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

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.

Photosynthesis in Arabidopsis Is Unaffected by the Function of the Vacuolar K+ Channel TPK3

“…sequencing (Jupe et al, 2019) and represents to our knowledge the only publicly available line with an insertion in the TPK3 coding region. Subsequently, we isolated homozygous tpk3-1 (SALK_049593) individuals by PCR and confirmed a T-DNA insertion in the first exon by sequencing ( Fig.…”

Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing

“…Read-lengths are limited only by input DNA lengths, and validated reads as long as 2,272,580 bases have been reported [2]. Nanopore sequencing has been used for various applications, including genome sequencing of Arabidopsis and a wild tomato relative [3, 4], resolving complex T-DNA insertions [5], and disease resistance gene enrichment sequencing [6].…”

The RNA Export Factor ALY1 Enables Genome-Wide RNA-Directed DNA Methylation

“…We explored the coverage of this locus in our MethylC-seq data (data not shown) and found a 12.6-kb deletion present in the aly1-2 mutant line encompassing the six genes AT4G22285 to AT4G22310 (gene annotations provided in Supplemental Figure 4A). Deletions can occur during the process of transgenesis, which was used to generate the aly1-2 line (Nacry et al, 1998;Jupe et al, 2019). We found that this deletion did not cause the loss of RdDM phenotype in aly1-2, as the deletion was not present in the aly1-1 mutant line (Supplemental Figure 4B), which displays decreased RdDM ( Figures 1A and 1C).…”