Experimental reconstruction of double‐strand break repair ......Non-Mendelian inheritance of the...

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This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may

lead to differences between this version and the Version of Record. Please cite this article as

doi: 10.1111/tpj.13769

This article is protected by copyright. All rights reserved.

DR DONG WANG (Orcid ID : 0000-0003-4278-2728)

Article type : Original Article

Experimental reconstruction of double-strand break repair-mediated

plastid DNA insertion into the tobacco nucleus

Dong Wang1,2*

, Jinbao Gu2, Rakesh David

3, Zhen Wang

2, Songtao Yang

4, Iain R. Searle

3,

Jian-Kang Zhu2,5

and Jeremy N. Timmis3

1. Key Laboratory of Molecular Biology and Gene Engineering in Jiangxi Province, College

of Life Science, Nanchang University, Jiangxi, 330031, China

2. Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences, Shanghai 201602, China

3. Department of Genetics and Evolution, School of Biological Sciences, The University of

Adelaide, South Australia, Australia 5005.

4. Crop Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, 610066,

China

5. Department of Horticulture and Landscape Architecture, Purdue University, West

Lafayette, IN 47907, USA

Dong Wang: [email protected]

Jinbao Gu: [email protected]

Rakesh David: [email protected]

Iain R. Searle: [email protected]

Zhen Wang: [email protected]

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Songtao Yang: [email protected]

Jian-Kang Zhu: [email protected]

Jeremy N. Timmis: [email protected]

* Corresponding author: Dong Wang ([email protected]) telephone number: (86) 791

83969537

Summary

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes.

DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were

engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation

regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei

has not been performed until the present analysis. Here we introduced double-strand DNA

breaks into the nuclear genome of tobacco through inducible expression of I-SceI, and

showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through

double-strand DNA break repair. Microhomology-mediated linking of disparate segments of

chloroplast DNA occurs frequently during healing of induced nuclear DSBs but the resulting

nuclear integrants are often immediately unstable. Non-Mendelian inheritance of a selectable

marker (neo), used to identify plastid DNA transfer, was observed in the progeny of about

50% of lines emerging from the screen. The instability of these de novo nuclear insertions of

plastid DNA (nupts) was shown to be associated with deletion not only of the nupt itself but

also of flanking nuclear DNA within one generation of transfer. This deletion of pre-existing

nuclear DNA suggests that the genetic impact of organellar DNA transfer to the nucleus is

potentially far greater than previously thought.

mailto:[email protected]

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Introduction

A pivotal feature of the evolution of eukaryotes is the formation of mitochondria and plastids,

the cytoplasmic organelles which originated from endosymbiotic prokaryotes. DNA from the

endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a

precursor of the eukaryotic host (Bock and Timmis, 2008; Timmis et al., 2004). This resulted

in the relocation of many organellar genes to the nucleus, and numerous nuclear sequences

were created by insertion of organelle DNA (Martin et al., 2002; Noutsos et al., 2007). While

functional gene transfers from organelle to nucleus are relatively rare, non-functional

sequence transfer happens very often and most nuclear genomes are extensively punctuated

with organelle genome sequences (Matsuo et al., 2005). Real time DNA migration from

organelle to nucleus has been demonstrated in yeast (Thorsness and Fox, 1990) and tobacco

(Huang et al., 2003; Sheppard et al., 2008; Stegemann et al., 2003), and the frequency of this

movement is unexpectedly high. Organellar DNA fragments incorporate into nuclear DNA

through double-strand break (DSB) repair in yeast (Ricchetti et al., 1999) and tobacco (Wang

et al., 2012) but the molecular mechanisms responsible for insertion remain unclear.

Bioinformatic analysis suggested that nonhomologous end joining (NHEJ) is involved in

chloroplast DNA incorporation during DSBs repair (Wang and Timmis, 2013), but direct

experimental evidence was lacking.

