RESUMO
We have generated more than 100 transgenic alfalfa plants, via Agrobacterium-mediated gene transfer, from genotypes selected from five alfalfa cultivars. These plants express the genes for kanamycin resistance and for the coat protein of alfalfa mosaic virus (AMV). The strongest expressers accumulated nearly 500 ng coat protein per mg soluble leaf protein. AMV inoculation of protoplasts from these strong expressers indicated that they were resistant to infection by AMV, while protoplasts from plants containing about a hundred-fold less coat protein and from control untransformed plants were not. Transgenic alfalfa plants containing large amounts of coat protein were, likewise, resistant to AMV. These plants did not develop systemic infections following inoculation with up to 50 micrograms/ml AMV, while inoculated control plants developed systemic infections following inoculation with as little as 10 micrograms/ml AMV. These results demonstrate that expression of the AMV coat protein gene confers resistance to AMV infection in transgenic alfalfa plants.
Assuntos
Capsídeo/genética , Medicago sativa/genética , Vírus do Mosaico/crescimento & desenvolvimento , DNA Viral/análise , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Resistência a Canamicina/genética , Medicago sativa/microbiologia , Vírus do Mosaico/genética , Doenças das PlantasRESUMO
Copies of alfalfa mosaic virus (AMV) RNA 4 synthesized by transcription in vitro were shown to be biologically active in protoplasts when inoculated together with a mixture of AMV RNAs 1, 2, and 3. A 5' cap was essential for activity, whereas 15 nonviral nucleotides at the 5' end of the RNA transcripts did not detectably affect activity. Transcripts that terminated 65 nucleotides from the 3' end of the coding region and transcripts that lacked all or part of the 3'-noncoding region were effective messengers in a wheat germ cell-free translation system. None of the incomplete transcripts, however, were as biologically active in protoplasts or in Xenopus laevis oocytes as were complete transcripts. This suggests that the 3' end of RNA 4 is involved in translational stability.
RESUMO
Enzyme-linked immunosorbent assays and local lesion infectivity assays showed that recombinant leukocyte interferons, rIFN-alpha A and rIFN-alpha D, did not reproducibly affect the accumulation of alfalfa mosaic virus in tobacco leaf discs or in tobacco or alfalfa protoplasts when applied within 1 hr after inoculation with virus.
