Journal of Biotechnology 204 (2015) 17–24

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Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts Alessandro Nicolia a,b,1 , Estelle Proux-Wéra b,2 , Inger Åhman a , Nawaporn Onkokesung a , Mariette Andersson a , Erik Andreasson b,∗∗,3 , Li-Hua Zhu a,∗,3 a b

Department of Plant Breeding, Swedish University of Agricultural Sciences, Alnarp, Sweden Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden

a r t i c l e

i n f o

Article history: Received 13 December 2014 Received in revised form 24 March 2015 Accepted 27 March 2015 Available online 4 April 2015 Keywords: Potato Site-directed mutagenesis TALEN Gene editing Protoplast Transient gene expression

a b s t r a c t Potato is the third largest food crop in the world, however, the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depression are major difficulties for conventional potato breeding. The rapid development of modern breeding methods offers new possibilities to enhance breeding efficiency and precise improvement of desirable traits. New site-directed mutagenesis techniques that can directly edit the target genes without any integration of recombinant DNA are especially favorable. Here we present a successful pipeline for site-directed mutagenesis in tetraploid potato through transient TALEN expression in protoplasts. The transfection efficiency of protoplasts was 38–39% and the site-directed mutation frequency was 7–8% with a few base deletions as the predominant type of mutation. Among the protoplast-derived calli, 11–13% showed mutations and a similar frequency (10%) was observed in the regenerated shoots. Our results indicate that the site-directed mutagenesis technology could be used as a new breeding method in potato as well as for functional analysis of important genes to promote sustainable potato production. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Random mutagenesis techniques, based on physical or chemical mutagens, have been used in plant breeding for producing several thousands of new cultivars in the past 70 years (Shu et al., 2012). Such methods provide a cheap and simple way to induce new variability in germplasm which has already been adapted for cultivation (Ahloowalia et al., 2004; Waugh et al., 2006). However, these methods rely on an extensive phenotypic or genotypic screening to identify desirable mutants, and with many unwanted

Abbreviations: SDM, site-directed mutagenesis; DSB, double strand break; HR, homologous recombination; NHEJ, non-homologous end joining; TALEN, transcription activator-like effector nuclease; ALS, acetolactate synthase. ∗ Corresponding author at: Department of Plant Breeding, Swedish University of Agricultural Sciences, SLU, Box 101, 230 53 Alnarp, Sweden. ∗∗ Corresponding author at: Department of Plant Protection Biology, SLU, Box 102, 230 53 Alnarp, Sweden. E-mail addresses: [email protected] (E. Andreasson), [email protected] (L.-H. Zhu). 1 Present address: ENEA Research Centre – Casaccia, UTAGRI-GEN, Rome, Italy. 2 Present address: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden. 3 The authors contributed equally. http://dx.doi.org/10.1016/j.jbiotec.2015.03.021 0168-1656/© 2015 Elsevier B.V. All rights reserved.

mutations in a single genotype as the common outcome (Shu et al., 2012). On the contrary, site-directed mutagenesis (SDM) allows predictable and precise editing of specific DNA sequences. This mutation technology has potential applications ranging from gene therapy to basic research and plant breeding (Fichtner et al., 2014; Gaj et al., 2013; Kim and Kim, 2014). In general, SDM can be achieved through introducing a double strand break (DSB) at a given DNA sequence in cells. Then the targeted cell’s repair pathways, either based on homologous recombination (HR) or non-homologous end joining (NHEJ), is exploited for gene addition, gene correction or gene knock-out (Gaj et al., 2013). Zinc finger nuclease (ZFN), the first nuclease used in SDM approaches, along with homing endonucleases (HE or meganucleases) have been studied and used in plants (Curtin et al., 2012; Fichtner et al., 2014). However, not until the recent introduction of the Transcription Activator-Like Effector Nucleases (TALENs) (Christian et al., 2010) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) system (Jinek et al., 2012), has the technology reached a level of simplification and efficiency sufficiently high to allow a rapid application among laboratories. The TALEN architecture is characterized by a nuclease domain fused to a DNA binding domain derived from the effector proteins produced by the plant pathogens in the genus Xanthomonas. The

