Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

Dong Wang, Andrew H. Lloyd1, and Jeremy N. Timmis2

Discipline of , School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia

Edited by William Martin, University of Dusseldorf, Dusseldorf, Germany, and accepted by the Editorial Board January 4, 2012 (received for review October 31, 2011) Mitochondria and (photosynthetic members of the transplastomic reporter genes designed for nucleus-specific family of cytoplasmic ) in eukaryotic cells expression. originated more than a billion years ago when an ancestor of the nucleated cell engulfed two different prokaryotes in separate Results sequential events. Extant cytoplasmic organellar contain Transient and Integrative Expression of the gus Reporter Gene. A very few genes compared with their candidate free-living ances- nucleus-specific reporter gene was placed into the large single- tors, as most have functionally relocated to the nucleus. The first copy region of tobacco plastome (plastid ). In this ge- step in functional relocation involves the integration of inactive netic compartment, the reporter gene (gus) was not expressed, DNA fragments into nuclear , and this process because it was driven by the 35S promoter and terminator and continues at high frequency with attendant genetic, genomic, contained a nuclear intron (STLS2) (20), precluding translation and evolutionary consequences. Using two different transplas- in the plastid. In this transplastomic line (tp-gus), the reporter tomic tobacco lines, we show that DNA migration from chloro- gus gene is expressed only when transferred to the nucleus—an plasts to the nucleus is markedly increased by mild heat stress. In events that is readily monitored by histochemical staining to addition, we show that insertion of mitochondrial DNA fragments detect the gus product, visualized as blue sectors in cells and during the repair of induced double-strand breaks is increased by tissues. Using this system in preliminary experiments, we in- heat stress. The experiments demonstrate that the nuclear influx vestigated the effects of various stress conditions examined of organellar DNA is a potentially a source of mutation for nuclear previously, including heat (21), salt (22), hydrogen peroxide (23), genomes that is highly susceptible to temperature fluctuations and paraquat (24), on plastid-to-nucleus DNA transfer. Heat that are well within the range experienced naturally. stress was outstanding in increasing nuclear gus expression (Table 1, experiment 1). The number of blue-stained sectors on leaves and NA in the highly energetic cytoplasmic organellar genetic roots of seedlings grown at 25 °C for 3 wk, treated for 3 h at 45 °C, Dcompartments of is subjected to high levels of and allowed 2 d to recover at 25 °C was ∼2.5-fold greater than that stress damage from the oxygen free radicals produced during of control seedlings. Two independent experiments using 2-wk-old fi respiration and photosynthesis. Nonetheless, for various reasons, plants con rmed this result in leaves of tobacco seedlings, with less the rate of accumulation of mutations is slower in plant cyto- pronounced effects seen in cotyledons and roots (Table 1, experi- plasmic organelles than in the nucleus (1, 2). The vast majority of ments 2 and 3). Extending the range of exposure time to heat stress fi – mitochondrial and plastid () genes have vacated their showed a signi cant dose response in gus expression events, with ancestral prokaryote genetic compartment in favor of the nu- the number of blue sectors peaking in leaves after 5 h of heat stress (Fig. 1A). After longer heat stress, some blue sectors included large cleus (2), with very few remaining within extant organelles. The areas of the leaves (Fig. 1A). first step in gene relocation for the genomes is DNA Despite the nucleus-specific promoter in tp-gus, both sense escape, followed by insertion into nuclear chromosomes, a pro- and antisense gus are transcribed in the chloroplast from cess shown to occur remarkably frequently under normal physi- adjacent plastid promoters (25), but the nuclear intron (STLS2) ological conditions in tobacco (3, 4) and yeast (5), although an precludes plastid expression (20). Thus, the design of the nu- experimental screen in the unicellular alga Chlamydomonas cleus-specific gus gene allowed quantification of its spliced nu- reinhardtii did not detect any such transpositions (6). These ex- clear mRNA in the presence of excess unspliced chloroplast perimental studies, together with bioinformatic and genomic transcripts. A reverse primer, predominantly in the second exon analyses (7–14), demonstrated that DNA transfer from cyto- but spanning the splice junction with the last three 3′ plasmic organelles is a frequent and continuous process in es- in the first exon, together with an upstream primer, allowed sentially all eukaryotes examined, although it may be very rare in specific PCR amplification of transcripts from which the intron organisms with very low organelle numbers (15). Although DNA had been removed (Fig. 1B). Under this regime, quantitative transfer per se is very frequent in higher plants, genes that mi- PCR demonstrated that spliced gus mRNA accumulation in- grate are normally inactive in the nucleus because they lack the creased in leaf tissue in parallel with the time spent by the motifs required for nuclear expression. Nonetheless, on rare seedlings at 45 °C and correlated with the number and size of occasions nuclear integrants of organelle DNA (norgs) are acti- vated by genomic rearrangements (16, 17), that effectively dupli- cate the gene. Over evolutionary time, these frequent nonfunc- Author contributions: D.W., A.H.L., and J.N.T. designed research; D.W. and A.H.L. per- tional and rare functional DNA transfer processes have resulted in formed research; A.H.L. and J.N.T. contributed new reagents/analytic tools; D.W. and A.H.L. analyzed data; and D.W., A.H.L., and J.N.T. wrote the paper. the net loss of redundant genes in the cytoplasmic organelles and The authors declare no conflict of interest. have added to the genetic complexity of the nucleus. This article is a PNAS Direct Submission. W.M. is a guest editor invited by the Editorial Environmental factors are known to modify mitochondria-to- Board. nucleus DNA movement in yeast (5), and there is good evidence 1Present address: Institut Jean-Pierre Bourgin, UMR1318, Institut National de la Recherche that the normal aging process in rats (18) and yeast (19) increases Agronomique, 78026 Versailles, France. the rate of formation of nuclear integrants of mitochondrial 2To whom correspondence should be addressed. E-mail: [email protected]. (numts). Here we tested abiotic stress effects on cytoplasmic or- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ganelle DNA transfer to the nucleus in tobacco using sensitive 1073/pnas.1117890109/-/DCSupplemental.

