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Distinct mechanisms for genomic attachment of the 5′ and 3′ ends of Agrobacterium T-DNA in plants

Abstract

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant’s nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5′ end. Different from capture of the 3′ end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5′ attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5′ end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5′ end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3′ capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant’s own DNA repair machineries to transform it.

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Fig. 1: TRANSGUIDE reveals different characteristics for LB and RB T-DNA junctions.
Fig. 2: TMEJ and cNHEJ function redundantly in genomic capture of T-DNA’s 5′ end.
Fig. 3: MRE11 and TDP2 are required for 5′ attachment of T-DNA.
Fig. 4: Proposed model of T-DNA integration.

Data availability

Mapped sequences are available from NCBI SRA (accession code: PRJNA786733). Junction footprint data is provided in Supplementary Data 2 and 3. The pAC161 data in Extended Data Figs. 2 and 3 is based on previously published data9,12.

Code availability

The custom java program used for junction calling is available from GitHub (https://github.com/RobinVanSchendel/TRANSGUIDE).

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Acknowledgements

This work was in part funded by ALW OPEN grants (OP.393 and OP.269) from The Netherlands Organization for Scientific Research for Earth and Life Sciences to M.T.

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Authors and Affiliations

Authors

Contributions

L.E.M.K., S.d.P., P.J.J.H. and M.T. conceived the study. L.E.M.K., R.v.S. and S.L.K. developed the TRANSGUIDE method. L.E.M.K., H.S. and S.d.P. conducted experiments. L.E.M.K. and R.v.S. performed bioinformatic analyses. L.E.M.K. and M.T. wrote the manuscript with input from all authors, who read the manuscript and authorized its publication.

Corresponding author

Correspondence to Marcel Tijsterman.

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The authors declare no competing interests.

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Nature Plants thanks Anne Britt, Shunping Yan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Genomic position of wild-type junctions obtained with TRANSGUIDE.

Arabidopsis chromosomes (1-5) were divided into 0.4 mb bins, in which RB (purple) and LB (green) junctions were counted. The brightness of the colour indicates the number of junctions (note the scales). Centromere positions were rounded to the nearest border between bins. The shown data is from 11 wt samples, transformed with either pUBC, pCAS9, or pWY82.

Extended Data Fig. 2 Homology, filler, and T-DNA loss profiles for 4 different constructs.

Frequency of different lengths of microhomology (a - d), filler (e - h), or T-DNA loss (i - l) at RB (purple) and LB (green) junctions, for 3 constructs after somatic transformation (pUBC, pCAS9, and pWY82) and for 1 construct after germ-line transformation (pAC161). The overlap between LB and RB is indicated in olive-green. The medians (dashed lines), the number of observations (n), and shifts in the RB distribution relative to LB (s) are indicated. Wilcoxon rank-sum tests were performed to find the direction (one-sided tests) and the significance of the shifts (two-sided tests, phomology_pUBC = 7 × 10−68, phomology_pCAS9 = 7 × 10−24, phomology_pWY82 = 2 × 10−14, phomology_pAC161 = 8 × 10−4, pfiller_pUBC = 2 × 10−1, pfiller_pCAS9 = 9 × 10−1, pfiller_pWY82 = 3 × 10−1, pfiller_pAC161 = 7 × 10−3, pdeletion_pUBC = 4 × 10−222, pdeletion_pCAS9 = 1 × 10−63, pdeletion_pWY82 = 1 × 10−32, pdeletion_pAC161 = 1 × 10−11). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Extended Data Fig. 3 Seamless junctions.

Average percentages of RB and LB junctions without T-DNA loss and without insertions, after somatic transformation (pUBC, pCAS9, pWY82) and germ-line transformation (pAC161). The number in bold indicates the number of samples over which the mean and error bars (standard error of the mean) have been calculated; the number in italic indicates the total number of junctions amongst those samples that were scored for ‘seamlessness’. Two-sided Student’s t-tests have been performed to test whether the percentage of seamless junctions differed significantly between RB and LB junctions (ppUBC = 6 × 10−9, ppCAS9 = 6 × 10−3, ppWY82 = 9 × 10−2). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Extended Data Fig. 4 Homology, filler, and T-DNA loss profiles for cNHEJ mutants.

Frequency of different lengths of microhomology (a - d), filler (e - h), or T-DNA loss (i - l) at RB and LB junctions, comparing wt (yellow) with cNHEJ mutants ku70 and lig4 (blue). The third colour in each panel indicates the overlapping area. The medians (dashed lines), the number of observations (n), and shifts in the mutant distribution relative to wt (s) are indicated. Wilcoxon rank-sum tests were performed to find the direction and the significance of the shifts (phomology_ku70_RB = 7 × 10−38, phomology_ku70_LB = 4 × 10−1, phomology_lig4_RB = 2 × 10−14, phomology_lig4_LB = 5 × 10−1, pfiller_ku70_RB = 4 × 10−1, pfiller_ku70_LB = 4 × 10−1, pfiller_lig4_RB = 4 × 10−3, pfiller_lig4_LB = 4 × 10−1, pdeletion_ku70_RB = 3 × 10−28, pdeletion_ku70_LB = 5 × 10−8, pdeletion_lig4_RB = 4 × 10−21, pdeletion_lig4_LB = 1 × 10−12). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Extended Data Fig. 5 Homology, filler, and T-DNA loss profiles for mre11 mutant.