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DNA binding domain is a customizable modular structure featured by a repeat-variable diresidue (RVD) array assembled to achieve binding specificity. Dimerization of two nuclease domains, is generally required to obtain a DSB, thus at least a pair of TALEN has to be designed for each DNA target site. Both TALEN and CRISPR-Cas systems have recently been tested and proven to be functional in a broad range of organisms, including mammals, arthropods, and plants (Chen and Gao, 2013; Gaj et al., 2013). In diploid crops, such as barley (Gurushidze et al., 2014; Wendt et al., 2013), rice (Li et al., 2012; Zhang et al., 2014; Zhou et al., 2014), soybean (Curtin et al., 2011; Haun et al., 2014), tomato (Brooks et al., 2014; Lor et al., 2014) and maize (Liang et al., 2013; Shukla et al., 2009), SDMs have been successfully employed for genome editing purposes. However, a number of important food crops are polyploid, such as wheat (Triticum aestivum L.) and potato (Solanum tuberosum L.). The major challenges in SDM of polyploid plants are the presence of multiple alleles of a certain gene, as well as frequent gene duplications resulting in a high number of possible targets in a single cell. The reports on successful applications of TALEN or CRISPR-Cas techniques in polyploid species are very limited. Recently, three homoalleles of the MLO gene in the allohexaploid wheat were successfully targeted employing TALEN and CRISPR-Cas (Wang et al., 2014), through a transgenic approach with stable integration. Moreover, Sawai et al. (2014) reported the mutation of all four alleles of the St SSR2 gene involved in glycoalkaloid synthesis via stable transformation of a TALEN construct in potato. Potato is the third most important staple crop with excellent yield potential and high nutritional value (Barrell et al., 2013). Potato cultivars are normally autotetraploid (2n = 4x = 48), but ploidy level can range from diploid (2n = 2x = 24) to pentaploid (2n = 5x = 60) (Spooner et al., 2005). Breeding efforts for improved cultivars are considerable, started more than 100 years ago; however, progress has been relatively slow and breeding is largely based on phenotypic selection among a large number of offspring. The high heterozygosity (Uitdewilligen et al., 2013) and tetrasomic inheritance in autotetraploid potato are the major drawbacks in potato breeding, because unfavorable alleles are continuously unmasked at each breeding cycle. Generally, it takes about 100,000 new genotypes to generate one new variety. Breeding programs based on ploidy reduction and selfing (to discard otherwise hidden unfavorable alleles) have been carried out, but the severe inbreeding depression and self-incompatibility of diploid germplasm have hampered these attempts (Lindhout et al., 2011). Breeding to improve resistance to late blight (Phytophthora infestans) in potato using traditional methods has been very slow (Lindhout et al., 2011), although considerable progress has been achieved in introduction of resistance to nematodes, viruses and potato wart (Mackay, 2003). On the other hand, new plant varieties bred by modern breeding techniques, such as gene transformation, are tightly regulated in the European Union (EU) for commercial use. Genome editing may be an alternative, as mutated lines can be generated by a transgenic approach with stable integration of T-DNA carrying TALEN or CRISPR-Cas, which can subsequently be segregated out, and thus resulting in a mutant indistinguishable from classically bred varieties (Hartung and Schiemann, 2013). However from the legislative and the technical point of view, it would be more favorable to use a transient expression system (Podevin et al., 2013) in which no T-DNA is integrated into the plant genome and the generated mutants are of immediate interest as potential new varieties. This approach is particularly attractive for vegetatively propagated crops such as potato. In this study, we have successfully established a pipeline for tetraploid potato targeted gene mutation where TALEN was transiently expressed in protoplasts using acetolactate synthase gene (ALS) as the target. This gene encodes for a critical enzyme involved in the biosynthesis

