RESUMEN
Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
ARN Polimerasa Dependiente del ARN/genética , Virus del Mosaico del Tabaco/enzimología , Tobamovirus/inmunología , Secuencia de Bases , Cromosomas , ADN Viral , Inmunidad Innata , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Plantas Tóxicas , Plásmidos , ARN Polimerasa Dependiente del ARN/metabolismo , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/microbiología , Virus del Mosaico del Tabaco/genética , Virus del Mosaico del Tabaco/patogenicidad , Tobamovirus/genéticaRESUMEN
We engineered cDNA of tobacco mosaic tobamovirus (TMV) into Agrobacterium tumefaciens for inoculation of plant cells. The resulting bacterial strains were used to transfect tobacco (Nicotiana tabacum cv. Xanthi and Xanthi/nc) with wild type and a defective virus. Lesion formation on Xanthi/nc tobacco was used to measure the timing and efficiency of transfection. Infections mediated by Agrobacterium produced lesions an average of two days later than infections produced by inoculation with virions. The addition of approximately 80 bp of non-viral sequences to the 5'-end of TMV transcripts abolished transfection. Transcripts with non-viral sequences at the 3'-end initiated infections, while precise transcript termination with a synthetic ribozyme sequence increased transfection frequencies two-fold. Culture conditions reported to induce genes of the vir region of the Agrobacterium Ti plasmid also increased the transfection frequency approximately two-fold. Therefore, in addition to the pararetroviruses and geminiviruses previously described, 'agroinoculation' may be used to infect plants with plus-sense RNA viruses.
Asunto(s)
Agrobacterium tumefaciens/genética , Nicotiana/genética , Plantas Tóxicas , Virus del Mosaico del Tabaco/genética , Transfección/métodos , Factores de Virulencia , Proteínas Bacterianas/genética , Secuencia de Bases , Vectores Genéticos/genética , Datos de Secuencia Molecular , Plásmidos/genética , ARN Catalítico/genética , ARN Viral/genética , Nicotiana/microbiologíaRESUMEN
alpha-Trichosanthin, a eukaryotic ribosome-inactivating protein from Trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (HIV) in vitro. The alpha-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant RNA viral vector. Two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated alpha-trichosanthin to levels of at least 2% of total soluble protein. The recombinant alpha-trichosanthin was purified and its structural and biological properties were analyzed. The 23-amino acid signal peptide was recognized by N. benthamiana and the processed enzyme caused a concentration-dependent inhibition of protein synthesis in vitro. The high level of heterologous gene expression observed in these studies is due to the unique features of the RNA viral-based transfection system.