Frequency of different lengths of microhomology (a, b), filler (c, d), or T-DNA loss (e, f) at RB and LB junctions, comparing wt (yellow) with the mre11 mutant (light red). The overlapping area is indicated in orange. The medians (dashed lines), the number of observations (n), and shifts in the mutant distribution relative to wt (s) are indicated. Wilcoxon rank-sum tests were performed to find the direction (one-sided tests) and the significance of the shifts (two-sided tests, phomology_RB = 2 × 10−6, phomology_LB = 6 × 10−1, pfiller_RB = 8 × 10−6, pfiller_LB = 3 × 10-1, pdeletion_RB = 9 × 10-8, pdeletion_LB = 8 × 10-4). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Extended Data Fig. 6 Regenerative ability.

Average percentage of calli with shoot tissue on non-selective plates. The number in italic indicates the total number of calli that were scored for that genotype. The number in bold indicates the number of experiments over which the mean (coloured bars) and standard error of the mean (error bars) were calculated. One-sided Student’s t-tests were performed to test for significant reductions in T-DNA integration efficiency of mutant compared to wt (pku70c = 9 × 10−1, plig4 = 7 × 10−1, ptdp2 = 7 × 10−1, pku70w = 6 × 10−1, pmre11 = 2 × 10−2, pmre11ku70 = 3 × 10−2, pmre11lig4 = 2 × 10−1, pmre11tdp2 = 8 × 10−4). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Mutants were compared to the wt of the same genetic background, except for mutants with a hybrid genetic background, which were compared to the Col-0 wt.

Extended Data Fig. 7 Comparison of junction numbers by competitive TRANSGUIDE.

Number of RB (a) and LB (b) junctions in competitive TRANSGUIDE, in which equimolar amounts of genomic DNA of two samples with differently barcoded T-DNA (barcode 1 in light grey, and barcode 2 in dark grey) were combined.

Extended Data Fig. 8 All tested genotypes show (transient) T-DNA expression.

Pictures show GUS-stained roots in well plates shortly after co-cultivation with Agrobacterium. The blue colour indicates expression of the T-DNA (pCAMBIA3301). Scale, 1 cm.

Extended Data Fig. 9 Homology, filler, and T-DNA loss profiles for tdp2 mutant.

Frequency of different lengths of microhomology (a, b), filler (c, d), or T-DNA loss (e, f) at RB and LB junctions, comparing wt (yellow) with the tdp2 mutant (cyan). The overlapping area is indicated in turquoise. The medians (dashed lines), the number of observations (n), and shifts in the mutant distribution relative to wt (s) are indicated. Wilcoxon rank-sum tests were performed to find the direction (one-sided tests) and the significance of the shifts (two-sided tests, phomology_RB = 7 × 10−11, phomology_LB = 2 × 10−1, pfiller_RB = 3 × 10−2, pfiller_LB = 7 × 10−1, pdeletion_RB = 2 × 10−24, pdeletion_LB = 6 × 10−2). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Extended Data Fig. 10 Relative genomic position of junctions.

Relative frequency of LB junctions after transformation with pWY82 (+ TRA, panels a-e) or pUBC (- TRA, panels f-j) along all chromosome arms, comparing wt (yellow) and mutants (other colours). Mutants were compared to wt of the same genetic background, with the exception of the hybrids (mre11 lig4 and mre11 tdp2), which were compared to the Col-0 wt. 0 % indicates centromeric position and 100 % telomeric; n indicates the number of mutant junctions. Wilcoxon rank-sum tests were performed to find the direction (one-sided tests) and significance level (two-sided tests) of the shifts (s) in relative position (plig4+TRA = 6 × 10−1, ptdp2+TRA = 4 × 10−1, pmre11+TRA = 4 × 10−1, pmre11lig4+TRA = 9 × 10−4, pmre11tdp2+TRA = 2 × 10−1, plig4-TRA = 3 × 10−3, ptdp2-TRA = 3 × 10−1, pmre11-TRA = 4 × 10−1, pmre11lig4-TRA = 7 × 10−1, pmre11tdp2-TRA = 2 × 10−3). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Only junctions that are represented by more than 20 different DNA molecules (thus representing events that are compatible with multiple cell divisions) were included in this analysis.

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Kralemann, L.E.M., de Pater, S., Shen, H. et al. Distinct mechanisms for genomic attachment of the 5′ and 3′ ends of Agrobacterium T-DNA in plants. Nat. Plants 8, 526–534 (2022). https://doi.org/10.1038/s41477-022-01147-5

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