Mechanisms of unintended transfer of Agrobacterium chromosomal DNAs (AchrDNAs) to plants and their extent, consequences, detection and elimination
Zusammenfassung der Projektergebnisse
Agrobacterium-mediated crop genome engineering is the most important and fastest growing plant biotechnology tool in the world and plants engineered with this technology are cultivated in several countries for food, feed and other industries. It was recently discovered that, besides the desired genes on T-DNA, sometimes other large bacterial chromosomal DNA fragments (AchrDNAs) 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 genetically engineered plants. Furthermore, the unnoticed transfer of large AchrDNA sequences to transgenic crops implicates important biosafety risks, when releasing transgenic plants to nature. In this work, the underlying mechanisms behind this undesired and potentially risky transfer of AchrDNA to plants were investigated. Two independent mechanisms were discovered leading to transfer of AchrDNAs to plants. These were T-DNA dependent and T-DNA independent mechanisms. The A. tumefaciens insertion sequence IS426 is one of the most frequently found AchrDNAs in plant cells but the way it is transferred was unknown. We found that IS426 can repeatedly transpose itself into T-DNA vectors using a copy and paste mechanism. IS426 cannot transfer to plant cells without the simultaneous transfer of a T-DNA or entire T-DNA plasmid. Additionally, its ability to control neighboring gene expression by insertional mutation or transcriptional activation in Agrobacterium was described. The two intact chromosomal copies of IS426 were sequentially deleted by homologous recombination in Agrobacterium strain A136. Thus, the first step towards a safer plant transformation strain was made. To understand how other AchrDNA fragments are transferred from Agrobacterium to plants, hot spots and non-hot spots regions on Agrobacterium chromosomes were tagged by inserting the coding sequence of the green fluorescent protein (gfp). The gfp-tagged bacteria were used for transient plant transformation assays. GFP expressing plant cells could be observed from most of the hot spot tagged regions but not from the control regions, indicating that the gfp gene was transferred from the bacterial chromosomes to plants and that this transfer occurs very specifically. Several subsequent molecular, genetic and biological experiments indicated that transfer of gfp tagged regions on Agrobacterium chromosomes are T-DNA independent but was completely dependent on the TypeIV secretion channel because no gfp transfer was observed from Agrobacterium deletion strains lacking the pTihelper (contains all the genes encoding the components of the TypeIV secretion channel). In order to determine whether the gfp tagged hot spots are cleaved and transferred with the help of VirD2 pilot protein or by other unknown mechanisms, new sets of experiments were initiated. A. tumefaciens mutant strain having the pTi helper with and intact TypeIV secretion system but lacking the virD2 gene was used in tagging the Agrobacterium linear chromosome hot spot-1 with gfp. Transient plant transformation assay with this engineered strain failed to show GFP expression in plant cells indicating that transfer of gfp to plant cells did not take place. This finding let to the most important clue that transfer of other AchrDNAs are virD2 dependent but T-DNA independent. Therefore, we narrowed down our searches to VirD2 cleavable sequences in and around the hot spots on Agrobacterium chromosomes. 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 AchrDNA to plant cells in a VirD2 dependent manner. The deletion of oriT-like sequence on hot spot1 from the genome of A. tumefaciens eliminated the transfer of both hot spot1 and hot spot2 tagged regions on linear chromosome. This indicated that the transfer of both these hots spots were initiated from the same element. Hence, it was shown for the first time that, apart from the Ti-plasmid borders, the A. tumefaciens genome contains additional sequences from which transfer of DNA can be effectively initiated. In future, the generation of Agrobacterium strains lacking these four trouble elements leading to un-intended and un-desired transfer of Agrobacterium chromosomal DNAs to plants would eliminate the associated biosafety risks and should re-gain the trust of researchers, Ag-biotech industry, regulators and public to this great technology. Further study of Agrobacterium genes located on hots spots transferred from Agrobacterium to plants should uncover whether such DNA transfers play additional roles in Agrobacterium-plant interactions.
Projektbezogene Publikationen (Auswahl)
-
PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications (2014). Scientific Reports 4: 5052
Hommelsheim, Carl Maximilian; Frantzeskakis, Lamprinos; Huang, Mengmeng & Ülker, Bekir
-
Precision genetic modifications: a new era in molecular biology (2014). Planta, 239 (4): 921-939
Fichtner, Franziska; Urrea Castellanos, Reynel & Ülker, Bekir
-
Unexpected chromosomal DNA transfer from Agrobacterium tumefaciens to plant cells: mechanisms and solutions. Ph.D. Thesis, May 2015
Tobias Berson
