Unexpected chromosomal DNA transfer from <em>Agrobacterium tumefaciens</em> to plant cells

Abstract

The soil bacterium and plant pathogen <i>Agrobacterium tumefaciens</i> has the ability to transfer a defined part of its genome, the so called transferred DNA (T-DNA), to plant cells. This transfer has been successfully exploited in modern plant biotechnology and today researchers utilize the Agrobacterium-mediated plant transformation to gather important information about all aspects of plant biology. Furthermore, crop modification by Agrobacterium-mediated genome engineering is the fastest growing croptechnology in the world and transgenic crops are cultivated in several countries with applications in food, feed and other industries.<br /> It was recently discovered that, besides genes located on T-DNA, sometimes other large bacterial chromosomal DNA fragments (<i>A</i>chrDNAs) are unintentionally transferred from bacteria to plants by an unknown mechanism. This additional DNA transfer to plant cells added a new aspect to our understanding of horizontal gene transfer and genome evolution but also major complications to the generation and analysis of transgenic plants. Furthermore, the unnoticed transfer of large <i>A</i>chrDNA sequences to transgenic crops implicates important biosafety risks, when releasing transgenic plants to nature.<br /> In this work, the underlying mechanisms behind the undesired transfer of <i>A</i>chrDNA to plants were investigated with the eventual goal to find ways to prevent them and thereby to improve the safety and reliability of Agrobacterium-mediated plant transformation. In the course of this study, the <i>A. tumefaciens</i> transposable element IS<i>426</i> was found to integrate itself repeatedly into T-DNA vectors. IS<i>426</i> is one of the most frequently found <i>A</i>chrDNAs in plant cells but the way it is transferred is unknown. In this work it was shown that IS<i>426</i> cannot transfer to plant cells without the simultaneous transfer of a T-DNA. Additionally, its ability to control neighbouring gene expression was described.<br /> The two chromosomal copies of IS<i>426</i> were sequentially deleted by homologous recombination in Agrobacterium strain A136. Thus, the first step towards a safer plant transformation strain was made.<br /> The transfer of <i>A</i>chrDNA to plant cells was successfully visualized by inserting the gene encoding the green fluorescent protein (gfp) into different regions of the bacterial genome. The tagged bacteria were used for plant transformation. GFP expressing plant cells could be observed, indicating that the gfp gene was transferred from the bacterial chromosomes to plants.<br /> Using an <i>A. tumefaciens</i> mutant strain led to the finding that VirD2, one of the most important proteins involved in T-DNA transfer, is also involved in the transfer of <i>A</i>chrDNA to plant cells. Thus, it was hypothesized that VirD2 can bind to bacterial chromosomal regions and from there mediate the transfer of <i>A</i>chrDNA to plant cells. By using bioinformatics, cloning and transient tobacco transformation assays, it was shown that a cryptic origin of transfer-like sequence (oriT-like), as well as a cryptic T-DNA border-like sequence (RB-like), were responsible for transferring <i>A</i>chrDNA to plant cells in a VirD2 dependent manner.<br /> The deletion of one of these sequences from the genome of <i>A. tumefaciens</i> drastically reduced this transfer. Hence, it was shown for the first time that, apart from the Ti-plasmid borders, the <i>A. tumefaciens</i> genome contains additional sequences from which a transfer of DNA can be initiated.