Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection. A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens. Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses. Resistance was effective against both mechanical and aphid transmission. Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection. Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses. To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic lne. Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

The herbicide glyphosate is a potent inhibitor of the enzyme 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in higher plants. A complementary DNA (cDNA) clone encoding EPSP synthase was isolated from a complementary DNA library of a glyphosate-tolerant Petunia hybrida cell line (MP4-G) that overproduces the enzyme. This cell line was shown to overproduce EPSP synthase messenger RNA as a result of a 20-fold amplification of the gene. A chimeric EPSP synthase gene was constructed with the use of the cauliflower mosaic virus 35S promoter to attain high level expression of EPSP synthase and introduced into petunia cells. Transformed petunia cells as well as regenerated transgenic plants were tolerant to glyphosate.

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Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP‐antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP‐mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism.

Ricin is a ribosome inactivating protein that catalytically removes a universally conserved adenine from the α-sarcin/ricin loop (SRL) of the 28S rRNA. We recently showed that ricin A chain (RTA) interacts with the P1 and P2 proteins of the ribosomal stalk to depurinate the SRL in yeast. Here we examined the interaction of RTA with wild type and mutant yeast ribosomes deleted in the stalk proteins by surface plasmon resonance. The interaction between RTA and wild type ribosomes did not follow a single step binding model, but was best characterized by two distinct types of interactions. The AB1 interaction had very fast association and dissociation rates, was saturable and required an intact stalk, while the AB2 interaction had slower association and dissociation rates, was not saturable and did not require the stalk. RTA interacted with the mutant ribosomes by a single type of interaction, which was similar to the AB2 interaction with the wild type ribosomes. Both interactions were dominated by electrostatic interactions and the AB1 interaction was stronger than the AB2 interaction. Based on these results we propose a two-step interaction model. The slow and ribosomal stalk nonspecific AB2 interactions concentrate the RTA molecules on the surface of the ribosome. The AB2 interactions facilitate the diffusion of RTA towards the stalk and promote the faster, more specific AB1 interactions with the ribosomal stalk. The electrostatic AB1 and AB2 interactions work together allowing RTA to depurinate the SRL at a much higher rate on the intact ribosomes than on the naked 28S rRNA.Ricin is a ribosome inactivating protein (RIP) isolated from the castor bean plant, Ricinus communis that consists of a Ricin Toxin A chain (RTA) and a galactose-binding B chain (RTB). RTA and RTB are linked by a disulfide bond between the Cys 259 near the C-terminus of RTA and the Cys 4 of RTB (1). The holotoxin is not active on ribosomes (2-6). The enzymatic activity of ricin is due to RTA, which is an N-glycosidase that specifically cleaves the Nglycosidic bond at A4324 of the 28S rRNA of rat ribosomes (7) and inhibits the elongation factor dependent ribosomal functions (8-10). RTA can also cleave the A2660 in naked 23S rRNA from E. coli but not the ribosomes from E. coli (11)(12)(13)(14). Both A4324 of rat 28S rRNA and A2660 of E. coli 23S rRNA are located at a universally conserved rRNA stem loop called the α-sarcin/ricin loop (SRL) named after the toxins (9). The SRL is the site where elongation factors interact with the ribosome and is involved in peptide translocation during protein synthesis (15). RTA depurinates the 28S rRNA on rat ribosomes about 5000 times faster than the naked rat 28S rRNA (7), indicating that the conformation of the rRNA on intact ribosomes and the ribosomal proteins play an important role in ribosome depurination by ricin.The α-sarcin/ricin loop (SRL) is located on the 60S subunit in close proximity to the ribosomal stalk, which is a lateral flexible structure on the large subunit. The stalk region was ...

SummaryRibosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the a-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the a-sarcin/ ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DP1 and DP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.

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Background: Ricin A chain (RTA) uses the ribosomal stalk to access the sarcin/ricin loop (SRL). Results: Arginine residues at the interface of RTB are critical for RTA to bind to the stalk and to stimulate depurination of the SRL. Conclusion: Stalk binding stimulates depurination by orienting RTA toward the SRL. Significance: We propose a model that describes how RTA accesses the SRL.