An Insight into T-DNA Integration Events in Medicago sativa
Abstract
:1. Introduction
- The ssT-DNA molecules can be directly integrated according to the model of Tinland [17]; however, such model does not explain the production of complex T-DNA insertions. Therefore, a model whereby the ssT-DNA is converted into double stranded T-DNA (dsT-DNA) prior to integration was proposed [5,18,19,20].
- The presence of the so-called filler DNA, that is, DNA sequences from unknown sources often found between tandemly repeated copies of T-DNA or between the borders and the gDNA, points to a role of the double strand break (DSB) repair machinery in T-DNA integration [5]. The dsT-DNA, that seems to be abundant in Agrobacterium infected plant cells [16], can be recruited by the plant cell’s own DSB repair machinery, thus leading to end joining between ds-molecules and/or integration; this pathway could represent the most likely route for T-DNA integration [21]. Notably, Singer et al. [16] observed the formation of complex T-DNA circular structures in infected cells resembling the observed complex patterns of integration. Recently, van Kregten et al. [22] demonstrated the involvement of polymerase theta (Pol θ), a DSB repair enzyme, in T-DNA integration in Arabidopsis: the primer–template switching ability of this polymerase can explain the presence of filler DNA.
- The availability of DSBs could be a limiting factor in the integration of T-DNA; however, a role of other types of lesions, such as single strand breaks (SSBs) cannot be excluded [4]. DSBs can be repaired either by the non-homologous end joining (NHEJ) pathway or by the homologous recombination (HR) pathway; the former seems to be the most frequent in plants, although the results are contradictory and a potential influence of the method of transformation used and of the cell type and/or developmental stage have been suggested. The evidences on the chromatin modifications that are essential for the DSB repair response and important for T-DNA integration have been reviewed, and models of integrations proposed [4].
2. Results
2.1. Isolation of Sequences Flanking T-DNA Insertions
2.1.1. LB Junctions
2.1.2. RB Junctions
2.2. Polymerase Chain Reaction (PCR) Detection of Vector Backbone Sequences
2.3. Southern Blot Analysis
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Isolation of Sequences Flanking T-DNA Insertions
4.3. PCR Detection of Vector Backbone Sequences
4.4. Southern Hybridization Analysis
5. Conclusions
- (a)
- Launching the T-DNA from the Agrobacterium chromosome may reduce the risk of transferring sequences belonging to the binary vector (including antibiotic resistance genes for bacterial selection); however, engineering the bacterial chromosome adds complication to the procedures, and a decrease in the transformation efficiency may result [27]; it should also be considered that rare cases of transfer of sequences belonging to Agrobacterium chromosome are documented [10,11,12];
- (b)
- (c)
- Using plant-derived sequences for vector construction (e.g., plant-derived SMGs, cisgenesis) [68,69,70] can relieve the perceived risk of genetically modified plants. The application of new breeding techniques such as genome editing is also offering new tools for precise modification of the alfafa genome.
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Species | Agrobacterium Strain | Vector | VB % * | References |
---|---|---|---|---|
Arabidopsis | EHA101, GV3101, LBA4404 | pTF101.1, pTF::Bin19, pTF::UCD2, pTF:ri, pSDM1550, pITC15, pMAW2035HYG | 0–68 | [9,27,28] |
Barley | AGL0 | pVec8-GFP | 48 | [29] |
Barrel medic | EHA105 | pSIM843 | 56 | [30] |
Canola | ABI | pMON67438 | 15 | [31] |
Creeping bentgrass | EHA101 | pPMI-GFP, pUHVA1, pAHVA1 | 3 | [32] |
Corn | ABI | pMON92726, pMON65153 | 30–33 | [31,33] |
Cotton | AGL1 | pPZP-GFP | 31 | [34] |
Grapevine | LBA4404 | pGA643, pBH710 | 29–50 | [35,36] |
Maize | EHA101, GV3101, LBA4404 | pTF101.1, pTF::Bin19, pTF::UCD2, pTF:ri | 18–55 | [27] |
Petunia | LBA4404 | pFLG5972 | 22 | [37] |
Potato | LBA4404 | pSIM108 | 72 | [38] |
Rice | LBA4404, AGL1, EHA105 | pCXa21K, pC30063, pGreen/pSOUP, pSK100/200, pEU334NA/NB, pNU393B2, pGA2144 | 4–60 | [39,40,41,42] |
Sorghum | LBA4404, AGL1 | PHP32269 | 4–26 | [43] |
Soybean | ABI | pMON83326 | 40 | [31] |
Strawberry | LBA4404 | pBINPLUS, pGUSINT | 67–90 | [44] |
Tobacco | LBA4404, GV3101, EHA105 | pBSG-1/BSG-2, pBH710 | 75–80 | [8,36] |
Tomato | LBA4404 | pBH710 | 67 | [36] |
Wheat | AGL1 | pCG181-1G+pCS167-1B, pCG185-1G+pCS167-1B, pCG185-2G+pAL154, pCG185-3G+pAL154, pCG185-4G+pAL154 | 8–62 | [45,46] |
Plant Group | No. of Events | Agrobacterium Strain a | Binary Vector | FS b | VB |
---|---|---|---|---|---|
A | 15 | LBA4404 | pPZP-nptII-hemL | 6 (40.0%) | 4 (26.6%) |
B | 13 | LBA4404 | pPZP-hemL + pZPZ-nptII | 8 (61.5%) | 4 (30.4%) |
C | 9 | LBA4404 | pPZP-nptII | 4 (44.4%) | 3 (33.3%) |
D | 9 | AGL1 | pPZP-MsGSAgr | 6 (66.6%) | nt |
Total | 46 | 24/46 (52.2%) | 11/37 (29.7%) |
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Nicolia, A.; Ferradini, N.; Veronesi, F.; Rosellini, D. An Insight into T-DNA Integration Events in Medicago sativa. Int. J. Mol. Sci. 2017, 18, 1951. https://doi.org/10.3390/ijms18091951
Nicolia A, Ferradini N, Veronesi F, Rosellini D. An Insight into T-DNA Integration Events in Medicago sativa. International Journal of Molecular Sciences. 2017; 18(9):1951. https://doi.org/10.3390/ijms18091951
Chicago/Turabian StyleNicolia, Alessandro, Nicoletta Ferradini, Fabio Veronesi, and Daniele Rosellini. 2017. "An Insight into T-DNA Integration Events in Medicago sativa" International Journal of Molecular Sciences 18, no. 9: 1951. https://doi.org/10.3390/ijms18091951