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Tracking and Targeting a T-DNA Vector for Precise Engineering of Plant Genomes

Final Report Summary - TRACTAR (Tracking and Targeting a T-DNA Vector for Precise Engineering of Plant Genomes)

T-DNA integration into the genome is the most commonly used tool for plant genome manipulation. However, little is known about the dynamics and regulation of the T-DNA integration to the genome. The journey of the T-DNA to the genome starts with infection by the bacteria Agrobacterium tumefaciens which transfers the T-DNA into the host cell as a single stranded molecule. The T-DNA is targeted to the nucleus where it becomes double stranded and integrates. Despite its wide use, many questions regarding T-DNA transformation have been left un-answered. How does it move in the cytoplasm or in the nucleus? when is it converted from single to double strand DNA? How many copies enter the nucleus? Does it integrate randomly? can we target it to specific loci in the genome?
Our goal was to address the above questions, using tools from molecular biology, structural chemistry, imaging, and bioinformatics. Our main scientific achievements are listed below.

- We show that it takes 6 hours or less, between the infection of Arabidopsis roots and T-DNA integration.
- T-DNA integrates in a rather random pattern of integration, with a slight enrichment in heterochromatic regions.
- The apparent bias in favor of euchromatic regions seen in T-DNA integrations selected for transgene activity is probably due to selection for gene activity rather than an integration bias.
- Live imaging of single T-DNA molecules was achieved, for both single and double-stranded T-DNA in tobacco and in Arabidopsis. Foci in the cytoplasm for sst-T-DNA and in the nucleus for dst-T-DNA were immobile and could not be tracked.
- There were 4-5 foci per cell, on average, of LacO repeats bound to LacI-mRFP proteins.
- The structure of VirE2 in complex with ssDNA was clarified by combined methods including cryo-electron microscopy, molecular modeling, and mass spectrometry. The modes of binding were investigated by surface plasmon resonance imaging.
- VirE2 is rapidly removed from ssDNA in Arabidopsis nuclear extract, independently of proteolysis.
- A comparative study showed and quantified the high efficiency of the CRISPR-Cas for genome editing in plants, compared to earlier technologies (ZFNs and TALENs).
- We can target any genomic locus for DNA break induction and mutagenesis in tomato using CRISPR-Cas making it an ideal system for genome editing.
- Ongoing work will determine the efficiency of CRISPR-Cas mediated DNA break induction in applications such as gene targeting or targeted crossover induction.

This project has received broad international exposure, with 7 publications, and 16 lectures presented at international conferences or in seminars. It was also presented to the general public (lectures in schools, to teachers, to the general public, TV interviews). We have trained a total of 13 students and postdoc who took active part in the project as well as 2 undergraduates.