2444–2448 | PNAS | February 14, 2012 | vol. 109 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1117890109 Downloaded by guest on September 26, 2021 Table 1. Plastid-to-nucleus DNA transfer in heat-stressed tobacco seedlings n Mean SD Min Max P

Experiment 1 Leaf Control 34 2.41 2.01 0 8 *** Heat 34 6.05 5.31 1 19 Cotyledon Control 34 3.05 2.10 0 9 NS Heat 34 4.41 1.81 1 8 Root Control 34 5.5 4.03 0 14 *** Heat 34 9.41 4.43 3 19 Experiment 2 Leaf Control 39 0.76 1.06 0 5 *** Heat 37 2.48 2.54 0 10 Cotyledon Control 39 3.84 1.82 0 9 NS Heat 37 3.89 1.69 0 7 Root Control 39 2.69 1.68 0 7 *** Heat 37 4.40 2.56 0 11 Experiment 3 Leaf Control 43 0.93 1.26 0 5 ** Heat 42 2.76 4.28 0 23 Cotyledon Control 43 4.32 1.82 0 9 NS Heat 42 3.5 2.14 0 8 Root Control 43 3.72 2.41 0 10 NS Heat 42 4.59 2.93 0 11

The results of three independent experiments are shown. Transplastomic (tp-gus) plants grown at 25 °C for 3 wk (experiment 1) or 2 wk (experiments 2 and 3) were treated at 45 °C for 3 h and allowed to recover for 2 d under normal growth conditions before histochemical staining for gus expression. SD and P values (two-sample Independent t test) are shown. n, seedling Fig. 1. Plastid-to-nucleus DNA transfer in heat-stressed tobacco seedlings: numbers counted; mean, the mean number of blue sectors per seedling; time course of heat treatment. (A) Histochemical staining of whole seed- min, the minimum number of blue sectors per seedling; max, the maximum lings. Seedlings were grown for 2 wk at 25 °C, treated at 45 °C for the in- number of blue sectors per seedling. *P < 0.05; **P < 0.01; ***P < 0.001; NS, dicated times, and allowed to recover at 25 °C for 2 d before staining for gus not significant. expression. (B) RT-PCR of gus mRNA with (+) and without (−) reverse tran- scriptase after heat stress (hr). The positive control gs1.1 (20) is a plant line containing a nuclear copy of gus. rpl25 mRNA RT-PCR was used as a technical the blue areas after staining (Fig. 1C). We conclude that the and loading control. (C) Spliced gus mRNA levels relative to rpl25 mRNA. Gus mRNA was significantly increased in the 5 h sample compared with the 0 h reporter gene escaped from the chloroplast into the nucleus, < where it was transcribed and spliced, and the mRNA was control (P 0.05, two-sample Independent t test). (D) The memory of heat stress. The 2-wk-old plants with two leaves and two cotyledons were treated translated on 80S ribosomes in the cytoplasm. at 45 °C for 0 or 5 h and allowed to recover for 1 mo before GUS staining. Young leaves in plants treated for 5 h at 45 °C were almost (Scale bars: 3 mm.) completely blue-stained (Fig. 1A), suggesting that at the time of heat treatment they were at a developmental stage particularly susceptible to stress. This high expression of gus might be related DNA to the nucleus as described above for gus, resulting in a few to incorporation of reporter genes into the nuclear genome, cells producing NPTII protein, which protects the entire seedling resulting in mitotic lineages of transformed cells, or possibly to to some extent. To