Regulation of Oncogene Expression in T-DNA-Transformed Host Plant Cells

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.

Silencing of Agrobacterium tumefaciens oncogenes ipt and iaaM induces resistance to crown gall disease in plum but not in apricot

“…Infecting plant cells by Agrobacterium involves transferring T‐DNA from the bacteria to the plant cell nucleus and integrating it into the plant genome . Oncogenes such as ipt (isopentenyl transferase), iaaM (tryptophan monooxygenase) and iaaH (indole‐3‐acetamide hydrolase) are found in the T‐DNA, and their expression is responsible for the overproduction of cytokines and auxins, resulting in the initiation of uncontrolled cell division and growth and producing a tumour …”

“…11 Oncogenes such as ipt (isopentenyl transferase), iaaM (tryptophan monooxygenase) and iaaH (indole-3-acetamide hydrolase) are found in the T-DNA, and their expression is responsible for the overproduction of cytokines and auxins, resulting in the initiation of uncontrolled cell division and growth and producing a tumour. 12 Different biotechnological approaches have been developed to control crown gall disease. For example, truncated genes involved in T-DNA transfer have been used in transformation experiments to induce resistance to crown gall.…”

Unmasking host and microbial strategies in the Agrobacterium-plant defense tango

“… Ditt et al (2006) reported no difference in gene expression between Agrobacterium -infected and mock-infected Arabidopsis at 4–24 h post-infection (hpi), although their microarray analysis did reveal distinct sets of up-regulated defense genes, as well as down-regulated cell-proliferation genes, at 48 hpi. In contrast, other studies (e.g., Veena et al, 2003 ; Lee et al, 2009 ; Zhang et al, 2015 ) uncovered a variety of alterations in the host transcriptome at earlier time points. Given the rapidity of the basal defenses described here, it seems likely that Ditt et al (2006) missed many of the changes in gene expression that may well have returned to pre-infection levels by 4 hpi.…”

“…The three major Agrobacterium -derived cancer-causing transgenes encode enzymes that direct the production of the phytohormones auxin and cytokinin ( Lacroix and Citovsky, 2013a ). Zhang et al (2015) discovered that although the auxin-production genes IaaH and IaaM are constitutively expressed in Arabidopsis , the promoter for the cytokinin synthesis gene Ipt contains several W-boxes and is activated by the mutually interacting trio of WRKY18, WRKY40 and WRKY60. WRKY40 and WRKY60 are induced within 2 h of Agrobacterium infection, while WRKY18 is turned on slightly later.…”

“…WRKY40 binds directly to the Ipt promoter, and its ability to activate expression is synergistically enhanced by auxin. Predictably, mutants deficient in any of the three WRKY genes form smaller tumors than wild-type plants ( Zhang et al, 2015 ). These three host factors apparently normally function to dampen host defenses to bacterial and fungal pathogens, perhaps as part of the plant’s feedback mechanism to prevent the deleterious effects of over-reaction to a pathogen ( Pandey et al, 2010 ).…”