Type-1 Ribosome-inactivating Protein Research Articles

Isolation, Characterization and Biological Action of Type-1 Ribosome-Inactivating Proteins from Tissues of Salsola soda L.

Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new single-chain (type-1) RIPs named sodins were isolated from the seeds (five proteins), edible leaves (one protein) and roots (one protein) of Salsola soda L. Sodins are able to release Endo’s fragment when incubated with rabbit and yeast ribosomes and inhibit protein synthesis in cell-free systems (IC50 = 4.83–79.31 pM). In addition, sodin 5, the major form isolated from seeds, as well as sodin eL and sodin R, isolated from edible leaves and roots, respectively, display polynucleotide:adenosine glycosylase activity and are cytotoxic towards the Hela and COLO 320 cell lines (IC50 = 0.41–1200 nM), inducing apoptosis. The further characterization of sodin 5 reveals that this enzyme shows a secondary structure similar to other type-1 RIPs and a higher melting temperature (Tm = 76.03 ± 0.30 °C) and is non-glycosylated, as other sodins are. Finally, we proved that sodin 5 possesses antifungal activity against Penicillium digitatum.

Structure and Activity of a Cytosolic Ribosome-Inactivating Protein from Rice.

Ribosome-inactivating proteins (RIPs) are cytotoxic enzymes that inhibit protein translation by depurinating ribosomal RNA. Although most plant RIPs are synthesized with leader sequences that sequester them away from the host ribosomes, several RIPs from cereals lack these signal peptides and therefore probably reside in the cytosol near the plant ribosomes. More than 30 RIP genes have been identified in the rice (Oryza sativa spp. japonica) genome, many of them lacking a signal peptide. This paper focuses on a presumed cytosolic type-1 RIP from rice, referred to as OsRIP1. Using 3D modeling it is shown that OsRIP1 structurally resembles other cereal RIPs and has an active site that meets the requirements for activity. Furthermore, localization studies indicate that OsRIP1-eGFP fusion proteins reside in the nucleocytoplasmic space when expressed in epidermal cells of Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Finally, OsRIP1 was recombinantly produced in Escherichia coli and was demonstrated to possess catalytic activity. Interestingly, this recombinant RIP inactivates wheat ribosomes far less efficiently than rabbit ribosomes in an in vitro system. These findings raise some interesting questions concerning the mode of action and physiological role of OsRIP1. This is the first time a RIP from rice is investigated at protein level and is shown to possess biological activity.

Ribosome-inactivating proteins from apple have strong aphicidal activity in artificial diet and in planta

The apple (Malus domestica) genome contains gene sequences encoding type-1 and type-2 ribosome-inactivating proteins (RIPs). Both types of proteins contain a RIP domain with N-glycosidase activity, but the type-2 RIPs possess an additional domain with lectin activity. Here we investigated the activity of RIPs from apple against two sucking-piercing aphids that are important in agriculture, in particular pea aphids (Acyrthosiphon pisum) and green peach aphids (Myzus persicae).When the recombinant RIPs were dosed in an artificial liquid diet against pea aphids a strong aphicidal activity was observed. Based on LC50s, the type-2 RIP (33 mg/L) was about 10 times more active than the type-1 RIP (341 mg/L), and the LC50 of the recombinant protein corresponding to the type-2 RIP lectin domain alone was 106 mg/L. In in planta experiments with transgenic tobacco plants expressing the type-1 RIP or the type-2 RIP and infected with green peach aphids, mortalities of the nymphal stages amounted to approximately 50% for both types of transgenic lines when compared to wild type plants. In addition, significant sublethal effects were observed in the surviving aphids with a reduction in fecundity, intrinsic rate of increase, net reproductive rate and doubling time of the insect population. The insecticidal activity of the type-1 RIP and type-2 RIP from apple is discussed in relation to the domain structure of the RIPs and potential use in plant protection.

