Formation of Complex Extrachromosomal T-DNA - Plant Physiology

Formation of Complex ExtrachromosomalT-DNA Structures in Agrobacteriumtumefaciens-Infected Plants1[C][W][OA]

Kamy Singer*, Yoel M. Shiboleth, Jianming Li, and Tzvi Tzfira2

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan48109–1048

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration oftransferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form inA. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules cancircularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencinganalysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patternsinclude illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown “filler”sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant’s DNAdouble-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomalcomplex T-DNA structures that subsequently may integrate.

The soil pathogen Agrobacterium tumefaciens geneti-cally transforms plants. This natural and unique abilityof transkingdom gene transfer has made A. tumefaciens-mediated transformation one of the most importanttools in plant biology research and the preferredmethod for producing transgenic plants. The trans-formation process is initiated by the transfer of asingle-stranded (ss) DNA segment derived from theA. tumefaciens tumor-inducing (Ti) plasmid into theplant cell nucleus. This transferred DNA (T-DNA) isdelimited by two 25-bp direct imperfect repeats knownas the right border (RB) and left border (LB), which arerequired for its transfer. The process also requires a setof bacterial chromosomal (chv) and Ti plasmid viru-lence (vir) genes as well as various host genes (for

review, see McCullen and Binns, 2006; Citovsky et al.,2007; Gelvin, 2010; Tzfira and Citovsky, 2010). Innatural A. tumefaciens strains, the T-DNA carries thegenes responsible for the formation of tumor growthon plants, known as the “crown gall” disease. In dis-armed laboratory strains, the native T-DNA is re-moved from the Ti plasmid. Instead, a gene of interestcan be placed between the two T-DNA borders and betransferred from a smaller binary plasmid (for review,see Gelvin, 2003; Tzfira and Citovsky, 2006).

To study the last step in the transformation process,T-DNA integration into the plant genome, T-DNA in-sertion events have been extensively characterizedby sequencing of the T-DNA/chromosome junctions.These studies have shown that T-DNAs are insertedrandomly throughout the plant genome (Forsbach et al.,2003; Kim et al., 2007). They have also shown that theintegration is “illegitimate” (i.e. not sequence specific)but may include overlapping microhomologies of ap-proximately 2 to 7 bp (Gheysen et al., 1991; Mayerhoferet al., 1991). Furthermore, T-DNA integration can resultin complex structures. These may include truncations ofT-DNA ends and multicopy T-DNAs arranged asinverted or direct repeats (Kwok et al., 1985; Spielmannand Simpson, 1986). Complex structures may also in-clude non-T-DNA bacterial sequences (Martineau et al.,1994; Ulker et al., 2008), DNA from an unknown source(“filler”), and plant sequence duplications (Gheysenet al., 1987, 1991; Mayerhofer et al., 1991). More recentstudies have further characterized these integrationpatterns under different experimental settings, thusproviding more insight into the transformation process(Kumar and Fladung, 2002; Meza et al., 2002; Stahl

1 The work was supported by the U.S.-Israel Binational Agricul-tural Research and Development Fund (grant no. BARD US–4150–08to T.T.) and the U.S.-Israel Binational Science Foundation (grant no.2007140 to T.T.).

2 Present address: Department of Life Sciences, Ben-Gurion Uni-versity of the Negev, Beer Sheva 84105, Israel.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Tzvi Tzfira ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a subscrip-

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et al., 2002; Forsbach et al., 2003; Kim et al., 2003;Windels et al., 2003; Thomas and Jones, 2007; Zhanget al., 2008; De Buck et al., 2009). However, the mecha-nism of T-DNA integration is still poorly understood (forreview, see Tzfira et al., 2004; Ziemienowicz et al.,2010).

Although complex T-DNA insertions are undesired intransgenic plants for commercial or research purposes,they are a relatively frequent outcome of transformation(Windels et al., 2003, 2010; Zhang et al., 2008). Under-standing how complex T-DNA insertions form is im-portant to better understand the mechanism behindT-DNA integration. De Neve et al. (1997) proposed thatcomplex T-DNA structures, such as those that involveT-DNA repeats, formwhen two or more double-stranded(ds) T-DNA intermediates ligate in the plant nucleusprior to integration. In contrast, other models proposethat T-DNA repeats form via ss T-DNA intermediatesthat ligate during (Krizkova and Hrouda, 1998) or priorto (Stahl et al., 2002) integration. Whether T-DNA inte-grates as an ss or ds intermediate is a fundamentalquestion related to T-DNA integration. According to themodel of Tinland (1996), conversion of ss T-DNA into dsT-DNA occurs only during its incorporation to the ge-nome. Similar ss-based models have been proposed(Brunaud et al., 2002; Kumar and Fladung, 2002; Mezaet al., 2002; Thomas and Jones, 2007; Teo et al., 2011).Conversely, the ds T-DNA model suggests that con-version to ds T-DNA occurs in plants prior to integra-tion (Mayerhofer et al., 1991). This is supported byevidence that at least some of the nonintegrating “free”T-DNA molecules in infected plants are ds (Janssen andGardner, 1990; Offringa et al., 1990; Narasimhulu et al.,1996). Also supporting the ds T-DNA model are datasuggesting that ds T-DNA integrates into genomicdouble-stranded breaks (DSBs; Salomon and Puchta,1998; Chilton and Que, 2003; Tzfira et al., 2003).

While a common approach to studying the mecha-nism of T-DNA integration is to characterize patternsof postintegration events in plants, analyzing T-DNAtransfer events in plants prior to integration offers aneffective alternative approach. Bakkeren et al. (1989)described a virus-based infection system to recovertransfer events indirectly. That method is based on thehypothesis that ds T-DNAs occasionally circularize inplanta. However, the experimental setup of that indi-rect virus-based system, which may have resultedfrom recombination by viral components within theT-DNA molecules, did not allow the detection of po-tential complex T-DNA structures.

