Formation of Complex Extrachromosomal T-DNA Structures in <i>Agrobacterium tumefaciens</i>-Infected Plants

Singer, Kamy; Shiboleth, Yoel Moshe; Li, Jianming; Tzfira, Tzvi

doi:10.1104/pp.112.200212

Cited by 44 publications

(58 citation statements)

References 68 publications

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“…Tandem repeat junctions with short overlapping microhomology sequences at junctions between two T-DNA units exist in cotton (Zhang et al 2008), aspen (Kumar and Fladung 2000) and wild tobacco (Singer et al 2012). In our study, identity existed between the single joining nucleotides from the RB sequences and the filler DNA in T-DNA junctions in some of our recovered clones.…”

Section: Discussionmentioning

confidence: 53%

“…These data suggested that the formation of complex repeats in our transgenic plants potentially involved homologous recombination. Singer et al (2012) noticed the possible involvement of homologous recombination in the formation of circular T-DNA molecules in the plant transformation. In our study, homology between filler sequences in the junctions and plant genomic DNA existed in some clones (e.g.…”

Section: Discussionmentioning

confidence: 98%

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Quantitative and structural analyses of T-DNA tandem repeats in transgenic Arabidopsis SK mutant lines

Shu

Song

et al. 2015

Plant Cell Tiss Organ Cult

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Complex T-DNA repeat structures are a frequent outcome of Agrobacterium-mediated plant transformation and often lead to co-suppression of gene expression, even gene silencing. Thus, they are undesired in transgenic plants for commercial or research purposes and frequently need to be efficiently identified in a large transgenic plant population. Application of conventional Southern blot analysis is limited because it is laborious and time consuming. In this study, a new advancement that enables for high throughput determination of T-DNA repeat copy numbers in large scale screening of transgenic plant populations was developed by improving on the standard addition quantitative PCR method with specific reference plasmids. The plasmids contained the Arabidopsis single copy gene encoding high mobility group A and either a T-DNA direct repeat or T-DNA BAR gene. The improvement measured complex T-DNA repeat numbers quickly and accurately. Discrepancies in qPCR detected T-DNA copy number versus Southern detected T-DNA insertion number were largely a result of complex T-DNA repeats. Sequencing results revealed the existence in the repeat junctions of similar T-DNA insertion patterns commonly found in transgenic plants, including precise RB structure and LB deletion. Moreover, the end-joining nucleotides from both RB and LB in many repeat junctions were exactly at sites immediately adjacent to a homologous segment in the two borders, suggesting possible involvement of homologous recombination in the repeat formation. Our study demonstrates that this new advancement in high throughput quantification of T-DNA tandem repeats is useful for a large scale transgenic plant population analysis and in elucidating the mechanism of T-DNA tandem repeat formation.

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Section: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 98%

Quantitative and structural analyses of T-DNA tandem repeats in transgenic Arabidopsis SK mutant lines

Shu

Song

et al. 2015

Plant Cell Tiss Organ Cult

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“…It is thus possible that T-strand conversion to dsT-DNA may be, at least in part, a limiting factor in T-DNA integration. Nevertheless, recent evidence suggests that dsT-DNA intermediates can undergo inter/intra-molecular cyclization and form circular molecules that may become incapable for integration 30 . Therefore, dsT-DNA formation and its accessibility to integration may be regulated in a more comprehensive manner.…”

Section: Discussionmentioning

confidence: 99%

In vivo formation of double-stranded T-DNA molecules by T-strand priming

Liang

Tzfira

2013

Nat Commun

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During plant genetic transformation, Agrobacterium transfers a single-stranded DNA (T-strand) into the host cell. Increasing evidence suggests that double-stranded (ds) T-DNA, converted from T-strands, are potent substrates for integration. Nevertheless, the molecular mechanism governing T-strand conversion to dsT-DNA is unknown. Integrated T-DNA molecules typically exhibit deletions at their 3 0 end as compared with their 5 0 end. We hypothesize that this may result from asymmetric polymerization of T-DNA's ends. Here we show that b-glucuronidase (GUS) expression from sense T-strands is more efficient than from antisense T-strands, supporting asymmetric conversion. Co-transfection with two partially complementary, truncated GUS-encoding T-strands results in GUS expression, which suggests functional hybridization of the T-strands via complementary annealing and supports the notion that T-strands can anneal with primers. Indeed, red fluorescent protein (RFP) expression from mutated T-strand can be restored by delivery of synthetic DNA and RNA oligonucleotides with partial wild-type RFP sequence, implying the involvement of plant DNA repair machinery.

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“…Therefore, one can use oligonucleotides such as triplex forming oligonucleotides (TFOs), short ssDNA or dsDNA donors such as T-DNA to induce HR. It should be noted that while T-DNA of Agrobacterium is iniltrated in the plant cell as a ssDNA coated by VirE2, it turns into dsDNA shortly after and probably integrates into the plant genome as dsDNA [19,20]. While unassisted HR levels are very low, and it is highly induced by speciic genomic DSBs [21].…”

Section: Principles Of Genome Editingmentioning

confidence: 99%

Plant Genome Editing and its Applications in Cereals

Weinthal¹,

Gürel²

2016

Genetic Engineering - An Insight Into the Strategies and Applications

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Recently developed methods for genome editing, representing a major breakthrough in the ield of genetic engineering, will enable researchers to produce transgenic plants in a more convenient and safer way. Double-strand breaks (DSBs) are triggered by synthetic nucleases that later induce DNA repair mechanisms known as nonhomologous-end joining (NHEJ) or homology-directed repair (HDR) in the presence of a donor DNA. Gene targeting (GT) was earlier demonstrated in rice and maize genomes by exploiting several genes (Acetohydroxyacid synthase, waxy, ALS, OS11N3 etc.), while zinc inger nucleases (ZFNs) were used to modify IPK1 gene in maize. Clustered regularly interspaced short palindromic repeats (CRISPR-CAS) system has been shown to be eicient for targeted mutagenesis in wheat that has a hexaploid complex genome, rice, maize, and recently in barley. The CRISPR system is considered as advantageous over previous approaches due to its easy use and eiciency, however, needs to be improved for high of-target efects.

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Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Cited by 44 publications

References 68 publications

Quantitative and structural analyses of T-DNA tandem repeats in transgenic Arabidopsis SK mutant lines

Quantitative and structural analyses of T-DNA tandem repeats in transgenic Arabidopsis SK mutant lines

In vivo formation of double-stranded T-DNA molecules by T-strand priming

Plant Genome Editing and its Applications in Cereals

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