De novo experimental nuclear DNA integrants originating from plastid genomes (nupts) are

often unstable. Non-Mendelian inheritance of the selectable marker (neo) used to identify

ptDNA transfers was observed in progeny of about half of the lines emerging from screens

(Huang et al., 2003; Wang et al., 2012). A detailed analysis demonstrated the physical loss of

the neo gene as the cause of instability of kanamycin resistance (Sheppard and Timmis,

2009). No explanations of instability other than deletion have been forthcoming.

In this study, we constructed a reporter system to examine whether DSB repair was involved

in chloroplast DNA insertion into the nuclear genome of tobacco. Three independent

chloroplast integrants were identified at repaired nuclear DSBs. DNA found at these loci

contained multiple disparate DNA sequences from the chloroplast genome linked together by

a process involving microhomology. Furthermore, the instability observed at these de novo

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nupts was shown to be associated with deletion, within one generation of chloroplast DNA

transfer. Deletions occurred not only in the insert itself but also included flanking native

nuclear DNA.

Results

A genetic screen for nuclear DSB repair events that incorporate chloroplast DNA

In a pilot experiment to investigate the effect of induced DSBs on the frequency of plastid

DNA migration, the transplastomic tobacco (Nicotiana tabacum) line (tpGUS) containing a

gus gene driven by the 35S promoter (Sheppard et al., 2008) was irradiated with UV-C. In

tpGUS, the reporter gus gene is expressed only when it transfers to the nucleus and this event

can be monitored readily by histochemical staining to detect the gus product. The number of

blue sectors resulting from nuclear gus expression observed in seedlings treated with UV-C

was greater than that of control seedlings (Table S1). This tentatively suggested that

increasing DSB frequency increased plastid DNA insertion into the nucleus but the lack of

significance necessitated further experimentation. Therefore, to detect DSB repairs that

mediate chloroplast DNA insertion, an experimental system was developed in tobacco

(Figure 1A). The transplastomic line tp-neo (Huang et al., 2003) contains, in its plastid

genome, a neo gene driven by a eukaryotic promoter. It was therefore poorly expressed in

chloroplasts but strongly activated after transfer to the nuclear genome, conferring kanamycin

resistance. To the nucleus of this line was added an inducible DSB system (Lloyd et al.,

2012; Wang et al., 2012). In this added cassette, the gene encoding the rare cutting

endonuclease I-SceI was controlled by an alcA:35S promoter expressed after ethanol

treatment to cut two adjacent engineered target sites (Figure 1B). Consequently, the plant line

containing both nuclear and plastid transgenes was named pISceI-neo. Two lines of

pISceI-neo: pISceI-neo-9 and pISceI-neo-10, containing low copy numbers of T-DNA

monitored by bar gene quantification (see Figure S1), were used in a screen to monitor

insertion of neo from the transplastome at cut and repaired I-SceI sites (Figure 1). In

pISceI-neo-10, it was also determined that there was only a single nuclear locus containing

the two I-SceI sites (Table S2).

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To evaluate the impact of DSB repair on the frequency of plastid-to-nucleus DNA transfer,

pISceI-neo-13 harbouring more than one locus was also used in the screen (Table S2). Leaf

explants of wild type (WT) tobacco were used as negative controls. These were all bleached

within two months when grown on regeneration medium supplemented with 400 mg/L

kanamycin (Figure 1C). Two week old pISceI-neo seedlings were sprayed with 0.7 M ethanol

and 4 days allowed for I-SceI expression, to generate DSBs and DSB repair (Lloyd et al.,