To study the formation of complex T-DNA struc-tures in plants, we developed a novel method to di-rectly isolate transfer events from infected plants. Themethod adopts a plasmid-rescue approach to recoverds T-DNA molecules that have undergone in plantacircularization. When sequencing these molecules, wediscovered various complex circular structures withpatterns similar to those previously reported in trans-genic plants. This suggests that complex extrachro-mosomal ds T-DNA structures may form in plants

prior to integration via ligation between ds DNA in-termediates. We propose that some of these ds DNAextrachromosomal complex structures may then inte-grate into the genome.

RESULTS

The Experimental Approach

To test the extrachromosomal T-DNA ligation modelfor the formation of complex T-DNA structures (De Neveet al., 1997), we hypothesized that extrachromosomalcomplex T-DNA molecules will occasionally circularizevia the same DNA-repair pathway responsible for theirproposed formation. To recover complex circular T-DNAstructures potentially forming in A. tumefaciens-infectedplants, we developed a direct plasmid-rescue method inwhich circular T-DNA molecules (T-circles) are capturedas plasmids in Escherichia coli.

To this end, we constructed AMP-ORI and KAN-ORI T-DNAs containing a pBR322 origin of replication(ori) for E. coli and a bacterial selectable marker, eitherthe ampicillin resistance gene (AmpR; bla gene) or thekanamycin resistance gene (KanR; aph gene; Fig. 1A;Supplemental Fig. S1). A KAN T-DNA construct thatdoes not contain an ori was also prepared for ad-ditional experiments (Fig. 2). The T-DNA constructswere cloned into a pRCS2 binary plasmid (Tzfiraet al., 2005). The backbone of this binary plasmid con-tains the bacterial spectinomycin and streptomycinresistance gene (SpR/SmR; aadA gene) and the pVS1ori for DNA replication in A. tumefaciens and E. coli(Fig. 1B, 1). The AMP-ORI and KAN-ORI constructswere introduced separately into plant leaves viaagroinfiltration (Fig. 1B, 2). At 4 to 8 d post agro-infiltration, we extracted DNA from the infected leaves(Fig. 1B, 3) and transformed E. coli cells with thisDNA (Fig. 1B, 4). Colonies resistant to either kana-mycin or ampicillin were isolated on medium sup-plemented with the corresponding antibiotics (Fig. 1B,5). In order to distinguish between E. coli colonies thatacquired binary plasmids from the A. tumefaciens inthe infected plant tissue and E. coli colonies that poten-tially acquired T-circles from plant cells, each colonywas tested for spectinomycin/streptomycin resistance(Fig. 1B, 6). As the SpR/SmR sequence is located onthe binary backbone, outside the T-DNA borders, col-onies sensitive to spectinomycin/streptomycin wereruled out as containing binary plasmids and thuswere further analyzed (Fig. 1B, 7). The frequency ofspectinomycin/streptomycin-sensitive colonies amongampicillin- or kanamycin-resistant colonies varied inagroinfiltration experiments using different plant species.While 5% to 10% of the colonies were spectinomycin/streptomycin sensitive when using tobacco (Nicotianatabacum; Supplemental Fig. S2), more than 30% ofthe colonies were spectinomycin/streptomycin sensi-tive when using Nicotiana benthamiana (see additionaldata below).

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Restriction Digestion Reveals T-Circles of Various Sizesand Structures

The plasmids recovered from spectinomycin/streptomycin-sensitive colonies (T-circles) were ana-lyzed by restriction digestion and gel electrophoresis.A ScaI site is present within the AmpR gene of theAMP-ORI construct (Fig. 1A). Thus, the T-circles con-ferring E. coli resistance to ampicillin are expected tobe digested at least once by ScaI. A second ScaI site ispresent in the pRCS2 binary backbone sequence 113 bpfrom the T-DNA’s right border (Fig. 1A). Thus, theT-circles digested twice with ScaI may contain an ad-ditional sequence that may be either a second T-DNAfragment or a binary backbone sequence. The latter mayderive from incorrect processing of T-DNAmolecules inthe bacterium (Kononov et al., 1997; Wenck et al., 1997).A random selection of six T-circles isolated from

tobacco agroinfiltrated with the AMP-ORI construct isshown in Figure 2A (T-1–T-6). Out of the six treatedwith ScaI, four T-circles generated a single band in asize ranging between 2.2 and 3.4 kb, suggesting thatthey consisted of a single copy of T-DNA with somedeletions or additions of DNA (T-1–T-4). The T-circlesT-5 and T-6 produced two bands after ScaI digestion,suggesting a complex structure in these T-circles.

Using Different T-DNA Constructs Simultaneously viaCoagroinfiltration Generates Complex T-Circles ThatContain Both T-DNAs

Cotransforming plants with different T-DNA con-structs simultaneously often results in the integrationof different T-DNAs at the same location (De Neveet al., 1997; Radchuk et al., 2005). To test the extra-chromosomal T-DNA ligation model, we hypothe-sized that when different T-DNA constructs are

delivered to plants simultaneously, they ligate to eachother, prior to integration, and occasionally generateT-circles. To this end, we introduced the AMP-ORIconstruct and either the KAN-ORI or the KAN con-struct simultaneously into plants by “coagroinfiltra-tion.” The different A. tumefaciens strains containingeach of the T-DNA constructs were mixed immedi-ately before coagroinfiltration.

