Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3' terminal nucleotides ofthe TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 ,ug/ml or 300 ,ug/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 ,g/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to S5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequencespecific RNA transcript of the expected size, but no protein product was detected.The organization of the tobacco mosaic virus (TMV) genome is fairly well understood. However, one aspect of the genome strategy that has not been fully elucidated is the exact nature of the replicase enzyme responsible for the synthesis of the genomic and subgenomic RNAs. It is generally accepted that the virus codes for four proteins, two of which are translated from the genomic RNA and two from individual subgenomic RNAs (see review in ref. 1). The 5' proximal region of the genomic RNA encodes two coinitiated proteins, the 126-kDa and 183-kDa proteins, considered to be components of the replicase (2). The 183-kDa protein is generated by a readthrough of the UAG stop codon of the 126-kDa protein. (8), although on occasion we have seen inconclusive faint bands in the region of the gel where such a protein would be expected. This is in spite of the fact that our antiserum is capable of precipitating the 54-kDa protein generated from in vitro translation products of either TMV RNA or T7 transcripts of the 54-kDa gene.In an effort to attribute a function to the 54-kDa protein, we have transformed tobacco with the coding sequence for this nonstructural viral protein. Unexpectedly, these plants showed a complete resistance to replication of the Ul strain of TMV from which the 54-kDa gene sequence was derived. These findings and their implications for the production of virus-resistant plants are discussed. MATERIALS AND METHODSPlants and Virus Strains. TMV strain U1 was purified from infected Nicotiana tabacum cv. Turkish Samsun as described by Asselin and Zaitlin (9). Unless otherwise indicated, subsequent use of the term TMV implies strain U1. Before use, preparations of TMV strain U2 (10) and L (11), which had been stored as laboratory stocks at 40C, were pelleted at 75,000 X g for 25 min and resuspended in water to enrich for full-length virions. A preparation of freshly prepared cucumber mosaic virus (CMV) strain Fny was a gift of M. J. R...

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Here, we describe a novel clade within Ensifer meliloti and consider how geographic and ecological isolation contributed to the limited distribution of this group. Members of the genus Ensifer are best known for their ability to form nitrogenfixing symbioses with forage legumes of three related genera, Medicago L., Melilotus Mill., and Trigonella L., which are members of the tribe Trifolieae. These legumes have a natural distribution extending from the Mediterranean Basin through western Asia, where there is an unsurpassed number of species belonging to these genera. Trigonella suavissima L. is unusual in that it is the only species in the tribe Trifolieae that is native to Australia. We compared the genetic diversity and taxonomic placement of rhizobia nodulating T. suavissima with those of members of an Ensifer reference collection. Our goal was to determine if the T. suavissima rhizobial strains, like their plant host, are naturally limited to the Australian continent. We used multilocus sequence analysis to estimate the genetic relatedness of 56 T. suavissima symbionts to 28 Ensifer reference strains. Sequence data were partitioned according to the replicons in which the loci are located. The results were used to construct replicon-specific phylogenetic trees. In both the chromosomal and chromid trees, the Australian strains formed a distinct clade within E. meliloti. The strains also shared few alleles with Ensifer reference strains from other continents. Carbon source utilization assays revealed that the strains are also unusual in their ability to utilize 2-oxoglutarate as a sole carbon source. A strategy was outlined for locating similar strains elsewhere.IMPORTANCE In this study, we employed a biogeographical approach to investigate the origins of a symbiotic relationship between an Australian legume and its nitrogen-fixing rhizobia. The question of the ancestral origins of these symbionts is based on the observation that the legume host is not closely related to other native Australian legumes. Previous research has shown that the legume host Trigonella suavissima is instead closely related to legumes native to the Mediterranean Basin and western Asia, suggesting that it may have been introduced in Australia from those regions. This led to the question of whether its rhizobia may have been introduced as well. In this study, we were unable to find persuasive evidence supporting this hypothesis. Instead, our results suggest either that the T. suavissima rhizobia are native to Australia or that our methods for locating their close relatives elsewhere are inadequate. A strategy to investigate the latter alternative is proposed.KEYWORDS Ensifer, rhizobium, Sinorhizobium meliloti, Trigonella, biogeography, clade, symbiotic nitrogen fixation

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