of branched-chain amino acids in plants (Tan et al., 2005) and has been used in experiments of targeted gene mutation in other species (Endo et al., 2006; Townsend et al., 2009; Zhang et al., 2013). 2. Materials and methods 2.1. TALEN design and construct preparation About 50 million paired-end reads from S. tuberosum cv Desirée (2n = 4x = 48) obtained by RNA-seq were mapped against the two ALS gene sequences retrieved from S. tuberosum Group Phureja (clone DM 1–3) chromosome 7 (PGSC0003DMG400034102) and chromosome 10 (PGSC0003DMG400007078), using TopHat2 v. 0.8b (Kim et al., 2013). The read coverage was estimated using bedtools 2.20.1 (Quinlan and Hall, 2010) and the alignments imported in IGV (Robinson et al., 2011). The primer ALSFor (5 -GCCATCCCTCCACAATATGC-3 ) and ALSRev (5 GCAGCAGGTACGCCACAAG-3 ) were designed in the two regions sharing 100% homology with the sequences of Desirée (Fig. 2a and b). The corresponding amplicon of 458 bp was further evaluated for putative TALEN targets with the TALEN targeter (https://tale-nt.cac.cornell.edu/node/add/talen) and a region of 55 bp, containing two 20 bp long conserved TALEN binding sites separated by a 15 bp spacer, was chosen (Supp. materials, 1). The synthesis of the two repeat-variable diresidue (RVD) arrays for the chosen TALEN pair (Supp. material, 1) was performed with the GoldenGate TALEN kit 2.0 (AddGene, Cambridge, USA) as described by Cermak et al. (2011). The two RVD arrays were independently assembled in the vector pZHY500, carrying the N152/C63 truncated backbone. The released fragments by XbaI/BamHI were subsequently cloned into the vector pZHY013 (Zhang et al., 2013) containing the EL/KK FokI obligated heterodimer and the T2A ribosomal skipping peptide (Halpin et al., 1999). The assembled coding sequences for the ALS-TALEN pair were then cloned into the Gateway (Life Technologies, Carlsbad, USA) compatible vector pK2GW7 0 (Karimi et al., 2002) under the 35S promoter, resulting in the plant expression vector p35S-ALSTALEN with the final size of 16,010 bp (Supp. materials, 2). The plasmid extractions and purifications in this study were carried out using the Plasmid mini kit (Qiagen, Netherlands, EU). 2.2. Protoplast extraction and transfection Leaves from in vitro propagated shoot cultures of S. tuberosum cv. Desirée were excised under sterile conditions for isolating protoplasts as described by Cardi et al. (1990) with some modifications (Supp. materials, 5a). The viability of the isolated protoplasts was assessed by fluorescine diacetate (FDA) staining as reported by Larkin (1976). The purified protoplasts were transfected as described by Craig et al. (2005) with modifications (Supp. materials, 5a). Briefly, for each transfection, 1.6 × 105 protoplasts (100 ␮l) were mixed with PEG4000 (12.5%) and 10 ␮g of the vector DNA, coding either for the green fluorescent protein (GPF) or the ALSTALEN. The transfection reaction at room temperature (RT) was stopped after 3 min, followed by an incubation in the nutrient ½ Medium E (Supp. materials, 5b) for 24 h at RT. To determine transfection efficiency, two different vectors carrying the GFP gene were used: pGFP1 (pCW498-35S-GFiP-OcsT, 14,743 bp, Wood et al., 2009) and pGFP2 (pHBT-sGFP-NosT, 4230 bp, CD3-911, Chiu et al., 1996) (Supp. materials, 3). The GFP expression was evaluated after 24 h and the digital images of the transfected and non-transfected protoplasts were acquired using the stereoscope (LEICA M165 FC) and the microscope (LEICA DMLB100T) equipped with digital camera (LEICA DFC450C), GFP filter with excitation range 450–490 nm and fluorescent illumination system (PRIOR-Lumen 200). The

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number of protoplasts was manually counted for calculation of the transfection efficiency. 2.3. TALEN validation in potato protoplasts Two protoplast transfections were carried out with the p35S-ALS-TALEN vector (PEG+/pDNA+) together with a control transfection including PEG but no plasmid DNA (PEG+/pDNA−). After 24 h (Sup. materials 5b) the protoplasts were centrifuged, re-suspended in 50 ␮l of Millipore water, incubated at 94 ◦ C for 5 min and briefly spun down for the PCR analysis. Two ␮l of the supernatant were used as template in a PCR reaction containing 0.5 mM of the primer ALSFor and ALSRev, HF Buffer, 0.2 mM dNTPs and 1 U of Phusion HF polymerase (ThermoFisher scientific, Waltham, USA). The PCR reaction was carried out using the following program: 98 ◦ C for 30 s, 35 cycles at 98 ◦ C for 10 s, 67 ◦ C for 20 s and 72 ◦ C for 10 s with Applied Biosystems 9700 thermal cycler (Life Technologies, Carlsbad, USA). The PCR product was purified with GeneJet PCR purification kit (ThermoFisher scientific, Waltham, USA), cloned in the pJET1.2 vector using CloneJet PCR Cloning Kit (ThermoFisher scientific, Waltham, USA) and electroporated into Escherichia coli (DH5␣). For each of the two PEG+/pDNA+ transfections and the control PEG+/pDNA−, 192 colonies were sequenced (GATC, Constance, Germany) with the primer ALSRev. The sequences were aligned with CLC Main Workbench 7 (CLCbio, Germantown, USA) and the efficiency of mutation was estimated through calculating the ratio between the number of mutated sequences and the total number of sequences obtained. The cdhit-est v 4.5.4-2011-03-07 (Fu et al., 2012) with 100% identity was used to cluster separately the sequences obtained from the two PEG+/pDNA+ transfections (171 and 193 sequences respectively). Only the first 314 bp out of 458 bp of the cloned PCR fragment were used and sequences of low quality were excluded for cluster generation. The number of sequences with ALS-TALEN induced mutations, fitting in each of the generated clusters (the average value from the two PEG+/pDNA+ transfections), was subsequently divided by the average cluster size (the number of sequences in each cluster) and the ratio used as estimation of ALS-TALEN targeting efficiency.

