Center for Biotechnology and Department of Plant Pathology, University of Nebraska, Lincoln 68583-0722, USA

Background and objectives
The interferon family of cytokines induces an antiviral state in cells of higher vertebrates. Interferons bind to specific cell-surface receptors, activating Jak-STAT signal-transduction pathways which induce interferon-stimulated genes. The proteins encoded by these genes are responsible for the biological effects of interferons, including the antiviral responses. The 2-5A system is one such interferon-induced, antiviral pathway present in cells of reptiles, avians and mammals. Two types of enzymes are essential for a functional 2-5A system: (i) any one of several 2-5A synthetases, which require dsRNA to produce 2-5A, a series of 5'-phosphorylated, 2',5'-linked oligoadenylates; and (ii) the 2-5A-dependent RNase L. 2-5A activates RNase L which cleaves viral and cellular single-stranded RNAs, predominantly after UpNp sequences. Because virus-infected cells often contain dsRNA activators of 2-5A synthetase, for instance as replicative intermediates of RNA viruses, RNA degradation by RNase L frequently occurs in interferon-treated, virus-infected cells. Plants appear to lack an equivalent of the mammalian 2-5A system. Accordingly, the present work was undertaken to genetically engineer virus-resistant plants by expression of a mammalian 2-5A system consisting of both 2-5A synthetase and RNase L.

Results and conclusions
To express a human 2-5A system in plants, a binary plant transformation-expression vector was constructed containing both the 2-5A synthetase and RNase L cDNAs under control of the cauliflower mosaic virus 35S promoter and the chlorella virus adenine methyltransferase gene promoter, respectively. Young tobacco seedlings were transformed using the Agrobacterium co-cultivation method. The expression of RNase L in transgenic tobacco was monitored by probing Western blots with a monoclonal antibody to human RNase L. All transgenic plants expressing RNase L mRNA also expressed the nuclease protein. To determine if 2-5A synthetase was present in the transgenic tobacco plants, we performed functional assays for this enzyme. Production of 2-5A oligonucleotides from ATP was determined by the ability to activate recombinant human RNase L. To monitor viral resistance, leaves were manually inoculated with three different types of plant viruses: tobacco etch virus (TEV), a member of the potyvirus group; tobacco mosaic virus (TMV), a member of the tobamovirus group; and alfalfa mosaic virus (AMV), a member of the bromovirus group.

Infection of plants expressing both proteins induced necrotic local lesions; in contrast, plants expressing only 2-5A synthetase, only RNase L, and control plants containing the empty plasmid vector, produced typical systemic infections. In nature, TEV and AMV are not known to produce local lesions in tobacco plants. TMV does produce local lesions in tobacco plants containing the N or N' gene. However, the tobacco cultivar that was used for current experiments (SR-1) does not contain the N gene and hence is a systemic host for TMV. The most likely interpretation of these data is that viral dsRNAs activated the 2-5A synthetase, resulting in RNA decay by RNase L and thus limiting viral replication. Activation of the 2-5A system in plants could conceivably be achieved either by extensive secondary structure in viral RNAs or by double-stranded replicative intermediates formed during viral replication. We therefore conclude that the viruses used in this study caused the destruction of the infected cells in the co-expressing transgenic plants, leading to the formation of necrotic lesions and the observed antiviral effects. The 2-5A system is predicted to provide resistance to any plant virus that produces dsRNA in the infected cell. Most plant viruses, including those in the economically important potyvirus group, are positive-sense, single-stranded RNA viruses which produce a dsRNA intermediate during their replicative process. Furthermore, because plant viruses are naive to the 2-5A system, they are likely to be even more vulnerable to it than viruses of higher vertebrates.