Cytological and Subcellular Response of Cells Exposed to the Type-1 RIP Curcin and its Hemocompatibility Analysis

AbstractCurcin, a type 1 ribosome inactivating protein (RIP) is investigated here for its cellular competence on six mammalian cell lines. Cells exposed to curcin (100 μg/ml) for 72 h exhibited significant cellular metabolic arrest, with the cancer cell lines being more sensitive. The viability assessment of the cancer cells in a 3D cell culture based assay revealed highly restricted sprouting and proliferation with near to complete dead cell population. Prominent mitochondrial dysfunction, elevated reactive oxygen species levels, nuclear degeneration, structural/mechanical destabilization and suppression of defense mechanisms were imminent with the RIP treated cells. Expression levels of nuclear factor κB (NF-κB), cytoskeletal focal adhesion kinases (FAK) and vinculin were significantly diminished. Vital cellular organelles as nucleus, mitochondria and actin were severely incapacitated on RIP exposure resulting in multimodal apoptosis and necrosis. The ability of curcin to impart comprehensive shutdown of the cells, especially cancer cells, complemented with its hemocompatibility, opens up possibilities of utilizing this ribotoxin as a prospective therapeutic candidate against cancers of diverse origins.

Novel animal defenses against predation: a snail egg neurotoxin combining lectin and pore-forming chains that resembles plant defense and bacteria attack toxins.

Although most eggs are intensely predated, the aerial egg clutches from the aquatic snail Pomacea canaliculata have only one reported predator due to unparalleled biochemical defenses. These include two storage-proteins: ovorubin that provides a conspicuous (presumably warning) coloration and has antinutritive and antidigestive properties, and PcPV2 a neurotoxin with lethal effect on rodents. We sequenced PcPV2 and studied whether it was able to withstand the gastrointestinal environment and reach circulation of a potential predator. Capacity to resist digestion was assayed using small-angle X-ray scattering (SAXS), fluorescence spectroscopy and simulated gastrointestinal proteolysis. PcPV2 oligomer is antinutritive, withstanding proteinase digestion and displaying structural stability between pH 4.0–10.0. cDNA sequencing and protein domain search showed that its two subunits share homology with membrane attack complex/perforin (MACPF)-like toxins and tachylectin-like lectins, a previously unknown structure that resembles plant Type-2 ribosome-inactivating proteins and bacterial botulinum toxins. The protomer has therefore a novel AB toxin combination of a MACPF-like chain linked by disulfide bonds to a lectin-like chain, indicating a delivery system for the former. This was further supported by observing PcPV2 binding to glycocalix of enterocytes in vivo and in culture, and by its hemaggutinating, but not hemolytic activity, which suggested an interaction with surface oligosaccharides. PcPV2 is able to get into predator’s body as evidenced in rats and mice by the presence of circulating antibodies in response to sublethal oral doses. To our knowledge, a lectin-pore-forming toxin has not been reported before, providing the first evidence of a neurotoxic lectin in animals, and a novel function for ancient and widely distributed proteins. The acquisition of this unique neurotoxic/antinutritive/storage protein may confer the eggs a survival advantage, opening new perspectives in the study of the evolution of animal defensive strategies.

A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity

Articulatin-D, a 66kDa ribosome inactivating protein (RIP) comprised of 29kDa A-chain linked to 35kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC–MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC50 against different cancer cell lines (Jurkat: 0.31±0.02nM, MOLT-4: 0.51±0.03nM, U-937: 0.64±0.07nM, HL-60: 0.79±0.11nM, Raji: 1.45±0.09nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.

Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein fromViscum articulatumand Its Biological Effects on Peripheral Blood Mononuclear Cells

Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB) showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

ATG-saporin-S6 immunotoxin: a new potent and selective drug to eliminate activated lymphocytes and lymphoma cells