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primer 35SFor (5 -CGACAGTGGTCCCAAAGATG-3 ) and N152Rev (5 -GCACCTTCGGTTTGATCTTC-3 ) (Supp. materials, 2), Phire PCR Buffer and 0.4 ␮l of Phire HSII polymerase (ThermoFisher scientific, Waltham, USA) with the thermal cycling: 98 ◦ C 5 min, 40 cycles at 98 ◦ C for 1 s, 64 ◦ C for 5 s and 72 ◦ C for 20 s. The shoot regeneration was subsequently induced from the calli on the shoot induction Medium H (Supp. materials, 5a). The gDNA was extracted from 20 randomly chosen in vitro plantlets and sequenced as above. 3. Results and discussion 3.1. The pipeline for tetraploid potato targeted gene mutation The pipeline is essentially composed of three steps (Fig. 1): (a) the putative target site of the target gene is explored either using sequences available in literature or de novo generated by gDNA sequencing or RNA-seq experiments of the target cultivar.

2.4. Regeneration of p35S-ALS-TALEN transfected protoplasts Ten different transfections were carried out with the p35S-ALSTALEN vector and the transfected protoplasts were incubated in the regeneration Medium E and placed under conditions allowing cell division and regeneration for 5–6 weeks (Supp. materials, 5a). Total genomic DNA (gDNA) was extracted as described by Edwards et al. (1991) from 15 and 18 calli randomly chosen among 2 transfections. One ␮l gDNA was used as template in a PCR reaction containing 0.5 mM of the primer ALSFor and ALSRev, HF Buffer, 0.2 mM dNTPs, 2% DMSO and 2 U of Phusion HF polymerase (ThermoFisher scientific, Waltham, USA) with the thermal cycling: 98 ◦ C for 30 s, 35 cycles at 98 ◦ C for 10 s, 67 ◦ C for 20 s and 72 ◦ C for 10 s. The PCR product was cloned into the pJET1.2 as described above and 32 E. coli colonies per callus were sequenced (GATC, Constance, Germany) with the primer ALSRev. The sequences were aligned with CLC Main Workbench 7 (CLCbio, Germantown, USA). The first 314 bp out of 458 bp of each amplicon were used to group the sequences obtained from each callus in the cluster structure previously generated. For each cluster, the ratio between the number of sequences with ALS-TALEN induced mutation and the size of the cluster was calculated and used as estimation of the targeting efficiency in calli (low quality sequences were discarded from calculation). In order to detect the possible stable integration of the vector p35S-ALS-TALEN, 1 ␮l of the DNA from each callus was used as template in the PCR reaction with 0.5 mM of