Following coagroinfiltration, we isolated T-circlesfrom the E. coli colonies resistant to both ampicillin andkanamycin. A ClaI site is present within the KanR genein KAN-ORI and KAN (Fig. 1A) but is not present inthe binary backbone itself. Accordingly, T-circles weredigested at least once with ClaI in addition to ScaI.Figure 2B shows a sample of T-circles digested withScaI and ClaI. Overall, the T-circles ranged in size be-tween 5 and 15 kb. KAN was coagroinfiltrated withAMP-ORI in T-7, T-8, T-10, and T-11, while KAN-ORIwas coagroinfiltrated with AMP-ORI in T-9 and T-12to T-16. Digestion of T-7 and T-8 with either ScaI orClaI revealed a single site for each enzyme and pro-duced a band of about 5 kb. This represents the ap-proximate fragment size expected from a ligation ofAMP-ORI and KAN. Sequencing analysis confirmedthe ligation between the two different T-DNA con-structs (Fig. 2C shows a schematic diagram of one ofthe junction regions of T-7 based on the sequencinganalysis shown in Supplemental Fig. S3). Digestion ofT-9 and T-12 also revealed single ScaI and ClaI sites.These T-circles were notably larger than expected fromthe joining of two T-DNAs and also conferred a re-sistance to spectinomycin/streptomycin, suggestingthat they included the aadA (SpR/SmR) sequence fromthe binary backbone. Similarly, T-15 and T-16 alsoconferred resistance to spectinomycin/streptomycin.Digestion of T-10, T-11, and T-13 to T-16 with ScaIrevealed two to three sites, while digestion of T-10 and

Figure 1. Constructs and experimental procedurefor the isolation of T-circles. A, Schematic dia-gram of the three T-DNA constructs used through-out the experiments: AMP-ORI, AmpR with ori;KAN-ORI, KanR with ori; KAN, KanR without ori. B,Illustration of the experimental procedure: 1,transformation of A. tumefaciens with a pRCS2binary plasmid harboring one of the three T-DNAconstructs (AMP-ORI is shown); 2, agroinfiltrationof plant leaves; 3, extraction of DNA from theagroinfiltrated leaves (4–8 d after infiltration); 4,transformation of E. coli with the DNA; 5, selectionof colonies resistant to ampicillin (or kanamycinif KAN-ORI or KAN is used); 6, detection ofspectinomycin/streptomycin-sensitive colonies;7, isolation of plasmids from each colony andanalysis by restriction digestion and DNA se-quencing. amp, Ampicillin; kan, kanamycin;spec, spectinomycin; strep, streptomycin. [Seeonline article for color version of this figure.]

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Complex Extrachromosomal T-DNA Structures




T-16 with ClaI revealed two sites (note the nearlyidentical size of the two ClaI bands in T-16), thussuggesting complex T-circles that contain multipleDNA fragments.

A. tumefaciens T-DNA Transfer Is Required forT-Circle Formation

To further study the origin of the T-circles, we testedthe effect of using a mutant A. tumefaciens strain defi-cient in T-DNA transfer. If the T-circles are producedin plant cells, then they should not be generated usingthis mutant. We used a virB92 A. tumefaciens mutantstrain that does not produce galls due to a defect in theT-DNA transfer apparatus (Beaupré et al., 1997). Wefirst tested whether T-DNA transfer can be detected inan agroinfiltration assay using this mutant strain. AT-DNA construct with the GUS sequence interruptedby an intron (GUS-int) was used to examine transientexpression in plants as an indication for T-DNA

transfer (Ohta et al., 1990). As expected, the virB92

strain did not induce GUS expression in N. benthamianaleaves (Fig. 3A), confirming that it is transfer deficientunder our experimental conditions. To complementthis strain, we used the pED37 plasmid, which con-tains virB9 under virB promoter control (Ward et al.,1991). GUS activity was restored in virB92/pED37,confirming that T-DNA transfer is restored by thisplasmid (Fig. 3A). Next, we transformed strainsvirB92, virB92/pED37, and control EHA105 with theAMP-ORI construct and agroinfiltrated plants in or-der to test the formation of T-circles (Fig. 3B). Whenthe virB92 strain was used, all of the E. coli coloniesselected on ampicillin (n = 675) were spectinomycin/streptomycin resistant, indicating that T-circles werenot generated. When the virB92/pED37 strain wasused, 44% of the colonies selected on ampicillin (n =107) were spectinomycin/streptomycin sensitive, anindication that a large proportion of the colonies con-tained T-circles. Similarly, the EHA105 control strainproduced 61% spectinomycin/streptomycin-sensitive

Figure 2. Restriction digestion reveals T-circles ofvarious sizes and structures. A, Agroinfiltration oftobacco with AMP-ORI (T-1–T-6). B, Coagroinfil-tration of N. benthamiana with AMP-ORI andKAN (T-7, T-8, T-10, and T-11) or AMP-ORI andKAN-ORI (T-9 and T-12–T-16). T-circles are shownon agarose gels as uncut (U; left lane), ScaI treated(S; middle lane), or ClaI treated (C; right lane). EachT-circle is marked by + or 2 (below the gel) toindicate the resistance or sensitivity (respectively)to an antibiotic (left side) it confers to E. coli. Thebinary plasmids AMP-ORI and KAN-ORI areshown (last two on bottom right). C, Schematicdiagram of T-7 shows a junction fragment of AMP-ORI and KAN. Numbers in parentheses representthe positions within the T-DNA relative to the LB(0 point) to indicate the deleted regions from eachside. [See online article for color version of thisfigure.]


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colonies (n = 288). Thus, T-DNA transfer is necessaryfor T-circle formation.