Anti-thymocyte globulins (ATG) are currently used to prevent graft-versus-host disease in haematopoietic stem cell transplants from alternative donors and to treat and prevent acute organ rejection after transplantation. Many recent studies have demonstrated that ATG can also be beneficial in patients with myeloma, lymphoma, leukaemia and myelodysplastic syndrome. This study showed, for the first time, that the cytotoxic effect of ATG can been enhanced by conjugation with saporin-S6, which is one of the most stable and active type-1 ribosome-inactivating proteins. The ATG-saporin-S6 immunotoxin showed a strong cytotoxic effect on five lymphoma- and leukaemia-derived cell lines as well as on activated lymphocytes while sparing non-haematological cell lines. ATG-saporin-S6 induced a time-dependent activation of caspase-3/7 in RAJI cells. The caspase inhibitor Z-VAD-fmk partially rescued the cells that were treated with ATG-saporin-S6, suggesting that multiple cell death pathways, some of which are caspase independent, play a role in ATG-saporin-S6 toxicity. In our experiments ATG increased the complement-independent cytotoxicity of activated lymphocytes by a magnitude of 3-5 logs after conjugation. These findings suggest that the ATG-saporin-S6 immunotoxin is a promising therapeutic tool for many pathological conditions involving T lymphocytes and T and B neoplastic cells.

Carbohydrate-binding activity of the type-2 ribosome-inactivating protein SNA-I from elderberry ( Sambucusnigra) is a determining factor for its insecticidal activity

In recent years, different classes of proteins have been reported to promote toxic effects when ingested. Type-2 ribosome-inactivating proteins (RIPs) are a group of chimeric proteins built up of an A-chain with RNA N-glycosidase activity and a B-chain with lectin activity. These proteins are thought to play a role in plant protection. Sambucus nigra agglutinin I (SNA-I) is a type-2 RIP, isolated from the bark of elderberry ( S. nigra L.). This study demonstrated the insecticidal potency of SNA-I on two Hemipteran insect species using two different methods. An artificial diet supplemented with different concentrations of the purified RIP reduced survival and fecundity of pea aphids Acyrthosiphon pisum. In addition, feeding of tobacco aphids, Myzus nicotianae, on leaves from transfected plants constitutively expressing SNA-I, resulted in a delayed development and reduced adult survival and also the fertility parameters of the surviving aphids were reduced, suggesting that a population of aphids would build up significantly slower on plants expressing SNA-I. Finally, a series of experiments with transgenic lines in which a mutant RIP was expressed, revealed that the carbohydrate-binding activity of SNA-I is necessary for its insecticidal activity. In a first set of mutants, the B-chain was mutated at one position (Asp231ΔGlu), and in the second set both carbohydrate-binding sites were mutated (Asn48ΔSer and Asp231ΔGlu). Mutation of one carbohydrate-binding site strongly reduced the insecticidal activity of SNA-I, whereas mutation of both lectin sites (almost) completely abolished the SNA-I effect on tobacco aphids.

Expression of Sambucus nigra agglutinin (SNA-I′) from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species

Tobacco plants (Nicotiana tabacum cv Samsun NN) have been transformed with the gene encoding the type-2 ribosome-inactivating protein (RIP) SNA-I' from elderberry (Sambucus nigra) under the control of the Cauliflower Mosaic Virus 35S promoter. Previous research confirmed that these plants synthesize, correctly process and assemble a fully active RIP. Variability in protein expression was observed within the transgenic lines. The effects of the type-2 RIP SNA-I' delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae) and Lepidoptera, the beet armyworm (Spodoptera exigua). In the experiment with aphids, significant effects were observed on the life parameters, such as survival, intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring, whereas with caterpillars significant reduction in fresh weight as well as retardation in development were observed. In addition, significant increases in mortality were noted for insects fed on the transgenic lines as compared to wild type plants. This information provides further support for RIPs having a role in plant resistance to insect pest species.

Isolation and Characterization of Ribosome‐Inactivating Proteins from Cucurbitaceae

Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.

Purification and characterization of abelesculin, a novel ribosome-inactivating protein from the mature seeds of Abelmoschus esculentus

A novel, type-1 ribosome-inactivating protein (RIP), abelesculin, from the mature seeds of Abelmoschus esculentus was successively purified to homogeneity using ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography. Abelesculin was found to have a molecular mass of 30 kDa and a pI of more than 10.1. The protein depurinates 28S rRNA in the ribosomes of rat liver with an EC50 (concentration causing 50% cleavage) of 62.8 pM. Study of the sequence of the 26 N-terminal amino acids of the protein revealed a close relationship with other RIPs. This study reports for the first time that RIPs exist in Malvaceae plants.