Fig. 1. Schematic representation of the pipeline proposed in this work for targeted gene mutation in tetraploid potato through TALEN transient expression in protoplasts. (a) The putative target site (in red) is explored using sequences available in the literature or de novo generated by gDNA sequencing/RNASeq experiments. The left and right TALEN RVD arrays (in green) are designed in order to allow dimerization of the two FokI domains (in yellow) and to introduce a DSB at the desired site; (b) protoplasts are extracted, under sterile conditions, from leaves of in vitro grown shoots and transfected through PEG-mediated transfection with a TALEN expression vector (in red); (c) following transient expression of TALEN in single cells, protoplasts-derived calli and shoots are regenerated and sequenced to identify those carrying mutations at the expected site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Genomic and transcriptomic analyses of the target region containing the ALS-TALEN target site (highlighted in red). (a) Structure and similarity of the two ALS genes (Ch7, Ch10) in the sequenced genotype (Phureja, clone DM1-3), the 458 bp region is delimitated by the primers ALSFor and ALSRev (left and right black arrows); (b) average number of RNA-seq reads of the tetraploid genotype cv. Desirée, mapped on the 458 bp region. SNPs are evidenced by inverted triangles and their relative positions are shown with the color codes as follows (triangles or asterisks): the SNPs detected in the Phureja clone DM1-3 ALS genes and sequence alignment are marked in orange, Desirée RNA-seq mapping in pink and the SNPs obtained from the analysis of the clustered Desirée gDNA sequences (first 314 bp) in black. The left and right arrays binding sites of the ALS-TALEN are indicated by horizontal green bars; (c) alignment of sequences with ALS-TALEN induced mutations obtained by gDNA sequencing of the target region in transfected protoplasts; mutations are marked in red as compared to the wild type sequence (Wt) (Fs: frame shift, s: substitution). Nucleotides corresponding to the left and right array binding sites of ALS-TALEN are marked in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The TALEN is subsequently synthesized and cloned into a suitable expression vector; (b) protoplasts are extracted from in vitro grown shoots and transfected with the TALEN expression vector in order to transiently express the nuclease in single cells; (c) protoplastderived calli and shoots are regenerated and sequenced to identify mutations at the expected target site. 3.2. Protoplast isolation and transfection The use of protoplasts isolated from leaves of various species has been reported as a versatile cell-based experimental system to transiently manipulate signal transduction, metabolic pathways, transcription and translation machinery (Yoo et al., 2007). While this type of transient assay has been adapted to several species, such as Arabidopsis (Yoo et al., 2007), maize (Sheen, 2001), tobacco (Fischer and Hain, 1995), tomato (Nguyen et al., 2010) and rice (Zhang et al., 2011), there are few reports on transient assays with potato (Jones et al., 1989), although protoplast extraction

protocols are reported in literature (Anjum, 1998 and references therein; Sharma et al., 2011 and references therein). Because of the lack of a published protocol for transient gene expression in potato protoplasts we first developed such a protocol based on the published works (Craig et al., 2005; Leucci et al., 2007; Yoo et al., 2007). For this purpose, two vectors harboring the reporter gene GFP were used (pGFP1, pGFP2; Supp. materials, 3). We first successfully isolated protoplasts of high viability (>98% at FDA test, not shown) from leaves of in vitro grown plants of Desirée. The protoplast yield ranged from 7.6 × 105 to 1.9 × 106 protoplasts per gram fresh weight of leaves, which is in accordance with previous reports (Foulger and Jones, 1986; Shepard and Totren, 1977; Tavazza and Ancora, 1986). We then transfected protoplasts with either the vector pGFP1 or pGFP2 and the transfected protoplasts showed clear GFP accumulation after 24 h under blue light compared to non-transfected controls (Supp. materials, 6), indicating their capability to sustain transient expression of the transgene. We observed similar transfection efficiency for both vectors

