Translation Start Sequences Affect the Efficiency of Silencing ofAgrobacterium tumefaciensT-DNA Oncogenes

Abstract

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′ 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.

Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines

“…Multiple T-DNA insertions are likely to be quite frequent in Arabidopsis TDNA-insertion libraries (Humann et al 2006; Jorgensen et al 1986; Lee et al 2003; Viss et al 2003). Insertions of inverted T-DNA repeats are common (Jorgensen et al 1986) and two or more of these will provide extended regions for homologous recombination regardless of the respective directions of insertion into plant DNA.…”

Salicylic Acid and Systemic Acquired Resistance Play a Role in Attenuating Crown Gall Disease Caused byAgrobacterium tumefaciens

“…However, this cross-protection is limited to only certain Agrobacterium strains. Alternate methods based on RNA interference to down-regulate auxin biosynthetic genes, including iaaM and ipt oncogenes (Escobar et al, 2001; Lee et al, 2003;Viss et al, 2003), were proposed for controlling the disease. However, these approaches are faced with limited practical application due to the need for developing transgenic lines.…”

Grapevine Crown Gall Suppression Using Biological Control and Genetic Engineering: A Review of Recent Research

“…The use of RNAs leads to the degradation of targeted RNA to nucleotides resulting in T-DNA oncogene expression prevention. Efficacy of gene silencing for high-level CG resistance was confirmed by several research experiments (Ebinuma et al 1997, Escobar et al 2001, Kovács et al 2003, Lee et al 2003.…”

“…A possible explanation for this phenomenon is that the 35S-iaaM transgene may have produced a chimeric RNA molecule that was unable to induce silencing of the A. vitis iaaM gene (Kovács et al 2003, Kovács, personal communication, 2009. It was demonstrated that the iaaM silencing construct must contain the translation start site of the gene's sense strand in order for silencing to be effective (Lee et al 2003).…”