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

The antiviral activity of the type-2 ribosome-inactivating protein (RIP) IRAb from Iris was analyzed by expressing IRAb in tobacco (Nicotiana tabacum L. cv. Samsun NN) plants and challenging the transgenic plants with tobacco mosaic virus (TMV). Although constitutive expression of IRAb resulted in an aberrant phenotype, the plants were fertile. Transgenic tobacco lines expressing IRAb showed a dose-dependent enhanced resistance against TMV infection but the level of protection was markedly lower than in plants expressing IRIP, the type-1 RIP from Iris that closely resembles the A-chain of IRAb. To verify whether IRIP or IRAb can also confer systemic protection against viruses, transgenic RIP-expressing scions were grafted onto control rootstocks and leaves of the rootstocks challenged with tobacco etch virus (TEV). In spite of the strong local antiviral effect of IRIP and IRAb the RIPs could not provide systemic protection against TEV. Hence our results demonstrate that expression of the type-1 and type-2 RIPs from Iris confers tobacco plants local protection against two unrelated viruses. The antiviral activity of both RIPs was not accompanied by an induction of pathogenesis-related proteins. It is suggested that the observed antiviral activity of both Iris RIPs relies on their RNA N-glycohydrolase activity towards TMV RNA and plant rRNA.

Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins.

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.

Molecular characterization and post-transcriptional regulation of ME1, a type-I ribosome-inactivating protein from Mirabilis expansa.

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, ME1, a type-1 RIP, was cloned and sequenced from storage roots of Mirabilis expansa (Ruiz & Pavon). The full-length cDNA sequence of ME1 has 1,129 nucleotides with an open reading frame of 951 nucleotides representing 317 amino acids. Nucleotide analysis revealed that the N-terminal region of ME1 was cleaved, and the mature protein started at amino acid 34. ME1 showed very close similarities to MAP and MAP-4 from Mirabilis jalapa. Southern blot analysis revealed the presence of two homologous genes for ME1 cDNA in M. expansa. Northern blot analysis showed high levels of ME1 transcripts in primary and storage roots. Interestingly, jasmonic acid induced ME1 transcript expression in cell suspension cultures of M. expansa; however, the production of ME1 protein was not enhanced as observed by Western blot analysis. Our data suggest that ME1 has the ability to depurinate its own mRNA, thus inhibiting its translation. These observations suggest a possible mechanism by which ME1 protein levels are post-transcriptionally regulated.

Type-1 ribosome-inactivating protein from iris bulbs: a useful agronomic tool to engineer virus resistance?

To study the in planta antiviral activity of a type-1 ribosome-inactivating protein from iris bulbs, called IRIP, Nicotiana tabacum cv. Samsun NN was transformed with the IRIP sequence expressed under the control of the 35S cauliflower mosaic virus promoter. Molecular analysis of the transgenic plants and characterization of the purified protein revealed that the recombinant IRIP from tobacco leaves has the same molecular structure and RNA N-glycosidase activity as the native protein from iris bulbs. The tobacco transformants show no apparent phenotypic side effects indicating that ectopically expressed IRIP is not cytotoxic for tobacco cells. No induction of PR-1 could be demonstrated in the transgenic plants expressing IRIP. The in planta antiviral activity of rIRIP was assessed using a bioassay with tobacco mosaic virus. All transformed lines showed a statistically significant lower number of lesions compared to the control plants. The fortunate combination of in planta antiviral activity and lack of cytotoxicity of the ectopically expressed IRIP in transgenic tobacco renders the iris RIP an interesting and useful model for the study and exploitation of the antiviral activity of type-1 RIPs.

