The ability to suppress transiently mRNA accumulation of specific genes in a high-throughput fashion is a powerful tool in a genomics-scale approach to assign biological function to uncharacterized genes. Virus induced gene silencing, or VIGS, is a method to transiently interrupt gene function through RNA interference. The exact mechanism by which VIGS operates is still unclear. It is known, however, that this approach harnesses the plant?s natural ability to suppress the accumulation of foreign RNAs by an RNA-mediated defense mechanism against plant viruses.38 The systemic signal by which this mechanism is induced is unknown, but it is thought to involve an RNA component.39 Recently it was shown that inoculation of transcript from cloned viruses capable of expressing host sequences in plants led to silencing of the homologous host gene.40 The infected plants displayed a phenotype representative of the loss of function of the host gene, and not of virus infection.

Should unique ESTs have no known role, it is possible to screen these sequences for function by using a virus vector to induce transient knockout of the gene in M. truncatula. We are evaluating a variety of virus candidates capable of infecting M. truncatula. Once identified, this virus (or viruses) will be used to construct VIGS-based gene knockout vectors. Putative function will be assigned to uncharacterized genes by screening suppressed plants at the visual, mRNA, and metabolite phenotypic levels. We anticipate that many of the M. truncatula uncharacterized genes are legume-specific, based on their absence from the genomes of mammalian, microbe, and other plant species. We hope that these unknown genes will give insight into the complex biochemical and physiological traits unique to the leguminous species.

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