2012). After that, 340 and 400 leaf explants (~25 mm2) from pISceI-neo-9 and pISceI-neo-10

respectively were placed on regeneration medium containing 400 mg/L kanamycin

(Stegemann et al., 2003). In contrast to the control plants, kanamycin resistant shoots

appeared in explants of pISceI-neo leaves (Figure 1C). Six and eight resistant shoots were

generated from pISceI-neo-9 and pISceI-neo-10 lines respectively (Table 1) and these shoots

were designated the kr7 series. Based on cell number estimations (Lloyd and Timmis, 2011),

the occurrence of kanamycin resistant shoots in the screen indicated 1 neo transfer to the

nucleus in approximately 2.1 million and 1.9 million cells in ISceI-neo-9 and pISceI-neo-10

lines respectively, consistent with the plastid-to-nucleus DNA transfer frequency observed in

somatic cells from previous studies (Wang et al., 2012). For pISceI-neo-13, eleven resistant

shoots were generated in 440 leaf explants, indicating a neo transfer to the nucleus of one in

1.5 million cells (Table 1). This frequency is slightly higher than that in pISceI-neo-10,

suggesting that the significant increase results from the higher incidence of DSBs in the

pISceI-neo-13 nuclear genome.

Chloroplast DNA incorporation at nuclear DSB sites

Considering that earlier experimental chloroplast DNA integrants were characteristically

large (tens of kilobases or more in size) and consequently too large to be amplified by the

conventional PCR (Huang et al., 2004; Lloyd and Timmis, 2011), genome walking based on

the nuclear sequences near I-SceI site (Figure 1B) was carried out to characterise DSB

repairs. As expected, associated with the acquisition of kanamycin resistance, transplastomic

DNA was present after I-SceI cleavage and DSB repair in three independent plants obtained

in the screen (Figures 2A, 3 and 4A).

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Sequences adjacent to the repaired I-SceI junctions in kr7.1, kr7.2 and kr7.3 lines (1092, 588

and 1209 nucleotides respectively) revealed complex mosaics of diverse chloroplast

fragments with perfect sequence conservation compared with the plastome or transplastome

(Figure 2A). This sequence fidelity is consistent with the hypothesis that escaped chloroplast

DNAs from pISceI-neo integrated into the nuclear genome at cleaved I-SceI sites during DSB

repair. Besides chloroplast DNA fragments, some unknown sequences presumed to originate

from nuclear DNA were also observed at two of the three repaired sites (Figure 2A). An

intact 35S promoter and part of the neo gene was present in kr7.3 that clearly originated from

the pISceI-neo transplastome (Figure 2A). To exclude the possibility that the other two

chloroplastic integrants from kr7.1 and kr7.2 originated from pre-existing nupts, PCR

amplification was carried out with the primer pairs described in Figure 2A. Corresponding

amplicons were generated only from DNA templates from seedlings containing the

chloroplast integrants and not from WT plants (Figure 2B), confirming that fragments of

chloroplast DNAs had inserted de novo into the nuclear genome at experimentally induced

DSBs.

Upon close examination, the fusion points between chloroplast DNA sequences inside the de

novo nupts suggested that short microhomology was involved in their formation (Figure 3).

Microhomolgy was observed at both chloroplast/chloroplast and chloroplast/nuclear junction

sites, consistent with bioinformatic analyses (Wang and Timmis, 2013).

Instability of chloroplast integrants

In previous experiments, genetic instability was observed within one generation of insertion

in approximately half the kanamycin-resistant lines in a similar screen (Sheppard and

Timmis, 2009). Therefore, the stability of kanamycin resistance was tested in the three kr7

lines reported here by backcrossing them to WT females to replace their transplastomes with

WT and growing the progeny of individual capsules on medium containing kanamycin. Only

one of the three lines (kr7.3) exhibited, in all 8 capsules tested, the 1:1 ratio of

resistant:sensitive expected for a nuclear hemizygote (Table 2). All capsules of kr7.1 and four

of six capsules of kr7.2 presented a very substantial paucity of kanamycin resistant seedlings

(Table 2). No capsules showed an excess of resistant progeny.