test for nuclear integration frequency, 2-wk- a large amount of plastid DNA entering the nucleus, resulting in old tp-neo plants were treated at 45 °C for 5 h and allowed to massive transient expression. To determine the longevity of the recover at 25 °C for 2 d, after which 400 leaf explants (each 30 elevation of gene transfer, 2-wk-old seedlings were treated for mm2) were placed on regeneration medium containing 400 μg/ 5 h at 45 °C and then returned to standard growth conditions for mL of kanamycin to overcome the low level of resistance in tp- 30 d. After this long recovery period, the first two leaves that neo (4). Most leaf explants from heat-treated plants remained were present at the time of heat stress showed markedly more green, and 45 resistant shoots were selected as soon as they blue sectors than controls (Fig. 1D), with no obvious increase in became visible over 120 d (Fig. 2). When a resistant shoot was leaves that initiated and expanded after the stress treatment. transferred to new medium, the entire explant was removed from This finding suggests that heat is a specific inducer of chloroplast the experiment, necessarily resulting in a gradual decrease in the DNA transfer. The observation of discrete leaf areas of persis- number of cells in the screen. All leaf explants from an equal tently elevated gus expression strongly implies nuclear in- number of non–heat-treated control seedlings were bleached tegration and stable expression, which suggests that considerable much more quickly, except for 13 resistant shoots that emerged transient expression of the reporter gene explains the large, more over the same 120 d period (Fig. 2). diffuse, blue areas seen after 5 h of heat stress (Fig. 1A). How- Based on cell number estimates (17), the frequency of kana- ever, it is very difficult to distinguish unequivocally between mycin-resistant shoots emerging from the screen equated to 1 transient expression and nuclear integration using tp-gus (20), neo transfer to the nucleus in ∼1.4 million cells in the control, EVOLUTION necessitating an alternative approach. compared with 1 in ∼400,000 cells after heat stress—an ∼3.5-fold increase. All of the resistant shoots were recovered, and the Integrative Expression of the neo Reporter Gene. To assess the ef- majority grew into mature plants, designated the kr6 series. fect of heat stress on nuclear integration, we used a second Back-crosses of mature kr6 genotypes to WT females were used transplastomic plant (tp-neo) containing a copy of neo-STLS2 to replace the transplastome with that of WT in the progeny, (3) encoding NPTII in both inverted repeats of the plastome. some of which were tested for segregation of the kanamycin These plants show a low level of kanamycin resistance compared resistance phenotype (Table S1). Of 14 kr6 lines tested geneti- with WT when grown under standard conditions (3, 4). This cally (7 from controls and 7 from heat-treated tissues), 9 showed partial resistance is probably due to the same leakage of plastid the expected 1:1 ratio in their progeny characteristic of a nuclear