T-Circles Form Prior to E. coli Transformation

To rule out the possibility that the T-circles are anexperimental artifact originating from ligation in E. coliduring the transformation step, we used PCR to ex-amine end joining between T-DNAs in DNA extractedfrom infected plants before the DNA was used totransform E. coli. We examined DNA samples fromleaves coagroinfiltrated with AMP-ORI and KAN-ORI(using different A. tumefaciens strains) and comparedthe results with control samples (Fig. 4). The primerswere designed to amplify different combinations ofT-DNA end-joining events (primers P1k, P2k, P1a, andP2a in Fig. 4A). Joining may occur between the ends ofseparate T-DNA molecules or between the ends of thesame T-DNA molecule. We used the constructs sepa-rately to agroinfiltrate N. benthamiana (I and II in Fig.4B) or used them together to coagroinfiltrate N. ben-thamiana (co-agro in Fig. 4B). As a control, a 1:1 mixedDNA sample was prepared from the DNA extractedfrom the infected plants in experiments I and II (mixin Fig. 4B). When DNA sample I or II was used asa template, the primer pair P1a and P2a or P1k andP2k, respectively, amplified fragments of the LB-RBjunctions (Fig. 4C), indicating that T-DNA ends werejoined in these samples. When the DNA sample “co-agro” was used as a template, PCR products wereobtained using each of all possible primer combina-tions (Fig. 4C). Importantly, PCR products wereobtained also with primers from different T-DNAs,

amplifying fragments of RB-RB, LB-LB, and LB-RBjunctions between AMP-ORI and KAN-ORI. To con-firm the sequences of the PCR products, we cloned andsequenced a few of them (Supplemental Fig. S4). Asexpected, PCR products involving primers from dif-ferent constructs were not obtained in the control“mix” sample (Fig. 4C).

We then further examined the type of plasmidspresent in each of the DNA samples. We transformedE. coli with each of the samples and selected colonieson medium plates supplemented with ampicillin,kanamycin, or both. As expected, DNA samples I andII generated colonies on either ampicillin or kanamy-cin, depending on the T-DNA construct used in eachexperiment, but not on medium supplemented withboth ampicillin and kanamycin. A portion of the am-picillin- or kanamycin-resistant colonies was specti-nomycin/streptomycin sensitive (39% in a and 27% ind in Fig. 5), indicating that T-circles were produced in Iand II. The DNA sample co-agro generated colonies onampicillin, kanamycin, and notably also on mediumsupplemented with both antibiotics. Among those se-lected on either ampicillin or kanamycin, a portion wasspectinomycin/streptomycin sensitive (38% in b and34% in e in Fig. 5). Among those resistant to both

Figure 4. T-circles containing both AMP-ORI and KAN-ORI constructsform in plants following coagroinfiltration. A, Schematic illustration ofthe locations of the primers designed for the AMP-ORI and KAN-ORIconstructs. The primers P1a, P1k, P2a, and P2k are positioned near theT-DNA ends (arrows indicate the directions of the primers). B, DNAsamples were extracted from N. benthamiana agroinfiltrated withAMP-ORI (I), agroinfiltrated with KAN-ORI (II), or coagroinfiltratedwith both AMP-ORI and KAN-ORI (co-agro). The fourth DNA sampleis a mixture of DNA from I and II (mix). C, PCR amplification using as atemplate DNA from the samples I, II, co-agro, mix, and (as a control)double distilled water (ddw). The primers used in each reaction areindicated on the right side of each gel. [See online article for colorversion of this figure.]

Figure 3. A. tumefaciens T-DNA transfer is required for T-circle forma-tion. A, A T-DNA construct with the GUS sequence interrupted by anintron was introduced into A. tumefaciens strains virB9–, virB9–/pED37,and EHA105 (the control strain). GUS staining was performed 3 d afteragroinfiltration. B, The AMP-ORI construct was introduced into each ofthe three A. tumefaciens strains, which were then agroinfiltrated intoN. benthamiana. Ampicillin-resistant E. coli colonies were selectedand tested for spectinomycin/streptomycin resistance. The numbers inthe pie charts are given as percentages of colonies. S, Spectinomycin/streptomycin; +, resistant; 2, sensitive. [See online article for colorversion of this figure.]






ampicillin and kanamycin, a higher proportion wasspectinomycin/streptomycin sensitive (75% [6% from8%] in b and 80% [8% from 10%] in e in Fig. 5). Thecontrol mixture did not generate resistant colonieson medium supplemented with both ampicillin andkanamycin, only on ampicillin or kanamycin sepa-rately. Thus, our experiments confirm that T-circles arepresent in the DNA prior to E. coli transformation andfurther support that they form in plants.

DNA Sequencing of T-DNA Junctions Reveals Details ofComplex T-Circles

To further analyze T-circle structures at the sequencelevel, we sequenced the T-DNA junctions in T-1 to T-9,T-11, and T-12 (Supplemental Fig. S3). According tothe junction sequences and the sizes of the bands afterrestriction digestion, we predicted the completestructure of the T-circles T-1 to T-5 (Fig. 6A). In T-1, an82-bp fragment from the LB side of the T-DNA wasmissing. However, the RB side was complete: it in-cluded the first three nucleotides of the RB sequence(Fig. 6A; Supplemental Fig. S3). We refer to a borderending at the third position of the 25-bp direct im-perfect repeat as a “precise” ending because it is thesite at which the VirD1-VirD2 complex is known tonick the lower T-DNA strand in A. tumefaciens (Wanget al., 1987). In T-2, both the LB and RB had preciseendings (Fig. 6A; Supplemental Fig. S3). On the otherhand, T-3 was missing 598 and 595 bp from the LB and