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In a previous study, deletion of neo rather than epigenetic silencing was found to be

responsible for the loss of resistance (Sheppard and Timmis, 2009). Therefore, the integrity

of neo in the kr7 backcross progenies was first checked by employing a PCR primer pair

targeting the neo gene. As a positive control, the offspring of kr7.3, the plant that showed

consistent Mendelian segregation, was analyzed first. Because half of its backcross progeny

were expected to be kanamycin sensitive, twelve seeds from a capsule characterised

genetically were grown on medium without selection and their DNAs assayed individually by

PCR. Eight of the 12 samples yielded an amplicon (Figure 4B), consistent with a 1:1 ratio (p

= 0.24813). A similar result was observed (p = 0.24813, Figure 4C top panel), for seedlings

from the fourth capsule of kr7.2 (Table 2). In contrast, DNA from seedlings from a capsule of

kr7.1, which showed high instability of kanamycin resistance, yielded no amplicons of neo

(Figure 4D). These results are consistent with the previous study that determined deletion of

all or part of neo as the cause of loss of kanamycin resistance (Sheppard and Timmis, 2009).

The presence of the engineered I-SceI sites and their known flanking sequences allowed us to

examine the cause of instability of these de novo nupts in greater detail than has been

achieved in previous studies. Primers bar FP and bar RP; ST FP and ST RP (Figure 4A),

designed to amplify neighbouring regions at chloroplast DNA integration sites in nuclear

DNA, were used to assess the extent of sequence deletion when neo was lost. Seedlings from

capsules of kr7.3 and kr7.2 presenting normal segregation of kanamycin resistance gave the

expected products from PCR targeted to sections adjacent to the insertion site (Figure 4A, B

and C top panel), confirming the stability of sequences of both the chloroplast DNA integrant

and its nuclear flanking region. In contrast, seedlings from the fourth capsule of kr7.1 (Table

2), which showed a complete lack of kanamycin resistant progeny, yielded no PCR products

using these same primers (Figure 4A and D). Surprisingly, the sequence missing revealed in

the eighth seedling from the fifth capsule of kr7.2 (Table 2) was found in regions neighboring

the chloroplast DNA insertion site rather than in the neo gene itself (Figure 4A and C bottom

panel). To explore the loss of nucleotides in the nupt lacking neo from kr7.2, one primer

located inside the chloroplast integrant combined with a primer positioned in its nuclear

flanking region were employed (Figure 4A middle panel, P3R for kr7.3 and P2R for kr7.2),

and no products were forthcoming (Figure 4C bottom panel). In contrast, amplicons

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generated by this strategy showed the same pattern as neo and other adjacent sequences

present in capsules exhibiting stable kanamycin resistance (Figure 4A, B and C top panel).

These results indicate that deletion occurs not only inside the chloroplast DNA integrant, but

also in the nuclear regions nearby. Finally, one more primer pair (DSBFP and DSBRP)

encompassing the positions of both I-SceI sites was used to further explore the nuclear

instability near the nupt (Figure 4A). In this strategy, amplicons were expected only in

pISceI-neo lines, not in plants of the kr7 lines showing kanamycin resistance, emphasizing

again that the sizes of these de novo nupts are extremely large. As for seedlings showing no

amplicons of neo or its nearby region, no PCR products were observed with the primer pair

DSBFP and DSBRP (Figure 4C bottom panel and D) suggesting, in common with all other

PCR results, that large deletions are possible in the nuclear regions flanking some nupts.

Discussion

In this study, we demonstrate that chloroplast DNA inserts into the nuclear genome during

DSB repair in tobacco and these inserts are comprised predominantly of complex mixtures of

disparate regions from the chloroplast genome. The integrants may include other, presumed

nuclear DNA sequences and all are joined together through microhomology (Figures 2A and

3). These findings support and extend the previous suggestion that end-joining of linear DNA

fragments creates mosaic integrants prior to, or during, insertion into the nuclear genome

(Noutsos et al., 2005). These novel chloroplast integrants are large such that they cannot be

amplified in their entirety by standard PCR methods (Figure 4B-D). In addition, three

chloroplast integrants inserted into an I-SceI site, and an intact I-SceI site remained

unchanged (Figure 4A). This observation is different from DSB repair-meditated

mitochondrial DNA insertion events (Wang et al., 2012), in that HincII was not used to

remove the stuffer that contains three HincII sites here as it was in the previous study (Lloyd

et al., 2012). After insertion, sequence loss may occur within the de novo nupts and also in

flanking nuclear DNA segments within one generation of their formation, explaining the

genetic instability that characterises about half of the de novo nupts.

