Genomic consequences associated with Agrobacterium-mediated transformation of plants

Abstract

Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium-mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant-microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long-read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium-mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large-scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.

Keywords: Agrobacterium; T-DNA concatenation; T-DNA integration; plant transformation; structural variants.

Figure 1:. Agrobacterium-mediated transformation of plants.

1. Plant signals stimulate VirA phosphorylation of VirG. 2. VirG then binds to the vir box to stimulate production of the other Vir proteins. 3. The relaxosome, containing VirC1, VirC2, VirD1, and VirD2, binds to the T-DNA borders. VirD1 and VirD2 cleave the minus strand from the T-DNA region and VirD2 covalently binds to the 5’end to produce the T-strand for transport to the plant cell. 4. The T-strand is shuttled to the plant cell via a Type IV secretion system composed of mostly VirB proteins. 5. The T-strand, with bound VirE2, has been found to interact with many potential nuclear targeting proteins. Importin has been found to interact with VirD2 and VirE2, and VirE2 interacts also with VIP1 and VIP2. 6. Once in the nucleus, the T-DNA incorporates into a random genomic location. ssT-DNA (T-strand) can be converted to dsT-DNA, presumably by the action of Pol θ, before integration to the genome. Both ssT-DNA and dsT-DNA can likely be used for plant genome integration. DSB = double-stranded DNA break.

Figure 2:. Key features of T-DNA insertion sites.

Five examples of T-DNA insertions (yellow) relative to the wild type genome loci. The notable characteristic of the insertion, along with name, species, binary vector, and reference are listed above each. A. A T-DNA inserted into Arabidopsis with 5 bp microhomologies (red) between the T-DNA and the genome at both insertion junctions (Mayerhofer et al., 1991). B. A T-DNA insertion in the Arabidopsis genome with filler sequence at the RB/genome junction, potentially sourced from the genome sequence immediately downstream (indicated in purple and orange fonts) (Mayerhofer et al., 1991). C. A tobacco T-DNA insertion which has resulted in a 158 bp duplication of sequence adjacent to the insertion site. Following insertion, the sequence occurs either side of the insertion. There is also a 33 bp filler sequence, possibly sourced from the genome downstream for the site of insertion (Gheysen et al., 1987). D. A T-DNA insertion into the genome of Arabidopsis which has caused a 832 bp deletion of genomic DNA disrupting two adjacent genes (Dickinson et al., 2023). E. A T-DNA insertion in the Arabidopsis genome which contains a fragment of the binary vector (>= 452 bp). Note that the orientation suggests that this backbone sequence initiated at the LB, and not with LB read through. There are also filler sequences at either junction (Dickinson et al., 2023).

Figure 3:. The roles of TMEJ/MMEJ/A-NHEJ and c-NHEJ in generating a variety of T-DNA-integration outcomes.

A. Pol θ is responsible for capturing the 3’end of the T-strand at a DSB site, often utilizing microhomologies between the T-strand and genomic DNA. B. The 5’end of the T-DNA can be processed and incorporated via TMEJ or c-NHEJ. TMEJ relies on VirD2 removal by the MRN complex (which generates a 3’ overhang), while c-NHEJ can be performed on blunt, TDP2-processed ends. C. Models for the incorporations of two T-DNAs at the same locus. Model 1 is a variation of panel A defined by the capture of a T-strand 3’ end by Pol θ at both genomic sides of a DSB. This leads to a RB-RB configuration, which has been shown to be a frequent occurrence. Less common is the formation of a dimer of T-DNA in the LB-LB configuration. This may occur in the absence of Pol θ (e.g., in polq mutants), and would rely on TDP2-mediating processing of VirD2, following by c-NHEJ-dependent capture of a T-DNA 5’end at both genomic sides of a DSB, and then ligation. D. Model for the genomic integration of T-DNAs as double stranded elements. Pol θ could be responsible for converting an extrachromosomal T-strand into dsT-DNA, either via random priming or failed capture of the T-DNA 3’ end. MRN processing and random priming/failed capture would be expected to generate 3’ overhangs at both ends of dsT-DNA, allowing it to incorporate in either orientation into the genome. E. Incorporation of an MRN-processed T-DNA into the genome could induce the capture of additional T-strands by Pol θ, using the T-DNA 3’end overhang. This process may lead to multiple cycles of T-strand integration (depending on cellular T-strand availability), creating opportunities for large T-DNA concatemers to arise. TDP2-mediated processing of VirD2 may serve to break this cycle, by generating a blunt end, thus potentially favoring genomic capture. Interestingly, MRN-processed, extrachromosomal dsT-DNA (as shown in panel D) may also serve as substrates, in either orientation, in this concatenation mechanism. It is also possible that concatenated T-DNA copies can be built, using this mechanism, before any genomic capture has occurred.

Figure 4:. Examples of structural variation associated with T-DNA insertion.

Five examples of structural variants that have resulted from T-DNA insertions (yellow). The structural variant as well as name, species, binary vector, and reference are listed above each. A. An Arabidopsis T-DNA insertion called hosoba toge toge, which caused a deletion of 75.9 kb of genomic DNA. The consequence of this insertion was the deletion or disruption of 19 genes (green and brown) including Fas1, FT, and AS2 (Kaya et al., 2000). B. The mgoun2 mutant was found to be linked to two T-DNA insertions which flank a >6.8 Mb inversion on chromosome 1. The genetic markers used to map and estimate the size of this inversion are annotated alongside. Note the markers which changed their relative association to one another in the mutant relative to the wild type chromosome are highlighted in red (Laufs et al., 1999). C. A pair of T-DNA insertions on chromosomes 1 and 5 of Arabidopsis which have caused the terminal ~5 Mb of chromosome 1 to be reciprocally translocated with the terminal ~3 Mb of chromosome 5 (Curtis et al., 2009). D. A complex insertion in rice caused by chromosomal duplications. While the insertion occurs in chromosome 8, the T-DNA is flanked by 29.8 kb of duplicated chromosome 9 adjacent to the LB and 6.6 kb of duplicated chromosome 3 next to the RB (Majhi et al., 2014). E. A complex series of five insertions following AMT of birch. There are a total of five T-DNA insertions causing three large chromosomal translocations, and three large deletions. Note that the second insertion in chromosome 2 resulted in the translocation of the remaining chromosomal segment to chromosome 8 and the deletion of the chromosomal segment between the first and second insertions. The chromosomal segment before the insertion on chromosome 8 has been translocated to the terminal end of the homologous chromosome 2. The terminal chromosomal segment of chromosome 8 has been translocated to the top of chromosome 9, where the original chromosome segment has been lost (Gang et al., 2019).