RB sides, respectively, and was attached to a 1.4-kbsequence derived from the binary plasmid (Fig. 6A;Supplemental Fig. S3). This binary fragment, whichdid not include SpR/SmR, was not a “read-through” ofthe binary sequence adjacent to T-DNA from its LB orRB side (Kononov et al., 1997; Wenck et al., 1997;Kim et al., 2003). T-9 also had a T-DNA with a non-read-through binary plasmid sequence. In this case,the binary backbone sequence that is immediately nextto the LB was attached to the RB side of a T-DNAin the T-circle (Supplemental Fig. S3). On the otherhand, in T-11 and T-12, the binary plasmid sequencewas a read-through of the T-DNA RB and LB, re-spectively (Supplemental Fig. S3). In T-4, the borderjunction included 5 bp of microhomology (GCGCC);thus, the exact ending point of each side can be any-where within this microhomology. The LB and RBsides lost from 363 to 368 bp and from 596 to 601 bp,respectively (Fig. 6A; Supplemental Fig. S3). T-5 con-tained two T-DNA fragments arranged in an invertedrepeat configuration. One T-DNA fragment includedthe AmpR gene and the ori. It was missing 612 bp of theLB side, whereas the RB was precise. The secondfragment was missing 1,088 and 1,465 bp from theLB and RB sides, respectively (Fig. 6A; SupplementalFig. S3). T-6 produced two fragments after digestionwith ScaI (Fig. 2A), BsaXI, or AatII (Fig. 6B). The sizesof the bands suggested that two relatively completeT-DNAs had joined in an inverted configuration. Toconfirm this, we cloned the junction fragments pro-duced by AatII and BsaXI (Fig. 6B) into the EcoRVsite of pBluescript KS1 (after blunt ending the frag-ments) and sequenced them. Sequencing the AatIIfragment showed LB-LB region joining (Fig. 6C, top;Supplemental Fig. S3). The junction included 3 bp ofmicrohomology (TAA). One LB side was missing be-tween 21 and 24 bp, whereas the other was missingbetween 261 and 264 bp. The BsaXI fragment includedRB-RB region joining (Fig. 6C, bottom; SupplementalFig. S3). It had a patchwork of unknown filler se-quence (“scrambled”) interrupted by short T-DNAsequences. The filler sequences from both ends of theinverted T-DNAs started at similar positions near theirBsaXI sites (2,640): positions 2,658 and 2,686. From theleft side, the T-DNA ending at 2,658 was interruptedby 225 bp of scrambled filler sequence. After this filler,there appeared a 37-bp sequence from a RB T-DNAregion (2,885–2,922). Interestingly, this short T-DNAsequence started 227 bp away from the position atwhich the previous T-DNA was interrupted by the225-bp filler, and it was in the same orientation. Afterthis T-DNA sequence, another scrambled filler DNAsequence began. We were not able to sequence theinternal region of the fragment, possibly due to theformation of secondary structures. Sequencing fromthe right side revealed 137 bp of scrambled filler DNAfollowed by an inverted T-DNA RB region fragment(greater than 199 bp). Additional sequences from T-7,T-8, T-9, T-11, and T-12 are shown in SupplementalFigure S3.

Figure 5. Antibiotic resistance of E. coli colonies transformed withDNA I, II, co-agro, and mix (the DNA sample types are indicatedabove the pie charts). Colonies were initially selected on either am-picillin or kanamycin (indicated to the left of the pie charts) and thentested for spectinomycin/streptomycin, ampicillin, and kanamycinresistance. The numbers in the pie charts are given as percentages ofcolonies. The numbers of colonies tested in each group are follows:a, n = 96; b, n = 478; c, n = 383; d, n = 120; e, n = 473; f, n = 477.A, Ampicillin; K, kanamycin; NC, no colonies; S, spectinomycin/streptomycin; +, resistant, 2, sensitive. [See online article for colorversion of this figure.]


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DISCUSSION

To our knowledge, extrachromosomal complexT-DNA structures have not been reported previouslyin plants or A. tumefaciens. To study T-DNA integra-tion and the formation of complex T-DNA insertions inplants, we developed a method to isolate complexT-DNA structures in the form of T-circles directly fromA. tumefaciens-infected plants. The T-circles resembledstructures commonly found in stable transgenic plants,which suggests that similar linear ds DNA structuresare present in plants prior to integration. If not inte-grated, ds DNA linear structures may circularize.

The Formation of Complex T-Circles in Plants

We first confirmed that our isolated T-circles areformed in the plant cells, because T-circles can poten-tially also form in A. tumefaciens during the infection.Although the circularization of a T-DNA in A. tume-faciens is a rare event with no apparent role in the chain

of events leading to transformation, a T-circle may beoccasionally released from its parent plasmid throughhomologous recombination between two direct re-peats that flank the T-DNA (Koukoliková-Nicola et al.,1985; Machida et al., 1986; Timmerman et al., 1988).Sequencing of end-joining junctions in our T-circlesrevealed that their ends had none or only a few basepairs of overlapping microhomology (SupplementalFig. S3), thus indicating that our T-circles did not formthrough homologous recombination, as occurs in A.tumefaciens, but through illegitimate end joining, whichis typical of the circularization of T-DNAs in infectedplants (Bakkeren et al., 1989; Zhao et al., 2003).Moreover, T-circles containing T-DNAs from differentA. tumefaciens strains were generated following co-agroinfiltration (Figs. 4 and 5), emphasizing the re-quirement of T-DNA transfer out of the bacteria priorto circularization. Finally, the requirement of T-DNAtransfer for T-circle formation was also demonstratedusing an A. tumefaciens strain deficient in the ability to

Figure 6. Schematic illustration of the T-circles T-1 to T-6 based on DNA sequencing. A, T-1 to T-5 are shown in a linear form,opened at the position of a junction, and aligned below a complete AMP-ORI construct. B, T-6 after digestion with BsaXI (leftlane) or AatII (right lane). The bands that include the junctions are indicated by arrows. C, Schematic illustration of the AatIIfragment (top; the LB-LB region junction of T-6) and the BsaXI fragment (bottom; the RB-RB region junction of T-6). [See onlinearticle for color version of this figure.]