Wang et al. PNAS | February 14, 2012 | vol. 109 | no. 7 | 2445 Downloaded by guest on September 26, 2021 Fig. 3. Confocal laser scanning microscopy images of tobacco guard cells. Epi- dermal strips with underlying mesophyll cells from control (A) and heat-treated (B) pMSK56 tobacco plants containing gfp in the chloroplast genome (27). GFP fluorescence (green, Left) and chlorophyll autofluorescence (red, Middle) superimposed on the interference contrast images (Right). (Scale bars: 25 μm.) Fig. 2. Heritable plastid-to-nucleus DNA transfer. Examples of plates showing the progression of heat-treated and control transplastomic (tp-neo) leaf explants regenerating under kanamycin selection. The 2-wk-old seedlings were treated for 0 h (Left)or5h(Right) at 45 °C and then maintained under repair events not involving insertion). Seven insertions were standard growth conditions for 48 h to allow recovery. Leaf explants (∼30 mm2) observed after heat stress, four of which were identical to clusters were placed on culture plates containing plant regeneration medium supple- of mitochondrial (mt) DNA from up to four disparate regions of mented with 400 μg/mL of kanamycin. As resistant shoots emerged over 120 the organellar genome (Table 2). In contrast, no insertions of d (left-hand labels), they were transferred to 0.5× MS medium to root and mtDNA were seen in DNA from an equal number of untreated subsequently transferred to soil. Plates are 11-cm Petri dishes. plants, suggesting that copious amounts of mtDNA were liber- ated into the cytoplasm after heat treatment, some of which were fi incorporated into nuclei. No integrants of plastid DNA were hemizygote, whereas the other 5 showed various signi cant observed; the remaining three insertions were not of cytoplasmic departures from expectation, consistent with previous experi- organellar origin (Table 2). ments (26). No differences were observed between the heat- Insertions D5 and G12 (Table 2) were patchworks of treated (kr6h) and control (kr6c) lines (Table S1). sequences originating from noncontiguous regions of mtDNA. It was thought that these could possibly be insertions of preexisting Anatomical Effects of Heat Stress. To identify the reasons for the numts, rather than de novo mtDNA; thus, PCR primers were large increase in plastid-to-nucleus DNA transfer induced by heat designed to amplify integrant D5 (Fig. S1A), with primer D5F stress, we examined the integrity of chloroplasts. A transplastomic located within mitochondrial sequence 57336–57413 and primer tobacco line pMSK56 (27) that encodes and expresses GFP in its D5R in mitochondrial sequence 29727–29810. No product was chloroplasts was treated at 45 °C for 5 h. In seedlings without heat amplified from WT DNA unless it was spiked with an equimolar treatment, GFP fluorescence colocalized with chlorophyll auto- fl amount (i.e., one copy per tobacco nuclear genome) of the uorescence in chloroplasts of stomatal guard cells and underlying original D5 smPCR product (Fig. S1B), indicating that the se- mesophyll tissues (Fig. 3A). In contrast, GFP and chlorophyll quence is not present in WT DNA. A control PCR using nu- distribution was widespread after heat stress, suggesting disruption cleus-specific primers confirmed the technical quality of DNA of chloroplast membranes and release of DNA into the cytosol samples (Fig. S1C). Therefore, D5 represents a de novo mtDNA and intercellular spaces, with a consequent potential for plastid integrant that was inserted at an experimentally induced I-SceI DNA fragments to transfect nuclei (Fig. 3B). DSB. Together, the lack of any mtDNA insertions in an equal number of single molecules from control plants and the patch- Use of Double-Strand Break Repair to Monitor Escape of Cytoplasmic working of the integrants suggest the availability of multiple Organellar DNA to the Nucleus. As an alternative to using trans- small fragments of mtDNA after heat stress that are regularly plastomic plants to screen for organellar DNA transfer to the incorporated during nuclear DSB repair. nucleus, we developed a method to determine the sequences at The reason why larger fragments of mtDNA were not seen may double-strand breaks (DSBs) repaired by nonhomologous end- be related to their relative difficulty of amplification. However, joining, which is involved in the majority of integration events PCR was demonstrably capable of amplifying considerably larger (17). In nuclear transgenic lines, we conditionally induced ex- inserts, suggesting that mtDNA was extensively fragmented. Thus, pression of the rare-cutting endonuclease I-SceI to cause DSBs it appears that chloroplast DNA is not present in such large at two restriction sites separated by a short spacer region that we amounts of small fragments, although it is clearly available as also introduced at a second nuclear locus. The spacer region large molecules sufficient to carry the entire neo and gus select- contained the dao1 gene, which potentially facilitates both pos- able markers (Figs. 1 and 2), as well as substantial flanking plas- itive and negative selection of seedlings in Arabidopsis (28), but tomic regions (17, 30). Most of these latter inserts would have selection was not used in the experiments reported here. Im- been too large to emerge from the smPCR screen. portantly, however, spacer excision enabled unequivocal recog- nition of digested and repaired loci after I-SceI digestion, and Discussion analysis of more than 300 unique DSB repair junctions was The experimental demonstration of DNA transfer from the achieved by single-molecule (sm) PCR. By diluting template chloroplast to the nucleus (3, 4) showed such a high frequency as DNA until each reaction received no more than one template to make its implications controversial (31, 32). Therefore, it is molecule, smPCR circumvented such problems as template even more surprising to find that the rate of transfer is increased jumping and preferential amplification of small products (29). up to 10-fold by mild temperature stresses applied over short This made it possible to amplify rare, large template molecules periods. Tobacco plants grow optimally at daytime temperatures (corresponding to insertion events) from a population of much of ∼25 °C, but crops regularly encounter much higher temper- smaller, more frequent template molecules (corresponding to atures in nature. The temperatures reached in leaf cells at