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(pGFP1: mean 39%, s.d. = 9.16, n = 3; pGFP2: mean 38%, s.d. = 7.69, n = 3), even if they differ in size (Supp. materials, 3). We also tested the different plasmid concentrations and found that 10 ␮g plasmid was the optimum concentration. Interestingly, we identified two critical parameters under our experimental conditions that differed from those reported in the previous studies (Sheen, 2001; Yoo et al., 2007; Zhang et al., 2011). The use of a lower concentration of PEG and a shorter incubation time retained the potato protoplast vitality with high reproducibility (Craig et al., 2005). We believe that the successful establishment of the potato transient expression system for targeted gene mutation in this study provides a technical platform for future research and mutation breeding in potato. 3.3. TALEN validation in potato protoplasts In order to design TALEN vectors for SDM in the tetraploid potato cv. Desirée, RNA-seq reads from this cultivar obtained in a former study (Ali et al., 2014) were mapped to the two ALS genomic DNA sequences of the sequenced genotype DM and a conserved region for primer design was identified (Fig. 2a and b). An ALSTALEN was synthetized to target both ALS genes in tetraploid potato Desirée with 8 potential targets per cell. Single nucleotide polymorphisms (SNPs) due to differences between the two ALS genes in DM, between Desirée and DM and differences among Desirée alleles are shown in Fig. 2b. In order to test the capability of our synthetized ALS-TALEN to induce DSB and mutations by NHEJ at the target site, the newly developed transient protoplast expression system was employed. The gDNA from the transfected protoplasts was isolated for sequencing 24 h after transfection. Sequence analysis of the target site showed mutations only in the protoplasts transfected with the vector p35S-ALS-TALEN (PEG+/pDNA+) (Fig. 2c), but not in the control transfection without the vector (PEG+/pDNA−). The predominant mutation type was deletions, ranging from 1 bp to 13 bp, while no insertions were detected. Notably, some single base substitutions were also present within the target region (Fig. 2c). Based on analysis of the mutated sequences, amino acid substitutions, frame shifts and protein truncations are expected (Fig. 2c). We obtained a mutation frequency of 8.2% (15 out of 182 sequences) and 6.9% (13 out of 189 sequences) with ALS-TALEN in two independent protoplast transfection experiments. Previous reports using TALEN in other plant species with a protoplasts based assay showed similar frequencies of mutation (Liang et al., 2013; Shan et al., 2013), or higher frequencies in some cases (Wang et al., 2014; Zhang et al., 2013). However, in these studies, the mutation frequency was estimated either through sequencing PCR products amplified from gDNA pre-digested with a restriction endonuclease (RE) cutting within the target region (Shan et al., 2013), or analyzing the intensity of the RE resistant gel bands obtained by the digestion of the PCR product amplified from the target region (Liang et al., 2013; Wang et al., 2014; Zhang et al., 2013). Our results are obtained through sequencing and therefore the mutation rates are not directly comparable to the abovementioned ones.

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Given the richness in SNPs of the first 314 bp out of the 458 bp region generated by protoplast DNA sequencing (Fig. 2b), we used this region as a tag to obtain information about target diversity within the two ALS genes in Desirée and to estimate the targeting efficiency. A total of 160 out of 171 sequences (92%) from the first protoplast transfection experiment and 170 out of 191 sequences (87%) from the second experiment grouped in 5 different clusters, corresponding to different alleles in ch7 and ch10 (Table 1; Supp. materials, 4). Three new SNPs (87, 120, 125) were identified compared to those evidenced from the previous RNA-seq mapping (Fig. 2b; Table 1), indicating that these sequences were likely amplified from non-expressed alleles of one or both of the two ALS genes in Desirée. From the two protoplast transfections with the vector p35SALS-TALEN we obtained sequences with ALS-TALEN-induced mutations in clusters that aligned with each of the 5 reference sequences; however the highest mutation frequency was observed in cluster B (18.1%, Table 1). Our results indicated that ALS-TALEN was able to induce mutations in both genes and in different alleles, although some were targeted more often than others, which might be due to the fact that the TALEN architecture has been reported to be sensitive to the chromatin structure and methylation state of the target region (Reyon et al., 2012; Valton et al., 2012). 3.4. Regeneration of p35S-ALS-TALEN transfected protoplasts In order to explore the feasibility of obtaining potato plants carrying mutations at the expected DNA site, we induced callus regeneration from the transfected protoplasts with the vector p35S-ALS-TALEN. After 6 weeks, gDNA was extracted from the regenerated calli. Sequence analysis of the target region showed that in two independent transfections we found 2 out of 15 (13%) and 2 out of 18 (11%) calli, carrying mutations at the expected target site; these frequencies were lower compared to previously reported figures in tobacco by Zhang et al. (2013) (32%). Further sequence analysis showed that the 4 mutant calli carried mainly deletions, but also base substitutions, and in one case a 1 bp insertion. Interestingly, in one callus there was an almost complete deletion of the TALEN region except the right array binding site (Fig. 3a). The analysis of mutated sequences confirmed the presence of amino acid substitutions, frame shifts and protein truncations, as previously evidenced in protoplasts (Fig. 3a). Notably, the observed diversity of mutations in single calli, is likely associated with the ploidy level of potato; however, TALEN is known to cause various type of mutations also in homozygous diploid plants of other species (Wendt et al., 2013; Zhou et al., 2014). In order to estimate the targeting efficiency in calli, we successfully grouped all the gDNA sequences obtained from each callus in the cluster structure previously generated (Table 2). The results showed that in all the analyzed calli only a limited number of clusters contained sequences with ALS-TALEN induced mutations and

