection by a number of different plant (Wyatt and Shepherd, 1969; Tomlinson et al., 1974;Chen et al., 1992) and animal viruses (Tomlinson et al., 1974; Ussery et al., 1977), including human immunodeficiency virus (Zarling et al., 1990). PAP also inhibits growth of tumor cells (Stirpe et al., 1992). Positive correlations were reported between RIP-catalyzed depurination of tobacco ribosomes and antiviral activity of exogenously applied RIPs (Taylor et al., 199...

Isolation and Characterization of a Novel Ribosome-Inactivating Protein from Root Cultures of Pokeweed and Its Mechanism of Secretion from Roots

“…Transgenic plants expressing nontoxic mutated PAP forms such as PAPx (active site mutant), PAPn (N-terminal mutant), and PAPc (C-terminal mutant) have shown that Glu-176, Gly-75, and 25 C-terminal amino acids are critical amino acid residues involved in ribosome depurination (Smirnov et al, 1997;Tumer et al, 1997;Zoubenko et al, 1997Zoubenko et al, , 2000Hudak et al, 2000). Comparison of cDNA sequences of PAP-H and PAP shows that PAP-H also contains those amino acid residues involved in ribosome depurination.…”

The type-1 and type-2 ribosome-inactivating proteins from Iris confer transgenic tobacco plants local but not systemic protection against viruses

“…Even though the induction of PR-proteins by the ectopic expression of PAP and several PAP mutants was initially thought to contribute to the in planta antiviral activity of PAP, subsequent experiments have failed to correlate the level of protection with the level of PR-proteins. Using reciprocal grafting experiments, Smirnov et al (1997) demonstrated that transgenic tobacco (N. tabacum L. cv. Samsun nn) rootstocks expressing PAP-v induced protection in both wild-type Samsun NN and nn scions in the absence of elevated levels of PR-proteins or salicylic acid.…”

“…Thus, once a virus is able to replicate and move symplastically into neighbouring cells, RIPs might be no longer effective. Although in the past several RIPs were shown to exhibit in planta antiviral activity against systemic viruses, it is still unclear whether these RIPs confer local as well as systemic protection (Lodge et al 1993;Lam et al 1996;Smirnov et al 1997;Wang et al 1998). In the present study, the (in)ability of RIPs to confer both local and systemic protection was assessed separately.…”

“…Therefore, RIPmediated cell death can only occur after the RIPs gain access to the cytoplasm. Third, RIPs may act indirectly through the activation of the plant's defence system resulting in a systemic resistance independently of salicylic acid or pathogenesis-related (PR)-protein accumulation (Smirnov et al 1997;Zoubenko et al 2000). At present, it remains unclear which of these three proposed mechanisms is operative in planta.…”

The cell death factor, cell wall elicitor of rice blast fungus (Magnaporthe grisea) causes metabolic alterations including GABA shunt in rice cultured cells

“…[1][2][3] Well-described phenomena such as the hypersensitive response (HR) with generation of reactive oxygen species (ROS), nitric oxide and salicylate, can be observed. [4][5][6] Proliferation of pathogens can be prevented by the endogenous synthesis of phytoalexins, pathogenesis-related (PR) proteins, cell wall components, phenol and antioxidants. [7][8][9][10][11][12] Elicitors such as oligosaccharides derived from pathogens induce plant cell death as well as pathogen infection.…”