Delivery of ribosome-inactivating protein toxin into cancer cells with shock waves

We report on the use of shock waves delivered by a shock-tube to permeabilize cancer cells and potentiate the cytotoxicity of the type-1 ribosome-inactivating protein, saporin. We studied human colorectal cancer HT29 and ovarian cancer OVCAR-5 cells, and used two different cytotoxicity assays, colony formation and loss of mitochondrial activity. A single shock wave and saporin (10 −9 M) produced significant toxicity not seen with either shock wave or drug alone. Increasing the number of shock waves up to five further increased cytotoxicity. Higher toxicity was seen with the clonogenic assay compared to MTT assay. Shock waves may have applications in promoting cytoplasmic delivery of toxins into cancer cells after intratumoral injection.

Sugar-mediated lattice contacts in crystals of a plant glycoprotein.

Pokeweed antiviral protein, PAP-S(aci), isolated from seeds of the Chinese pokeweed plant, Phytolacca acinosa, belongs to the family of type-1 ribosome-inactivating proteins (RIPs). Type-1 RIPs are approximately 30-kDa N-glycosidases that inactivate eukaryotic and prokaryotic ribosomes via a site-specific depurination of ribosomal RNA (rRNA). Here we describe the preliminary X-ray structure determination at 1.7 A resolution of one PAP isoenzyme from seeds, PAP-S1(aci), after crystallisation from a heterogeneous mixture of two isoenzymes. PAP-S1(aci) possesses a rare type of glycosylation, specifically, N-linked N-acetyl-D-glucosamine monosaccharide (GlcNAc) substitutions at canonical Asn-Xaa-Ser/Thr sequons. One GlcNAc residue was found to play a critical role in crystal lattice formation, forming a packing interface across a crystallographic two-fold with the identical sequon of an adjacent monomer. This observation suggests that deglycosylation protocols for the crystallisation of glycoproteins should be designed to allow for exploitation of the crystal packing potential of the innermost core sugar residue (N-linked GlcNAc or O-linked GalNAc).

A complex fruit-specific type-2 ribosome-inactivating protein from elderberry (Sambucus nigra) is correctly processed and assembled in transgenic tobacco plants.

Fruits of elderberry (Sambucus nigra) express small quantities of a type-2 ribosome-inactivating protein with an exclusive specificity towards the NeuAc(alpha2,6)Gal/GalNAc disaccharide and a unique molecular structure typified by the occurrence of a disulfide bridge between the B-chains of two adjacent protomers. A cDNA clone encoding this so-called Sambucus nigra fruit specific agglutinin I (SNA-If) was isolated and expressed in tobacco (Samsun NN) under the control of the 35S cauliflower mosaic virus promoter. Characterization of the purified protein indicated that the recombinant SNA-If from tobacco leaves has the same molecular structure and biological activities as native SNA-If from elderberry fruits, demonstrating that transgenic tobacco plants are fully capable of expressing and correctly processing and assembling a type-2 ribosome-inactivating protein with a complex molecular structure. None of the transformants showed a phenotypic effect, indicating that the ectopically expressed SNA-If does not affect the viability of the tobacco cells. Bioassays further demonstrated that none of the transgenic lines exhibited a decreased sensitivity to infection with tobacco mosaic virus suggesting that the elderberry type-2 RIP SNA-If does not act as an antiviral agent in planta.

The Sambucus nigra type-2 ribosome-inactivating protein SNA-I′ exhibits in planta antiviral activity in transgenic tobacco

Transgenic tobacco (Samsun NN) plants transformed with a cDNA clone encoding SNA-I′ from Sambucus nigra synthesize, and correctly process and assemble, a fully active type-2 ribosome-inactivating protein. Expression of SNA-I′ under the control of the 35S cauliflower mosaic virus promoter enhances the plant’s resistance against infection with tobacco mosaic virus. In contrast to type-1 ribosome-inactivating proteins, the expression of SNA-I′ does not affect the growth and fertility of the transgenic plants and is not accompanied by an increased expression of pathogenesis-related proteins indicating that its antiviral activity most probably differs from that of pokeweed antiviral protein.