2446 | www.pnas.org/cgi/doi/10.1073/pnas.1117890109 Wang et al. Downloaded by guest on September 26, 2021 Table 2. Characterization of sequences inserted after DSB repair Insertion Length, bp Insertion origin

A5 338 Mitochondrial (298061–298400)* D7 102 Nuclear; DSB flanking region † D5 358 Mitochondrial (57336-57413 [408086–408166] , 24465–24390, 56185–56304, 29727–29810)* F5 165 Nuclear; similar to copia-like retro transposon (e = 1.1 × 10−14) F8 229 Mitochondrial (187992–188220)* − G5 118 Nuclear; similar to tobacco EST EG649865.1 (e = 2 × 10 30) G12 211 Mitochondrial (152588–152685, 181465–181435, 164031–163950)*

*Numbers in brackets correspond to the equivalent nucleotides in the tobacco mitochondrial genome (46). † Alternative coordinates are given in square brackets.

ambient temperatures exceeding 40 °C may be even higher as together, these results suggest the presence of a large amount of a result of radiant heating effects, although these effects would available organelle DNA at the time of DSB repair after heat be countered somewhat by the evaporative cooling effects of stress. There is some previous evidence that mitochondrial transpiration. Nonetheless, the complex leaf canopy of any spe- membrane potential is perturbed in cucumber (41) and Arabi- cies is certain to provide for a large range of temperatures in dopsis (42) by much more extreme heat shock at 55 °C for short different microclimates, and we would expect to see equally periods, suggesting damage to mitochondrial ultrastructure that complex cellular responses, including cell-to-cell variation in may be associated with mtDNA release into the cytosol. plastid and mtDNA escape and insertion into the nucleus. Given that the environments studied are by no means extreme, Given our monitoring of the nuclear expression of entire these results have far-reaching consequences for nuclear evolu- functional nuclear reporter genes as they escape from the tion in organisms that must withstand ambient temperatures. transplastid, we necessarily expected all other parts of the plas- The ingress of cytoplasmic organellar DNA in tobacco grown in tome to be involved equally even though they remained invisible ideal environments is already considered remarkably high (31), to the screen. Therefore, an astonishing array of undetectable and it must be mutagenic at rates equivalent to or exceeding any transpositional events must occur that would substantially other causes (43). Clearly, eukaryotes evolved in the real world change the clonal nature of the somatic cells of individual plants of highly variable growth conditions, and they must have been and make them heterogeneous chimeras containing nuclei that subject to massive oscillations in the rate of bombardment with are highly genetically variable with respect to recent integrants of cytoplasmic organellar DNA. The possibility of approaching in- cytoplasmic organellar DNA. creased fluctuations in global environments points to the im- The sensitivity of the two transplastomic lines used in the portance of understanding the processes involved. present study was the key to revealing this unique facet of nu- clear mutation and evolution. Although these transplastomics Materials and Methods are initially able to show only DNA movement per se, sub- Plant Lines. Tobacco (Nicotiana tabacum) plants were grown under standard sequent rearrangements within the nucleus have been shown to conditions (16-h light, 8-h dark cycle at 25 °C) in sterile jars or 11-cm Petri dishes activate prokaryotic genes in ways that recapitulate real endo- on 0.5× MS medium (44) or in compost in growth rooms with a 14-h light/10-h symbiotic evolution (16, 