transfer T-DNA to plants (Fig. 3). However, it is im-possible to rule out completely that a small number ofT-circles are produced in A. tumefaciens. For example,T-2 had a precise border joining (Fig. 6); thus, poten-tially, it may have formed in A. tumefaciens throughhomologous recombination between the borders. Thenext possibility that we considered was that theT-circles are an artifact of the experimental procedure,forming through random ligation in E. coli during thetransformation step. We ruled out this possibility, first,because PCR of DNA extracted from coagroinfiltratedplants (before the DNA was used for E. coli transfor-mation) confirmed end joining between the AMP-ORIand KAN-ORI constructs (Fig. 4C; Supplemental Fig.S4). Second, T-circles containing both AMP-ORI andKAN-ORI could be isolated in E. coli only if trans-formed with DNA extracted from coagroinfiltratedplants, not in E. coli transformed with a DNA mixturefrom separate agroinfiltration experiments (Fig. 5).Thus, we conclude that the T-circles formed in theplants cells.

T-DNA Integration and the Formation of ComplexT-DNA Insertions

Our results support a model of integration thatpredicts that T-DNAs integrate as ds DNA molecules(Mayerhofer et al., 1991) and that complex transgenicstructures form when ds DNA fragments ligate ex-trachromosomally prior to integration (De Neve et al.,1997). Our conclusion is based on the finding of ex-trachromosomal ds T-DNA complex structures similarto complex patterns commonly found integrated intransgenic plants. The complex patterns that weidentified include T-DNA repeats, binary plasmid se-quences, filler DNA sequences, microhomologies, andT-DNA truncations.

T-DNA repeats are a frequent pattern found at in-sertion sites following A. tumefaciens-mediated trans-formation (Cluster et al., 1996; Krizkova and Hrouda,1998; Wolters et al., 1998; De Paepe et al., 2009).Moreover, following cotransformation using differentT-DNA constructs, cointegration of the T-DNAs oftenoccurs into the same site (De Buck et al., 2000; Radchuket al., 2005). Similarly, following agroinfiltration andcoagroinfiltration, we recovered T-circles with morethan one T-DNA of the same type or different types,respectively (Figs. 2, 5, and 6). The joining between theT-DNAs can be at their RB-LB, RB-RB, or LB-LB sides.Joining at the RB-RB or LB-LB side is particularly dif-ficult to explain by the ss model, because the ss T-DNAis always 59 at its RB terminus and 39 at its LB ter-minus. Thus, further supporting the idea that theT-DNAs in our T-circles were ds DNA molecules priorto their extrachromosomal ligation, sequencing re-vealed T-DNAs joined from their RB-RB and LB-LBsides (Fig. 6; Supplemental Fig. S3).

Binary plasmid sequences adjacent to the insertedT-DNAs is a pattern that has been reported in 30% to

70% of transgenic plant lines (Martineau et al., 1994;Kononov et al., 1997; Wenck et al., 1997; Kim et al.,2003; Lange et al., 2006). Similarly, our results indicatethat binary sequences were present in more than one-quarter of the T-circles containing both AMP-ORIand KAN-ORI, because one-quarter of them also con-ferred spectinomycin/streptomycin resistance (Fig. 5).A read-through transfer of a T-DNA together with anadjacent binary sequence occurs frequently when theVirD1-VirD2 endonuclease supposedly skips the pro-cessing of one of the borders in A. tumefaciens duringthe release of the T-DNA from its parent binary plas-mid (Kononov et al., 1997; Wenck et al., 1997). Becausethe SpR/SmR gene sequence in our system is located onthe binary backbone near the LB (approximately 0.5 kbaway), a read-through transfer of SpR/SmR sequencewith the T-DNA can be expected to occur frequently.For example, a read-through of the LB occurred in T-12(Supplemental Fig. S3), which also conferred specti-nomycin/streptomycin resistance (Fig. 2B). In addi-tion, other binary sequences that do not include SpR/SmR may have been present in other structures butwere not detected in the resistance assay. For example,the binary sequence adjacent to the T-DNA in T-3 didnot include SpR/SmR and was not a read-through fromeither side of the T-DNA (Fig. 6; Supplemental Fig. S3).Because complex T-DNA structures have never beenreported in A. tumefaciens, and in agreement with thereport of Kononov et al. (1997) that binary sequencesin plants can integrate independently of the T-DNA,the binary sequence in T-3 most likely entered theplant cell independently as ss DNA, then converted tods DNA, and finally ligated to a ds T-DNA.

Filler DNA is DNA sequence from an unknownsource that often flanks T-DNA insertions. Filler DNAis a pattern also found at genomic DSB-repair sitesin plants. It is believed to form through a modifiedsynthesis-dependent strand-annealing mechanism thatinvolves random template “switches” of the DNA poly-merase, resulting in a patchwork of different fillersequences (Gorbunova and Levy, 1997; Salomon andPuchta, 1998). Patterns of filler DNA at T-DNA inser-tion sites often consist of short sequences of T-DNA,plant genome, or scrambled DNA (Gheysen et al.,1991; Mayerhofer et al., 1991; Krizkova and Hrouda,1998; Forsbach et al., 2003; Windels et al., 2003). Wefound a similar pattern of filler DNA in T-6 (Fig. 6C;Supplemental Fig. S3). The RB-RB junction consisted ofa patchwork of scrambled filler DNA interrupted byshort sequences homologous to the RB region. Onefiller sequence had a length almost identical (225 bp) tothe T-DNA sequence it intercepted (227 bp). This mayindicate that a template switch of DNA polymerasehad occurred at this region. Alternatively, end joiningbetween “free-floating” ds DNA fragments may po-tentially result in patchworks.