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It appears that primary insertions of organelle DNA are initially large but, in many cases,

they are reduced to shorter stretches over very short time periods. However, it remains a

possibility that the large nupts are a feature of the experimental selection for the entire neo

gene and may not represent the full spectrum of events.

Nucleotide substitutions were not detected as all the nupt sequences remain identical to the

corresponding regions of the plastome. However, some nupts clearly survive for long periods

of evolutionary time and substitutions are seen at these older loci (Ayliffe and Timmis, 1992;

Richly and Leister, 2004; Michalovova et al., 2013; Yoshida et al., 2014; Chen et al., 2015).

Our results suggest that scrambled nuclear organellar DNAs are generated through deletion

during the early stages of post-integration rather than by longer term sequence decay. Of

course, several to many somatic cell divisions must have occurred before our attempts to

characterise nupt sequences and organization. Thus we may be reporting their organization

after an initial period of great flux.

The novel demonstration that incorporation of chloroplast DNA can be accompanied by

deletion of adjacent non nupt DNA, indicates that the process must be an important player in

dynamic nuclear genome evolution. Because analyses of instability within nupts in a previous

study relied on the kanamycin resistance provided by neo (Sheppard and Timmis, 2009),

deletion of non nupt DNA also indicates that the frequency of deletion occurring in nupts was

underestimated. Considering the high frequency of DNA movement from plastid to nucleus

(one event in between 11000 or 16000 pollen grains (Huang et al., 2003; Sheppard et al.,

2008)), high incidence of sequence deletion within nupts not only counterbalances the

resulting problem of increasing genome size, but also provides more chance for prokaryotic

plastid genes to gain the functional elements required for nuclear expression. Moreover, the

intraspecific variation of nupts observed in the progeny (Figure 4C bottom panel) suggests

that the majority of sequence deletion occurs during mitosis rather than meiosis. It is formally

possible that these deletions may be different in each seed of a capsule exhibiting kanamycin

sensitivity (Figure 4D), but it is more likely that local sequence contexts determine and

orchestrate a restricted range of deletion outcomes.

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Although the extent of deletion in nupts identified here was characterized, the underlying

mechanisms are still obscure. Direct repeats are thought to be associated with deletions

occurring in nupt DNA (Noutsos et al., 2005), and candidate short repeats were found in

kr7.3 (Figure 2A). Third generation sequencing technology with long read lengths is required

for a thorough understanding of the instability of these nascent nuclear organelle DNAs.

Experimental procedures

Plant growth conditions

Nicotiana tabacum plants were grown with 16 hour light/8 hour dark cycle at 25 °C in jars or

11 cm petri dishes containing half MS medium. Soil grown plants were grown in a growth

room with a 14 hour light/10 hour dark cycle and 25 °C day/18 °C night regime. UV-C

treatment was performed as previously described (Rosa et al., 2013).

Generation of pISceI-neo

pISceI-neo contains a previously-described chloroplast genome insert that is expressed as

kanamycin resistance only after transfer to the nucleus (Huang et al., 2003) and an inducible

DSB system in the nucleus (Figure 1A). The nuclear inducible DSB system was as described

(Lloyd et al., 2012) with the neo gene replaced by a selectable marker bar and a stuffer

fragment of known sequence (Figure 1B).

