Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

Abstract

Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.The soil bacterium Agrobacterium tumefaciens is a plant pathogen responsible for tumor induction on dicotyledonous plants through its ability to transfer DNA carrying plant-active oncogenes to the plant cell (16,25,36). Because of this ability, Agrobacterium is widely used for plant transformation (12). The transferred DNA (T-DNA), which in Agrobacterium resides on a large tumor-inducing (Ti) plasmid, is processed within the bacterium and is exported to the plant, where it is integrated into the plant genome (28,29,30). Proteins encoded by the virulence (vir) region of the Ti plasmid regulate T-DNA processing and transfer. Virulence proteins recognize 24-bplong imperfect direct repeats (border sequences) that define the T-DNA. In the presence of the VirD1 protein, VirD2 cleaves the border sequence in a site-and strand-specific manner and concomitantly becomes covalently attached to the 5Ј end of the nicked DNA (9, 13, 33, 35). The nicked DNA is then displaced 5Ј to 3Ј from the plasmid, producing single-stranded T-DNA (8). The T-DNA-VirD2 complex and the VirE2 protein are believed to be transferred to the plant with the help of a pilus-like structure containing the VirB and VirD4 proteins (2, 4, 10, 37). In the plant cell, T-DNA is coated with the single-stranded DNA (ssDNA)-binding protein VirE2 (8, 25), forming a T-DNA-protein complex that is imported into the nucleus, where the T-DNA is integrated into the nuclear genome. The VirD2 protein is transferred into the nucleus in conjunction with the T-DNA; it presumably remains attached to it up to the integration step.In higher eukaryotic organisms, such as plants, illegitimate recombination is the predominant mechanism of integration for naked DNA (20,22,24,26). Likewise, the T-DNA is integrated into the plant genome by illegitimate recombination, a mechanism in which two DNA molecules that do not share extensive homology are joined (11,18,19,31). It is not clear yet whe...

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…The functional conservation of TBP and CDK-activating protein kinase (CAK2) orthologs in eukaryotes indicates that these nuclear VirD2-binding factors may provide a link between T-DNA integration and transcription-coupled repair. Ziemienowicz et al (2000) demonstrated that VirD2 protein does not possess general ligase activity using an in vitro ligation assay. Enzyme(s) present in plant extracts, likely DNA ligase, and E. coli T4 DNA ligase were able to ligate the 5' end of T-DNA from an artificial T-DNA-VirD2 complex to a partly double-stranded oligonucleotide (Ziemienowicz et al, 2000).…”

“…Ziemienowicz et al (2000) demonstrated that VirD2 protein does not possess general ligase activity using an in vitro ligation assay. Enzyme(s) present in plant extracts, likely DNA ligase, and E. coli T4 DNA ligase were able to ligate the 5' end of T-DNA from an artificial T-DNA-VirD2 complex to a partly double-stranded oligonucleotide (Ziemienowicz et al, 2000). An Arabidopsis orthologue of the yeast and mammalian DNA ligase IV gene termed AtLIG4 (At5g57160) is required for the repair of DNA damage because seedlings of the Atlig4 mutant are hyper-sensitive to the DNA-damaging agents methyl methanesulfonate and X-rays (Friesner and Britt, 2003;van Attikum et al, 2003).…”

“…Integration and/ or expression of T-DNA AtLIG4 (DNA ligase IV) At5g57160 Ziemienowicz et al, 2000;Friesner and Britt, 2003;van Attikum et al, 2003;Zhu et al, 2003a;Nishizawa-Yokoi et al, 2012;Park et al, 2015KU80 At1g48050 van Attikum et al, 2001Friesner and Britt, 2003;Gallego et al, 2003;Li et al, 2005b;Nishizawa-Yokoi et al, 2012;Jia et al, 2012;Mestiri et al, 2014;Park et al, 2015KU70 At1g16970 van Attikum et al, 2001Li et al, 2005b;Nishizawa-Yokoi et al, 2012;Jia et al, 2012;Mestiri et al, 2014;Park et al, 2015 MRE11 (meiotic recombination 11) At5g54260 van Attikum et al, 2001;Jia et al, 2012; XRCC1 (homolog of X-ray repair cross complementing 1) At1g80420 Mestiri et al, 2014;Park et al, 2015 XRCC2 (homolog of X-ray repair cross complementing 2) At5g64520 Mestiri et al, 2014;Park et al, 2015 XRCC4 (homolog of X-ray repair cross complementing 4) At3g23100 Vaghchhipawala et al, 2012;Park et al, 2015 XPF/RAD1/UVH1 (ultraviolet hypersensitive 1) At5g41150 Nam et al, 1998;Mestiri et al, 2014;Park et al, 2015 PARP1 (poly(ADP-ribose) polymerases 1) At2g31320 Jia et al, 2012;Park et al, 2015 HTA1 (histone H2A) At5g54640 Nam et al, 1999;Mysore et al, 2000a, b;Yi et al, 2002Yi et al, , 2006…”

The Agrobacterium-Plant Cell Interaction. Taking Biology Lessons from a Bug

“…Because the T-DNA does not encode enzymatic activities required for integration, the protein components of the T-complex, i.e. VirD2 (Tinland et al, 1995; Mysore et al, 1998) and VirE2 (Rossi et al, 1996) and/or host nuclear factors, such as AtKu80 (Friesner and Britt, 2003) and plant DNA ligases (Ziemienowicz et al, 2000;Friesner and Britt, 2003), must provide these functions. T-DNA integration was proposed to initiate with ligation of the 5Ј end of the T strand to the genomic DNA followed by second strand synthesis by the plant DNA repair machinery (Tinland et al, 1995).…”

Site-Specific Integration ofAgrobacterium tumefaciensT-DNA via Double-Stranded Intermediates

“…Conversely, this model cannot account for the formation of several complex T-DNA integration patterns (e.g. Krizkova and Hrouda, 1998;McCormac et al, 2001), it relies on the ligase activity of VirD2 that may be dispensable for integration in vitro (Ziemienowicz et al, 2000), and it does not explain the fast kinetics of transient T-DNA expression (Janssen and Gardner, 1990;Tinland et al, 1994;Narasimhulu et al, 1996;De Neve et al, 1997) or rapid expression of T strands corresponding to the coding strand of the T-DNA (Narasimhulu et al, 1996), all of which imply that T strands can be converted to double-stranded molecules already early in the infection process.…”