Table 1 Clusters characterization and estimation of ALS-TALEN targeting efficiency, based on the alignment of the gDNA sequences obtained from the transiently transfected protoplasts. Cluster

A B C D E a b c

SNPsc 78

87

120

125

144

180

201

204

234

306

G A G A A

G A G A A

A G G G G

G G A G G

T T T C T

A T A T T

A G A A A

G G G G A

C T C T T

G G G G A

Cluster sizea

ALS-TALEN targeting eff.b (%)

53 32 19 16 20

8.5 18.8 7.9 6.3 5.0

Number of sequences populating each cluster (mean, n = 2). Percentage of the sequences with ALS-TALEN induced mutations (mean, n = 2) for each cluster. SNPs are numbered according to their relative position in the 458 bp gDNA region delimitated by the primers ALSFor and ALSRev.

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Fig. 3. Alignment of sequences with ALS-TALEN induced mutations (in red) obtained by gDNA sequencing of the target region in calli (a) and plantlets (b) regenerated from the transfected protoplasts (compared to the wild type sequence, Wt) (Fs: frame shift, s: substitution). Nucleotides corresponding to the left and right array binding sites of ALS-TALEN are marked in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2 Estimation of ALS-TALEN targeting efficiency in clusters generated by alignment of gDNA sequences obtained from regenerated calli. Callus

1 8 R2 R4

Clustera A

B

C

D

E

18.8 (3/16) 0.0 (0/15) 0.0 (0/8) 7.1 (1/14)

16.7 (1/6) 0.0 (0/9) 0.0 (0/7) 0.0 (0/8)

0.0 (0/5) 0.0 (0/2) 0.0 (0/2) 0.0 (0/1)

0.0 (0/6) 0.0 (0/0) 100.0 (3/3) 0.0 (0/2)

0.0 (0) 25.0 (1/4) 0.0 (0/5) 0.0 (0/4)

a For each cluster the targeting efficiency is expressed as % of the ratio in brackets (number of sequences with ALS-TALEN induced mutations/cluster size).

only in one callus the efficiency was 100%, for a specific cluster (Table 2). All the 4 mutant calli were PCR-negative (not shown) using a specific primer pair amplifying the vector p35S-ALS-TALEN, indicating, along with the high capacity of shoot induction from the calli obtained under our experimental conditions (about 100% shoot regeneration, unpublished results), the possibility of generating potato mutant plants deprived of the vector sequences. In order to evaluate the mutation efficiency of regenerated shoots from the transfected calli, we regenerated shoots and plantlets from the calli in vitro. We found 2 out of 20 (10%) plantlets carrying mutations at the expected target site, a frequency that is in agreement with our previous observation in calli. Further sequence analysis revealed 1 bp insertion and 1 bp deletion in the two plantlets, resulting in a protein truncation and a frame shift, respectively (Fig. 3b). Mutated regenerated plantlets were not phenotypically different from non-mutated ones (Fig. 4). Notably, in all the analyzed calli and plantlets, no full knock out mutants were detected, which might be explained by the fact that a functional ALS gene is essential for the plant metabolism (Tan et al., 2005), as confirmed also by our preliminary experiments on protoplasts regeneration in the presence of the ALS-inhibitor Imazamox (unpublished results). Therefore, the full knock-out events might have been probably lost, while consequently only protoplasts with

Fig. 4. Plantlets regenerated from transfected protoplasts not showing (Plantlet 12) and showing (Plantlet 20) ALS-TALEN induced mutations.

a limited number of targeted alleles (1 or 2) underwent regeneration, giving rise to calli and shoots. In conclusion, we have developed a pipeline for tetraploid potato genome editing based on transient expression of TALEN in protoplasts, but any other SDNs (e.g. CRISPR-Cas), can potentially be used. We have demonstrated the regeneration of both mutant calli and plantlets carrying mutations at the expected target site from the transfected protoplasts. We are convinced that, with the achieved mutation frequency and the diversity of target alleles employed under our experimental conditions, this method could be used in future research and breeding of potato and other related species without any use of stable transformation. Acknowledgements The authors thank the Mistra-Biotech program supported by the Swedish Foundation for Strategic Environmental Research