Truncations of T-DNA ends and microhomologiesof 2 to 7 bp at plant/T-DNA and T-DNA/T-DNA in-sertion junctions are common in transgenic plants(Tinland, 1996; Brunaud et al., 2002; Windels et al.,


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2003). They are also associated with the repair of ge-nomic DSBs in plants (Gorbunova and Levy, 1997;Salomon and Puchta, 1998). Similarly, T-DNA trun-cations were found in most of our T-circles (Fig. 6;Supplemental Fig. S3). Microhomologies of between1 and 5 bp were found at T-circles T-4, T-6, T-8, andT-9. In T-2, a precise junction contained 12 bp of theoverlapping border direct repeats. In T-11, AMP-ORIand KAN T-DNA were joined within an 85-bp over-lapping region (Supplemental Fig. S3). Truncationsand microhomology were also detected in AMP-ORI/KAN-ORI junctions amplified by PCR (SupplementalFig. S4).Because the ligation patterns in the T-circles de-

scribed above resemble the ligation patterns of re-paired DNA DSBs, it is likely that T-DNA integration,the formation of complex T-DNA structures, and cir-cularization share the same DNA-repair mechanism.That we isolated only circular but not linear ds DNAstructures is due only to the prerequisite of our de-tection system; circular configurations suggest thatsimilar linear configurations also exist in the plant cell.In support of the idea that termini of ds T-DNA arerecognized as DSBs, it has been shown previously thatgenomic DSBs may be “hot spots” for ds T-DNA inte-gration (Salomon and Puchta, 1998; Chilton and Que,2003; Tzfira et al., 2003). The major pathway to repairDSBs in plants is the nonhomologous end joining(NHEJ) DNA-repair pathway (for review, see Bleuyardet al., 2006). Supporting a role for the NHEJ pathwayin T-DNA integration, two components of the NHEJpathway, LigIV and KU80, have been reported to berequired for T-DNA integration (Friesner and Britt,2003; Li et al., 2005). Conversely, reports by Gallegoet al. (2003) that KU80 is dispensable for T-DNA in-tegration and by Park et al. (2011) that KU80 and other

NHEJ components limit integration suggest thatthe absolute role of the NHEJ pathway for T-DNAintegration is still inconclusive. It is possible that al-ternative DSB-repair pathways, such as the micro-homology-mediated end joining DNA-repair pathway(McVey and Lee, 2008), play a role in T-DNA inte-gration. However, the process of DSB repair in plantsis still not fully understood, underscoring the difficultyin understanding the mechanism behind T-DNA in-tegration. Nonetheless, given the high frequency ofcomplex T-DNA structures usually found in transgenicplants and our detection of similar complex ds DNAstructures prior to integration, we suggest that themajor route for integration is through the ds T-DNAand the DSB DNA-repair pathway. According to thismodel (Fig. 7), ss T-DNA molecules are first trans-ferred into the plant nucleus via the type IV secretionsystem (for review, see McCullen and Binns, 2006;Citovsky et al., 2007; Gelvin, 2010; Tzfira and Citovsky,2010). In addition to T-DNA molecules, other bacterialDNA may enter the plant cell, including binary plasmidDNA (Kononov et al., 1997; Wenck et al., 1997) and A.tumefaciens chromosomal DNA (Ulker et al., 2008).Once inside the nucleus, ss DNA molecules are con-verted into ds DNA molecules (Janssen and Gardner,1990; Offringa et al., 1990; Narasimhulu et al., 1996).The plant’s DSB DNA-repair machinery recognizes assubstrate for repair the exposed termini of the ds DNAmolecules. This repair can lead to end joining betweenfree-floating ds DNA molecules, thus creating circularand complex extrachromosomal molecules. Alterna-tively, the DSB DNA-repair machinery may also me-diate integration of the ds DNA molecules into theplant genome. VirD2, which is covalently attached tothe RB of the transferred ss T-DNAs, may also remainattached to the ds T-DNAs and thus play a role in the

Figure 7. A proposed model for T-DNA integration, the formation of complex T-DNA structures, and T-circles. 1, In A.tumefaciens, the T-DNA borders are nicked by the VirD1-VirD2 endonuclease complex and an ss T-DNA (T-strand) is excised(the T-DNAs are marked in red). A VirD2 protein remains attached to the 59 terminus of the T-strand (the RB). 2, The T-strand istransferred into the plant cell nucleus. Additional ss DNA molecules (derived from the A. tumefaciens binary plasmid orchromosomal DNA) may occasionally be transferred independently or together with a T-DNA by read-though linkage. 3, In thenucleus, ss DNAs are converted into ds DNAs (only ds T-DNAs are shown). 4, ds DNA may then integrate into the genome (firstoutcome) via VirD2 and the plant’s DSB-repair pathway. 5, Termini of a ds DNAmay ligate to each other (second outcome) andgenerate T-circles. 6, Termini of different ds DNA molecules may ligate to each other (third outcome) and form complexstructures. These structures may then integrate or form complex circular molecules. [See online article for color version of thisfigure.]









integration process. It is important to note that T-DNAmolecules may also integrate as ss molecules (Tinland,1996), yet the process by which such molecules mayform complex T-DNA structures may differ from ourproposed model.