Experimental induction of DSBs and kanamycin selection

Induction of I-SceI was described previously (Lloyd et al., 2012). Briefly, 2-wk-old seedlings

grown in jars were sprayed with 0.7 M ethanol and four days allowed for producing DSBs.

Leaf explants (~25 mm2) were then placed on 11 cm petri dishes containing regeneration

medium containing 400 mg/L kanamycin. Resistant shoots were moved to 1/2 MS medium to

root and later transferred to soil.

Seedling selection was performed by growing surface-sterilized seeds on half MS medium

supplemented with 150 mg/L kanamycin (Wang et al., 2012), and plates were placed at 25 °C

with a 16 hour light/8 hour dark cycle. The significance of deviation from the expected

Mendelian ratio was calculated by a χ2 test.

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DNA preparation, PCR assays and genome walking

DNA was prepared employing a DNeasy Plant Mini Kit (Qiagen), and PCR amplification

was performed using Taq DNA polymerase (Takara) according to standard protocols.

TaqMan real-time PCR was performed as described previously (Bubner and Baldwin, 2004).

Genome walking utilized a GenomeWalker Universal Kit (Clontech) in accordance with the

manufacturer’s instructions. Details of the primers used are listed in Table S3. Sequence data

for this article can be found in the GenBank data libraries under accession numbers from

KU240015 to KU240017.

Acknowledgments

We thank Mathieu Rousseau-Gueutin and Yuan Li for helping with plant transformation and

TaqMan real-time PCR, respectively. This work was funded by the National Science

Foundation of China (31401077), Natural Science Foundation of Jiangxi Province

(20171ACB20001) and a Nanchang University Seed Grant for Biomedicine

(9202-0210210807). The authors declare no conflict of interest.

Author Contributions

D.W. and J.N.T conceived the study, supervised the experiments, and wrote the article; D.W.,

J.B.G., Z.W., S.T.Y. and R.D. performed the experiments; I.R.S., D.W., J.K.Z and J.N.T.

contributed new reagents and analytic tools; D.W., J.B.G. and J.N.T analyzed data.

List of abbreviations

Double-strand break- DSB

nuclear insertions of plastid DNA- nupts

Nonhomologous end joining- NHEJ

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Short Supporting Information Legends

Figure S1. The comparative copy number of bar in pISceI-neo lines as determined by

real-time quantitative PCR. The least value of copy number is appointed as one.

Table S1. Plastid-to-nucleus DNA transfer in tpGUS seedlings.

Table S2. Segregation of herbicide Basta resistance of self crossed pISceI-neo-9,

pISceI-neo-10 and pISceI-neo-13 lines.

Table S3. Primers used in this study.

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Tables

Table 1. Frequency of DNA transfer from plastid to nucleus observed in pISceI-neo-9,

pISceI-neo-10 and pISceI-neo-13 lines.

Leaf explants Resistant shoots Estimated transfer rate

pISceI-neo-9 340 6 ~2.1×106

pISceI-neo-10 400 8 ~1.9×106

pISceI-neo-13 440 11 1.5×106

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Table 2. Segregation of kanamycin resistance of progenies from the three kr7 lines

backcrossed to female WT plants.

Lines Capsule Resistant Sensitive P

kr7.1 1 2 168 ***

2 3 88 ***

3 7 176 ***

4 1 164 ***

5 5 76 ***

kr7.2 1 21 96 ***

2 50 143 ***

3 29 110 ***

4 85 83 NS

5 9 53 ***

6 22 30 NS

kr7.3 1 58 55 NS

2 21 24 NS

3 47 43 NS

4 50 51 NS

5 49 43 NS

6 59 57 NS

7 44 42 NS

8 56 52 NS

Seeds from individual capsules were grown on half MS medium supplemented with 150 mg/L of

kanamycin. The p values signify deviation from a 1:1 ratio of resistant:sensitive (χ2 test). NS, not

significant; ***, p < 0.001.

