A Genomic Analysis of Tumor Development and Source-Sink Relationships in Agrobacterium-Induced Crown Gall Disease in Arabidopsis

Agrobacterium tumefaciens is a Gramnegative rod-shaped bacterium that is commonly found in the rhizosphere of many plants, where it survives on root exudates. It will infect a plant only through a wound site (which often occurs in nursery stock through transplanting and grafting and in vineyards through pruning). Galls typically form at the crown (the point at the soil line where the main root joins the stem) but may also develop on secondary or lateral roots and on the main stem and branches above the soil line (see figure). Agrobaterium is widely recognized for its ability to transfer foreign DNA into plant cells, whereby T-DNA becomes integrated into the plant genome. Certain phenolic compounds produced by the plant (including acetosyringone) cause the induction of agrobacterial virulence genes encoding, among other proteins, an endonuclease that excises T-DNA from the bacterial tumor-inducing plasmid. The T-DNA then becomes integrated into the plant genome, and T-DNA genes are expressed via the plants normal transcriptional and translational machinery. Some of the salient features of crown gall disease were reviewed by Nester et al. (1984), and a review concerning T-DNA transfer was presented by Gelvin (2003). Agrobacterium-based vectors are the most widely used tools for plant transformation in research and commercial applications. Plant transformation vectors are created in the laboratory by removing the tumorand disease-related oncogenes and retaining only those segments of DNA (the T-DNA border repeat sequences) responsible for T-DNA integration. T-DNA oncogenes include several encoding enzymes that synthesize the plant hormones cytokinin and auxin (indole-3-acetic acid) and novel plant metabolites known as opines (such as octopine, nopaline, and the agrocinopines). The synthesis and release of phytohormones and opines from transformed plant cells causes changes in gene expression and metabolism. Phytohormone overproduction results in the development and proliferation of tumors, and opines are small molecules that provide a carbon and energy source for the agrobacteria. The study of the development of crown gall disease in plants is important, not only because the disease affects a wide range of dicotyledonous plants (especially those in the rose family, including fruit trees and raspberries as well as roses), but also because of the nature of the developmental changes that occur. Understanding tumorigenesis and crown gall development could provide important insights into plant hormone signaling pathways, carbon and nitrogen metabolism, and source-sink relationships. In this issue of The Plant Cell, Deeken et al. (pages 3617–3634) provide an indepth functional genomics analysis of the developmental changes that occur during tumorigenesis in Arabidopsis infected with virulent agrobacteria. To get a comprehensive picture of a T-DNA–induced plant tumor, the authors combined genome-wide expression analysis (using Affymetrix ATH1 arrays) with direct analysis of metabolites in tumor tissue. Tumors were induced by inoculating the nopaline-using Agrobacterium strain C58 to the base of a wounded, very young inflorescence stalk, and tumor tissue was harvested 35 d after inoculation. Wounded but uninfected tumor-free inflorescence stalk segments of the same age served as reference tissue. The micorarray data were obtained from four independent biological replicates each from tumor tissue and nontumorous reference tissue, for a total of eight independent microarray hybridizations. The authors conducted several experiments to show convincingly that the results obtained reflect changes in plant, rather than bacterial, metabolism and that these changes are the result of plant transformation. Using in situ hybridization with antisense-RNA for the T-DNA oncogene Nopaline synthase (NOS) as probe, the