Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA On

Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA On

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Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes 1 Hyewon Lee, Jodi L. Humann, Jennifer S. Pitrak, Josh T. Cuperus, T. Dawn Parks, Cheryl A. Whistler, Machteld C. Mok, and L. Walt Ream* Departments of Microbiology and Horticulture, Oregon State University, Corvallis, Oregon 97331 Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene ( ipt ) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH ) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt . Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt , suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5 9 end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM . Silencing A . tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease. Crown gall tumors result from overproduction of auxin and cytokinin in plant cells transformed by Agrobacterium tumefaciens (Winans, 1992). These ab- normally high phytohormone levels result from ex- pression of three genes transferred stably into the plant genome from the A . tumefaciens tumor-inducing (Ti) plasmid: iaaM (Trp mono-oxygenase), iaaH (indole-3-acetamide hydrolase), and ipt (AMP iso- pentenyl transferase; Garfinkel et al., 1981; Ream et al., 1983). IaaM converts Trp into indole-3-acetamide, which IaaH converts into indole-3-acetic acid (auxin; Inze ´ et al., 1984; Schroeder et al., 1984; Thomashow et al., 1985, 1986; Van Onckelen et al., 1986). Loss of either enzyme prevents auxin production. Ipt con- verts AMP into isopentenyl-AMP, a cytokinin (Akiyoshi et al., 1984; Barry et al., 1984; Buchmann et al., 1985). Inactivation of ipt and either one of the two auxin biosynthesis genes abolishes crown gall forma- tion (Ream et al., 1983). Adequate means do not exist to control crown gall disease on grapes, fruit and nut trees, cane berries, chrysanthemum, rose, and other nursery crops. Inoc- ulation of plants with Agrobacterium radiobacter strain K84 affords some protection against specific strains of A . tumefaciens (Moore, 1988); however, crown gall disease remains a significant problem. Arabidopsis plants resistant to crown gall disease have been pro- duced by traditional genetic approaches (Nam et al.,
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Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA On

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