Nilgun E. Tumer

Engineering Herbicide Tolerance in Transgenic Plants

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.

Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA

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.

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants.

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid‐derived plant transformation vectors. The progeny of the self‐fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross‐protection may be a general method for preventing viral disease in plants.

Pokeweed Antiviral Protein Accesses Ribosomes by Binding to L3

Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein, catalytically removes an adenine residue from the conserved α-sarcin loop of the large rRNA, thereby preventing the binding of eEF-2·GTP complex during protein elongation. Because the α-sarcin loop has been placed near the peptidyltransferase center in Escherichia coli ribosomes, we investigated the effects of alterations at the peptidyltransferase center on the activity of PAP. We demonstrate here that a chromosomal mutant of yeast, harboring the mak8–1 allele of peptidyltransferase-linked ribosomal protein L3 (RPL3), is resistant to the cytostatic effects of PAP. Unlike wild-type yeast, ribosomes from mak8–1 cells are not depurinated when PAP expression is induced in vivo, indicating that wild-type L3 is required for ribosome depurination. Co-immunoprecipitation studies show that PAP binds directly to L3 or Mak8–1p in vitro but does not physically interact with ribosome-associated Mak8–1p. L3 is required for PAP to bind to ribosomes and depurinate the 25 S rRNA, suggesting that it is located in close proximity to the α-sarcin loop. These results demonstrate for the first time that a ribosomal protein provides a receptor site for an ribosome-inactivating protein and allows depurination of the target adenine.

A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template.

Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs.

Antiviral activity of ribosome inactivating proteins in medicine.

Pokeweed antiviral protein and several other ribosome inactivating proteins are effective against a broad range of viruses. Recent results have shown that their enzymatic activity is not limited to depurination of the large rRNA, they can depurinate other nucleic acids, including viral RNAs. Antiviral activity of RIPs is summarized here in light of their novel activities and recent developments in the field.

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. 'Russet Burbank'.

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. ‘Russet Burbank’. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.

The ribosomal stalk is required for ribosome binding, depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐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 α‐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 ΔP1 and ΔP2 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.

Pokeweed antiviral protein binds to the cap structure of eukaryotic mRNA and depurinates the mRNA downstream of the cap.

Several cap-binding proteins from both the nucleus and cytosol have been identified that mediate processes such as pre-mRNA splicing, translation initiation, and mRNA turnover. Here we describe a novel cap-binding protein, pokeweed antiviral protein (PAP), a 29-kDa type I ribosome-inactivating protein (RIP) isolated from Phytolacca americana. In addition to depurinating the sarcin/ricin loop of the large rRNA, an activity common to all RIPs, we have reported recently that PAP depurinates capped, but not uncapped RNAs in vitro. Here we characterize this activity further and, using affinity chromatography, show that PAP binds to the m7Gppp cap structure. PAP UV-crosslinks to m7GpppG-capped luciferase mRNA more efficiently than GpppG-capped luciferase mRNA, indicating specificity for the methylated guanosine. We present evidence that PAP does not remove the cap structure or depurinate the m7Gppp as shown by primer extension of capped and uncapped luciferase transcripts incubated with PAP. Modeling studies of cap interaction with PAP predict that the cap structure would bind to the active site of PAP in a similar manner to guanine. We map the depurination sites on the capped luciferase RNA and illustrate that depurination occurs at specific adenine and guanine residues throughout the RNA sequence. Incubation of isolated ribosomes with PAP and increasing molar concentrations of m7GpppG relative to PAP resulted in a decrease in the level of rRNA depurination. Therefore, at elevated concentrations, the methylated cap structure competes with the adenine or guanine for binding to PAP, even though the affinity of PAP for capped message is almost fourfold lower than for rRNA. These results demonstrate that the activity of PAP is not limited to rRNA depurination, but that PAP binds to the cap structure and depurinates mRNAs downstream of the cap in vitro. These findings may have implications for understanding PAP activity in vivo.

A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway.

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.