The complex architecture and epigenomic impact of plant T-DNA insertions.

Florian Jupe,S Timothy Motley,Mark Zander,Angeline C Rivkin,Joseph R Nery,Rosa Castanon,Huaming Chen,R Keith Slotkin,Joseph R Ecker,Todd P Michael,Justin P Sandoval,Jeremy Schmutz

doi:10.1371/journal.pgen.1007819

Abstract

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

Highlights

Plant genome engineering using the soil microorganism Agrobacterium tumefaciens has revolutionized plant science and agriculture by enabling identification and testing of gene functions and providing a mechanism to equip plants with superior traits [1, 2, 3]
Transgenics enables the study of gene function and allows the development of modern crop plants without the unwanted genetic baggage coming from natural crossing
We discovered that insertion of the anticipated DNA fragment occurred as multiple concatenated full and partial fragments that led in some cases to intra- and interchromosomal rearrangements

Summary

Introduction

Plant genome engineering using the soil microorganism Agrobacterium tumefaciens has revolutionized plant science and agriculture by enabling identification and testing of gene functions and providing a mechanism to equip plants with superior traits [1, 2, 3]. Targeted T-DNA sequencing approaches were conducted on approximately 325,000 of these lines to identify the disruptive transgene insertions and to link genotype with phenotype [4]. This wealth of sequence information, much of which has been made available prior to publication, is available at: http://signal.salk.edu/Source/AtTOME_Data_Source.html, has been iteratively updated since 2001, and accessed by the community over 10 million times by 2018. Agrobacterium-mediated transgene integration occurs through excision of the T-DNA strand between two imperfect terminal repeat sequences [5], the left border (LB) and right border (RB) [6], and translocation into the host genome (reviewed in Nester [7]). The phenomenon of long T-DNA concatemers has previously been attributed to the replicative T-strand amplification specific to the floral dip method, and which is less often observed after tissue explant transformation of roots or leave discs [22]

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Discussion

Conclusion