Abstract
Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation.
Highlights
Agrobacterium tumefaciens is a pathogenic bacterium that infects several plant species
The intergenic regions between the oncogenes function as promoters in plant cells To discover how the expression of the agrobacterial oncogenes indole-3-acetamide hydrolase (IaaH), IaaM and isopentenyl transferase (Ipt) is regulated in plant cells, we analyzed the structure of the transfer DNA (T-DNA) region of the nopaline-type tumor inducing (Ti) plasmid pTiC58
To prove whether the intergenic regions (IGRs) function as promoters in plant cells, the complete IGR sequences were fused with the coding sequence (CDS) of the green fluorescent protein (GFP) in a binary vector
Summary
Agrobacterium tumefaciens is a pathogenic bacterium that infects several plant species. The first is responsible for producing opines, so providing a carbon and nitrogen source for A. tumefaciens, with the second group expressing the oncogenes required for crown gall development. These oncogenes include IaaH, IaaM, Ipt, gene 6b and gene 5. IaaH and IaaM code for enzymes that catalyze biosynthesis of auxin and Ipt mediates cytokinin biosynthesis [5,6]. A. tumefaciens secretes auxin and cytokinin from the cells to initiate crown gall development [16] and pretreatment of plant tissues with auxin and cytokinin promotes A. tumefaciens-mediated transformation efficiency [14,17,18]. Very recently it was shown that cytokinins secreted by A. tumefaciens repress a Myb transcription factor in host plant cells, resulting in an enhanced transformation efficiency [18]
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