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(Mistra), the Swedish Foundation for Strategic Research (SSF) and the Swedish University of Agricultural Sciences (SLU) for the financial support and Ann-Sofie Fält and Pia Ohlsson for technical assistance. PlantLink is thanked for bioinformatical support. We also acknowledge Teodoro Cardi, Sergio Ochatt, Gabriella Piro, Debabrata Sarkar and Jochen Kumlehn for advice on potato protoplasts procedures, and Per Hofvander for discussions about genome editing and for providing the vector pCW498-35S-GFiP-OcsT and pK2GW7 0. The authors are grateful to Daniel Voytas for providing the vectors pZHY500 and pZHY013. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2015.03.021. References Ahloowalia, B.S., Maluszynski, M., Nichterlein, K., 2004. Global impact of mutation-derived varieties. Euphytica 135, 187–204, http://dx.doi.org/10.1023/ B:EUPH.0000014914.85465.4f. Ali, A., Alexandersson, E., Sandin, M., Resjö, S., Lenman, M., Hedley, P., Levander, F., Andreasson, E., 2014. Quantitative proteomics and transcriptomics of potato in response to Phytophthora infestans in compatible and incompatible interactions. BMC Genom. 15, 497, http://dx.doi.org/10.1186/1471-2164-15-497. Anjum, M., 1998. Effect of protoplast source and media on growth and regenerability of protoplast-derived calluses of Solarium tuberosum L. Acta Physiol. Plant. 20, 129–133, http://dx.doi.org/10.1007/s11738-998-0003-7. Barrell, P.J., Meiyalaghan, S., Jacobs, J.M.E., Conner, A.J., 2013. Applications of biotechnology and genomics in potato improvement. Plant Biotechnol. J. 11, 907–920, http://dx.doi.org/10.1111/pbi.12099. Brooks, C., Nekrasov, V., Lippman, Z., Van Eck, J., 2014. Efficient gene editing in tomato in the first generation using the CRISPR/Cas9 system. Plant Physiol., http://dx.doi.org/10.1104/pp.114.247577. Cardi, T., Puite, K., Ramulu, K., 1990. Plant regeneration from mesophyll protoplasts of Solanum commersoni. Plant Sci. 70, 215–221, http://dx.doi.org/ 10.1016/0168-9452(90)90136-C. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V., Bogdanove, A.J., Voytas, D.F., 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82, http://dx.doi.org/10.1093/nar/gkr218. Chen, K., Gao, C., 2013. Targeted genome modification technologies and their applications in crop improvements. Plant Cell Rep., http://dx.doi.org/ 10.1007/s00299-013-1539-6. Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., Sheen, J., 1996. Engineered GFP as a vital reporter in plants. Curr. Biol. 6, 325–330, http://dx.doi.org/ 10.1016/S0960-9822(02)00483-9. Christian, M., Cermak, T., Doyle, E.L., Schmidt, C., Zhang, F., Hummel, A., Bogdanove, A.J., Voytas, D.F., 2010. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761, http://dx.doi.org/10.1534/ genetics.110.120717. Craig, W., Gargano, D., Scotti, N., Nguyen, T.T., Lao, N.T., Kavanagh, T.A., Dix, P.J., Cardi, T., 2005. Direct gene transfer in potato: a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts. Plant Cell Rep. 24, 603–611, http://dx.doi.org/10.1007/ s00299-005-0018-0. Curtin, S.J., Voytas, D.F., Stupar, R.M., 2012. Genome engineering of crops with designer nucleases. Plant Genome J. 5, 42, http://dx.doi.org/ 10.3835/plantgenome2012.06.0008. Curtin, S.J., Zhang, F., Sander, J.D., Haun, W.J., Starker, C., Baltes, N.J., Reyon, D., Dahlborg, E.J., Goodwin, M.J., Coffman, A.P., Dobbs, D., Joung, J.K., Voytas, D.F., Stupar, R.M., 2011. Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol. 156, 466–473, http://dx.doi.org/10.1104/pp.111.172981. Edwards, K., Johnstone, C., Thompson, C., 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19, 1349, http://dx.doi.org/10.1093/nar/19.6.1349. Endo, M., Osakabe, K., Ichikawa, H., Toki, S., 2006. Molecular characterization of true and ectopic gene targeting events at the acetolactate synthase gene in Arabidopsis. Plant Cell Physiol. 47, 372–379, http://dx.doi.org/10.1093/pcp/pcj003. Fichtner, F., Urrea Castellanos, R., Ülker, B., 2014. