Analysis of the in planta antiviral activity of elderberry ribosome‐inactivating proteins

Abstract

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 polynucleotideadenosine 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 ribosomeinactivating proteins and the in vitro polynucleotideadenosine 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.Keywords: elderberry; polynucleotide-adenosine glycosylase activity; ribosome-inactivating protein; Sambucus nigra; viral protection.Ribosome-inactivating proteins (RIPs; EC 3.2.2.22) are a heterogeneous family of structurally and evolutionary related plant proteins sharing a common functional domain that catalytically removes a specific adenine residue from a highly conserved, surface-exposed stem-loop structure found in the large rRNA of prokaryotic and eukaryotic ribosomes [1,2]. At present, they are subdivided on the basis of the structure of the genes and the corresponding proteins into holo-RIPs and chimero-RIPs [3]. Whereas holo-RIPs consist exclusively of a single catalytically active protomer of either one (classical type-1 RIPs) or two smaller polypeptide chains (e.g. maize RIP b-32), chimero-RIPs are built up of chimeric protomers with an N-terminal catalytically active domain arranged in tandem with a structurally and functionally unrelated C-terminal domain (classical type-2 and type-3 RIPs).Biochemical and molecular studies have shown that the elderberry tree expresses a complex mixture of type-2 RIPs and/or lectins in virtually all tissues. In agreement with the chronological order of their discovery, these Sambucus nigra agglutinins (SNAs) are numbered SNA-I to SNA-V. The first elderberry lectin was identified in bark tissue and described as a NeuAc(a-2,6)Gal/GalNAc-specific agglutinin (called SNA-I) [4,5]. Although already discovered in 1984, SNA-I was recognized as a type-2 RIP only when the corresponding gene was cloned in 1996 [6]. Besides SNA-I, elderberry bark contains a second NeuAc(...

Ribosome-inactivating proteins: progress and problems

“…Indeed, some protection from some viruses was conferred to transfected plants by several type 1 RIPs [reviewed in refs 144,145]. and, although with less efficiency, by type 2 RIPs from S. nigra, SNA-I'[147] and SNA-V[148], but not by SNA-If from the same plant[149]. High-level expression of PAP was…”

Plant AB Toxins with Lectin Domains

“…Although type 2 RIPs are sequestered from the host ribosomes in plant cells in, e.g., vacuoles and intercellular spaces, it can be envisaged that once the plant is attacked by pathogens the type 2 RIPs Plant Toxins DOI 10.1007/978-94-007-6728-7_4-1 # Springer Science+Business Media Dordrecht 2015 could enter the plant cytosolic compartments. However, for several type 2 RIPs such as SNA-I, SNA-V, and SNLRP it was reported that they are inactive on plant ribosomes (Vandenbussche et al 2004a). …”

“…Although there is no doubt about the antiviral activity of RIPs, their mode of action has not been elucidated. With respect to the RIP antiviral activity, two major hypotheses have been proposed (Vandenbussche et al 2004a). (i) The RIPs can directly work on virus nucleic acids by their N-glycosidase activity or polynucleotide:adenosine glycosidase activity.…”

“…Analyses of the local lesions provoked by the Eranthis hyemalis lectin showed in vivo antiviral activity of this RIP against alfalfa mosaic virus infection (Kumar et al 1993). Several type 2 RIPs from S. nigra such as SNA-I, SNA-I', SNA-V (or nigrin b), and SNLRP showed the potential to protect transgenic tobacco plants against tobacco mosaic virus infection (Chen et al 2002;Vandenbussche et al 2004a). Furthermore, SNA-I, SNA-V, and SNLRP exhibit a potent N-glycosidase activity on tobacco mosaic virus RNA by multidepurination of the RNA chain (Tejero et al 2015).…”

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

“…In contrast to type-1 RIPs, the antiviral activity of type-2 RIPs has been far less documented. Several type-2 RIPs were demonstrated to exhibit in vitro or in vivo antiviral activity (Stevens et al 1981;Kumar et al 1993) but, so far in planta antiviral activity has been reported only for type-2 RIPs from Sambucus nigra (Chen et al 2002a;Vandenbussche et al 2004). …”