The Aspects of in Planta T-DNA Circularization

Because “free” ends are not available in T-circles,such molecules may be mostly “dead-end” productsthat do not integrate. Therefore, the extent of ds T-DNAcircularization is a relevant question when consideringthe efficiency of T-DNA integration. Previous studieshave shown that linearized plasmids recircularize inprotoplasts (Gorbunova and Levy, 1997; Gallego et al.,2003). However, circularization of ds T-DNAs in plantswas considered a relatively rare event until now, be-cause evidence for circularization was indirect andrequired a viral amplification system (Bakkeren et al.,1989; Zhao et al., 2003). Our ability to detect T-circlesdirectly from infected plants using a plasmid-rescueapproach suggests that circularization of ds T-DNAsmay not be as rare as previously thought. The rela-tionship between circularization and integration is anespecially interesting question, considering that dsT-DNAs seem to be abundant in infected plants. Theexistence of free ds T-DNAs in infected plant cellsimmediately after infection is evident from the earlyand broad transient gene expression in infected planttissues (Janssen and Gardner, 1990), from transientexpression using a nontemplate strand (Narasimhuluet al., 1996), from experiments involving homologousrecombination (Offringa et al., 1990), and from theisolation of extrachromosomal T-DNAs in the formof ds plasmids (this work). Further highlighting thisquestion, the majority the free ds T-DNA mole-cules do not integrate (Janssen and Gardner, 1990;Narasimhulu et al., 1996; Maximova et al., 1998; DeBuck et al., 2000), but their fate is unclear becausetransient gene expression that can be used to monitorthem is silenced usually a few days after infection(Johansen and Carrington, 2001).

Any outcome that is not integration of the deliveredT-DNA is probably unfavorable for the A. tumefaciens.Thus, it is tempting to speculate that A. tumefaciensevolved mechanisms that favor T-DNA integration overextrachromosomal ligation. It might also explain why A.tumefaciens-mediated transformation is more efficientwhile producing relatively fewer complex insertions com-pared with transformation by the particle-bombardmentmethod (Makarevitch et al., 2003; Travella et al., 2005).A potential candidate that may be involved in amechanism that prefers integration over extrachromo-somal ligation is VirD2, because it is bound to the 59 endof the RB (Herrera-Estrella et al., 1988) and has beensuggested to play a role, albeit unknown, in T-DNAintegration (Tinland et al., 1995; Mysore et al., 1998).

Understanding how complex T-DNA insertions formmay help identify ways to develop plants with single

perfect insertions, as generally desired for crop im-provements. Our DNA recovery system may be use-ful in future related studies. Furthermore, additionalstudies are needed to uncover the spectrum of factorsfrom plants and A. tumefaciens involved in T-DNA in-tegration and their specific roles in the process.

MATERIALS AND METHODS

Plant Material and Agroinfiltration

Tobacco (Nicotiana tabacum) and Nicotiana benthamiana plants were grownat 20°C under long-day conditions (14 h of light). Plants were grown on SuperGermination mix (Fafard) and agroinfiltrated when about 4 to 8 weeks old.Agroinfiltration was performed as described by Sparkes et al. (2006) withmodifications. Infected leaves were harvested and DNA extracted using amodified cetyl-trimethyl-ammonium bromide protocol as described by Doyleand Doyle (1987). For complete DNA extraction and agroinfiltration protocols,see Supplemental Texts S1 and S2, respectively.

Constructs, Bacterial Strains, Transformation, andTransient T-DNA Assay

The AMP-ORI T-DNA construct was prepared by cloning the backbone ofthe pSAT6 vector (Tzfira et al., 2005) into the pRCS11 binary plasmid (Zeeviet al., 2008). The KAN-ORI and KAN T-DNA constructs were prepared bycloning pET28c backbone sequences into the pRCS2 vector (Tzfira et al., 2005).The primers used for PCR and cloning the KanR gene are listed in SupplementalText S3. Cloning was performed using standard procedures. Binary vectors wereintroduced into EHA105, a disarmed stain of Agrobacterium tumefaciens (excep-tions are experiments involving the virB9– mutant [strain Ax42], which is derivedfrom the A348 background; Beaupré et al., 1997). To recover T-circles, we usedthe X-cell electroporation system (Bio-Rad) with DH5a, Top10 (Invitrogen), or10-b (New England Biolabs) Escherichia coli competent cells. The following an-tibiotic concentrations in Luria-Bertani medium were used for E. coli: ampicillin(100 mg mL21), kanamycin (50 mg mL21), and spectinomycin/streptomycin(50 mg mL21 each). A. tumefaciens cells were grown on yeast extract peptonemedium with the following antibiotic concentrations: spectinomycin/streptomycin(200 mg mL21 each) and rifampicin (10 mg mL21). After agroinfiltration with aGUS-expressing vector, leaves were stained using 5-bromo-4-chloro-3-indolyl-b-glucuronic acid as described by Li et al. (2005).

T-Circle Analysis

Molecular genetics methods for T-circle analysis, including the primersused for sequencing, are detailed in Supplemental Text S3.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. DNA Sequences of the T-DNA constructs AMP-ORI, KAN-ORI, and KAN.

Supplemental Figure S2. Frequency of spectinomycin/streptomycin sen-sitive colonies from agroinfiltrated tobacco plants.

Supplemental Figure S3. Sequencing of T-DNA junctions in T-circles T-1to T-9, T-11, and T-12.

Supplemental Figure S4. Sequencing of PCR products.

Supplemental Text S1. DNA extraction protocol for T-circle isolation.

Supplemental Text S2. Agroinfiltration protocol.

Supplemental Text S3. T-circle analysis.

ACKNOWLEDGMENTS

We thank all members of the Tzfira laboratory for their help. We thankAndrew Binns (University of Pennsylvania) for providing strain Ax42 and


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plasmid pED37. We thank Lyle Simmons and Tariq Akhtar (University ofMichigan) for helpful discussions and critical reading of the manuscript

Received May 13, 2012; accepted July 12, 2012; published July 13, 2012.

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Formation of Complex Extrachromosomal T-DNA - Plant Physiology