| The soil bacterium Agrobacterium tumefaciens is the causal agent of crown gall disease, a serious agricultural problem in many fruit, nut, and ornamental crops. A. tumefaciens pathogenesis is mediated by a unique process of horizontal gene transfer in which a fragment of virulent DNA, the T-DNA, is transferred from the bacterium into the genome of a plant cell. The expression of three T-DNA oncogenes, iaaM, iaaH , and ipt, in infected plant cells causes overproduction of the plant hormones auxin and cytokinin, leading to cell proliferation and the production of a crown gall. Using a biotechnology approach, we have generated de novo resistance to crown gall disease by initiating post-transcriptional gene silencing (PTGS) of A. tumefaciens oncogenes in Arabidopsis, tomato, and walnut. Self-complementary RNAs homologous to iaaM and ipt were found to be highly efficient inducers of silencing-mediated resistance, as more than 50% of transformed plants carrying inverted-repeat iaaM/ipt transgenes displayed abolition of tumorigenesis. Oncogene silencing was associated with accumulation of ∼25 nt siRNAs and a greater than 90% reduction in steady-state levels of iaaM and ipt mRNAs. Both macroscopic and microscopic indications of tumorigenesis were absent in oncogene-silenced lines, though A. tumefaciens populations did survive (albeit at reduced levels) in silenced tissue. Oncogene-silenced plants were resistant to tumor initiation by all tested biotype I, II, and III strains of A. tumefaciens, as was expected based upon the high level of iaaM and ipt sequence conservation across all studied strains. Interestingly, oncogene-silencing mediated resistance was not graft transmissible to susceptible plant genotypes, supporting the hypothesis that systemic silencing signals are generated exclusively by the “indirect” (sde1/sgs2-dependent) pathway of PTGS initiation. Oncogene-silenced walnuts are currently being assessed for resistance efficacy and durability under field conditions. Because resistance is based upon overall RNA sequence rather than highly stringent protein-protein interactions, high durability is expected. |