17). The profound global consequences dark and 25 °C-day/18 °C-night growth regime. The transplastomic tobacco of the processes that are made possible by initial DNA transfer lines were described previously: tp-neo (3), tp-gus (20), and pMSK56 (27). are fully revealed by bioinformatic analyses showing how endo- symbiotic evolution has contributed thousands of genes to the Heat Stress Treatments. In a pilot experiment, 3-wk-old seedlings were treated eukaryotic nucleus (9, 10, 33–35). This deluge of cytoplasmic for 3 h at 45 °C (21), followed by recovery at 48 h under standard conditions. organellar DNA into the nucleus (31) clearly results in a “trial- Subsequent experiments used 2-wk-old seedlings grown at 45 °C for various and-error” system of genomic rearrangements after which a few times (3, 4, 5, and 6 h), followed by recovery at 48 h or 30 d under standard integrants gain functionality. Thus, a large majority are quickly conditions. In all experiments, control plants were maintained at 25 °C. removed in whole or part (26), whereas a small minority have Analysis of GUS Activity. Histochemical GUS staining was done as described profoundly changed gene disposition in eukaryotes and added to previously (20). The numbers of blue spots from seedlings with and without the gene content and heterogeneity of the cell. stress treatment were scored blind and analyzed with the two-sample in- These transplastomic assays have shown that plastomic DNA dependent t test using SPSS software. Images were captured using an is transferred to the nucleus in very large, sometimes scrambled Olympus Provis AX70 microscope. (17), sections, and bioinformatic evidence shows that the process also involves small pieces of the DNA (8, 9, 11, 17). However, PCR Assays. RNA preparation was performed using an RNeasy Plant Mini Kit there is no experimental evidence of small de novo inserts of (Qiagen), and genomic DNA contamination was removed using an Ambion plastid DNA analogous to the mitochondrial sequences that we TURBO DNA-Free Kit (Invitrogen). Reverse-transcription (RT) was then per- demonstrated by DSB induction and smPCR. The lack of a sys- formed using an Advantage RT-for-PCR Kit (Clontech) with random hexamer tem for transforming the mitochondrial genome of plants pre- primers. All kits were used in accordance with the manufacturers’ cludes a screen for large mtDNA integrants, although there is instructions. PCR amplification was performed using Taq DNA polymerase EVOLUTION ample bioinformatic evidence of their existence (11, 36). (Invitrogen) in accordance with standard protocols. Standard RT-PCR am- fi ′ The insertion of organelle DNA at sites of DSB repair in yeast pli cation of gus cDNA was done using primers 5 -ATACCGAAAGGTTGGG- CAG-3′ and 5′-TTCACACAAACGGTGATACGTA-3′, and amplification of RPL25 was first reported more than a decade ago (37). Although in- cDNA was done using primers 5′-AAAATCTGACCCCAAGGCAC-3′ and 5′- direct evidence of the same process occurring in some higher GCTTTCTTCGTCCCATCAGG-3′ (17). Quantitative real-time PCR was per- organisms has been published (17, 38), the present study has formed as described previously (45). The primers used for gus quantitative been able to demonstrate directly that organelle DNA integrates PCR were 5′-ATACCGAAAGGTTGGGCAG-3′ and 5′-TTCACACAAACGGTGA- at DSBs in multicellular eukaryotes. In previous experiments (39, TACGTA-3′. For relative quantification, RPL25 mRNA, amplified with primers 40), in the absence of stress, no cytoplasmic organelle DNA 5′-CCCCTCACCACAGAGTCTGC-3′ and 5′-AAGGGTGTTGTTGTCCTCAATCTT-3′, insertions were observed at sites of nuclear DSBs. Taken served as an internal standard (45).