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Figure legends

Figure 1. A genetic screen for DNA transfer from chloroplast to nucleus through DSB repair.

(A) Structure of tp-neo. The neo and aadA genes are under CaMV 35S and psbA promoter

control, respectively (Huang et al., 2003). The black box represents the STLS2 intron. (B)

Schematic representation of the DSB system in the nucleus. The top panel presents vector

T-DNA harbouring a hygromycin selectable marker gene (hyg), an AlcR gene and the I-SceI

gene driven by an ethanol inducible promoter (Lloyd et al., 2012). The bottom panel shows

vector T-DNA holding a herbicide resistant gene (bar) and a stuffer fragment of known

sequence flanked by two I-SceI target sites. LB, left border; RB, right border of the

Agrobacterim transformation system. (C) Stable plastid-to-nucleus transfer. Leaf explants

(~25 mm2) were grown on regeneration medium supplemented with 400 mg/L kanamycin.

As resistant plants emerged (as presented in the right hand panel), they were transferred to

1/2 MS media to root and subsequently moved in soil. After 2 months, all leaf explants from

Petite Havana (WT) were totally bleached, and the screen ended at this time point. Plates are

11 cm petri dishes.

Figure 2. Chloroplast DNAs inserted at repaired I-SceI sites. (A) Schematic representations

of partial chloroplast integrants at DSB repair junctions. DNA from three seedlings contained

mosaic integrants comprised of different chloroplast and transplastomic DNA fragments

(different shades of green, shades are not scale). Numbers in the green boxes indicate

corresponding nucleotides of the native tobacco chloroplast genome (Shinozaki et al., 1986).

Grey boxes represent presumed nuclear DNA. The T-DNA vector flanking sequence of

I-SceI restriction site is shown in black. The three primer pairs shown (P1F and P1R, P2F and

P2R, P3F and P3R for kr7.1, kr7.2 and kr7.3, respectively) were designed to amplify specific

chloroplast integrants in the nucleus. (B) The de novo experimental nupts are not present as

nupts elsewhere in WT tobacco. One section of the tobacco nuclear genome amplified by

primers from a recent study (Lloyd and Timmis, 2011) was used as a positive control.

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Figure 3. Microhomology involved in nupt-mediated DSB repair. Alignments show nupts in

three kr7 lines, their corresponding nuclear sequence in pISceI-neo (pISceI-neo_N), and the

chloroplast DNA sequences (tabacum_pt) or transplastomic DNA sequence (pISceI-neo_tp).

Short microhomology at fusion points is highlighted in red, green is used to mark DNA

originating from chloroplast or transplastome, and the unidentified DNA sequence in nupts is

shown in blue. Numbers inside two slashes indicate nucleotides in nupts that are identical to

chloroplast DNA or unidentified DNA origins.

Figure 4. PCR analysis of kr7 lines showing genetic instability for kanamycin resistance. (A)

Schematic representations of nupt flanking sections. Primers used are labelled and their

direction shown arrowed. Primer pairs bar FP and bar RP, ST FP and ST RP cover most of

the bar gene and the stuffer fragment, respectively. The primer pair DSBFP and DSBRP

amplifies a region that includes the two I-SceI sites. P3R and P2R are primers specific for

chloroplast integrants in kr7.3 and kr7.2, respectively. Pnos and Tnos signify the nos

promoter and nos terminator respectively. (B, C and D) PCR analysis of backcross progeny

from three kr7 lines. DNAs prepared from the corresponding pISceI-neo seedlings were

applied in the reaction. The abbreviations kr7.3_K+, kr7.2_K+ and kr7.3_K+ stand for

kanamycin resistant seedlings from each line. The primer pair used as a control is described

in Figure 2B. Ve-, no template.

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Experimental reconstruction of double‐strand break repair ......Non-Mendelian inheritance of the...

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Transcript of Experimental reconstruction of double‐strand break repair ......Non-Mendelian inheritance of the...