Abstract
Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.
Highlights
The bacterial pathogen A. tumefaciens genetically engineers the host plant by transferring its T-DNA, a piece of DNA from the tumor-inducing (Ti) plasmid, into the plant genome
Crown gall tumor development requires expression of oncogenes, which are transferred on T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome
We found that oncogene expression was associated with an unmethylated oncogene sequence the promoters were susceptible to methylation. siRNA–mediated promoter methylation caused transcriptional silencing of oncogenes and prevented crown gall proliferation
Summary
The bacterial pathogen A. tumefaciens genetically engineers the host plant by transferring its T-DNA, a piece of DNA from the tumor-inducing (Ti) plasmid, into the plant genome. Expression of the T-DNA-encoded oncogenes IaaH, IaaM, and Ipt results in increased synthesis of both auxin and cytokinin [1,2]. High concentrations of these phytohormones facilitate proliferation of transformed plant cells, and differentiation of specialized cell types within the resulting crown gall tumor [3]. The massive changes in gene expression, together with the cooperative action of phytohormones, fulfill distinct roles in differentiation, pathogen defense, metabolic changes, and physiological adaptations in crown galls [3,7,8]. Environmental stresses have been shown to influence epigenetic processes, inducing the release of transcriptional silencing of transgenes and several endogenous A. thaliana gene loci
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