Wang et al. PNAS | February 14, 2012 | vol. 109 | no. 7 | 2447 Downloaded by guest on September 26, 2021 Fluorescence Microscopy. Leaves of pMSK56 were cut into sections, placed on mM dNTPs, 0.4 μM primers (DSBF1: 5′-GATAGTGACCTTAGGCGACTTTT- a glass slide, and viewed by confocal scanning laser microscopy (Leica SP5 GAACG-3′; DSBR1: 5′-TCCCCTGATTCTGTGGATAACCGT-3′), 0.2 U LongAmp spectral scanning confocal microscope). Images were captured as described taq DNA polymerase, 1× LongAmp buffer, and 110–130 pg of template DNA. previously (27). Reactions were overlaid with mineral oil to prevent evaporation. Cycle con- ditions were as follows: initial denaturation at 95 °C for 30 s; followed by 45 ∼ 2 Analysis of Kanamycin Resistance. Leaf explants ( 30 mm )fromplantsgrown cycles of 95 °C for 20 s, 59 °C for 20 s, and 65 °C for 4 min; followed by a final in sterile jars were placed in 11-cm Petri dishes containing plant regeneration extension at 65 °C for 10 min. After PCR, 18 μL of water was added to each re- medium supplemented with 400 μg/mL of kanamycin (4). Resistant shoots were action; 5 μL was analyzed by standard agarose gel electrophoresis, and the re- transferred to 0.5× MS medium to root and then transferred to soil in a con- mainder was used in subsequent sequencing. Insertion events (smPCR products trolled environment chamber with a 14-h light/10-h dark and 25 °C-day/18 °C- larger than ∼850 bp) were sequenced directly using primers DSBF1 and DSBR1. night growth regime. For seedling selection, surface-sterilized seeds were grown on 0.5× MS medium containing 150 μg/mL of kanamycin. All plates were fi incubated at 25 °C with 16-h light/8-h dark cycle. Significance of deviation from Insert D5 Ampli cation. PCR was performed using Taq DNA polymerase 2 ’ the expected Mendelian ratio was determined using the χ test. (Roche) in accordance with the manufacturer s instructions. The D5 insertion PCR used primers D5F (5′-GGAAGCGAGATTGGATTGACG-3′) and D5R (5′- Analysis of DNA DSB Repair Junctions. Seedlings of DSB induction lines D4A2 TGGGTCAGGGTCAGATACGGA-3′), with WT DNA as a template. As a positive and D14A2 were grown in tissue culture jars. At 2 wk after germination, control, WT DNA spiked with one copy per diploid genome of the original plants were heat-treated in a controlled temperature chamber for 5 h at 45 °C. D5 smPCR product was used as template. The L25 gene was amplified from Immediately after, or 1 d after heat stress, DSBs were induced by spraying ∼1– WT DNA using primers L25F (5′-AAAATCTGACCCCAAGGCAC-3′) and L25R 2 mL of 0.7 M ethanol into tissue culture jars. Plants were left for 4 d to allow (5′-GCTTTCTTCGTCCCATCAGG-3′) (17). Controls with no template DNA were time for I-SceI expression, creation of DSBs, and DSB repair. included for each primer pair. After recovery from heat stress, DNA was prepared from leaf tissue and used as template for single-molecule (sm) PCR (29). Single-molecule PCR was ACKNOWLEDGMENTS. We thank Dr. Mathieu Rousseau-Gueutin for helpful performed using LongAmp taq DNA polymerase (New England Biolabs) and discussions. This research was supported under the Australian Research HincII (which cuts three times within the spacer region) digested DNA as Council’s Discovery Projects funding scheme (Project DP0986973). D.W. is a template. Reactions were conducted in a volume of 2 μL containing 0.3 supported by the China Scholarship Council.

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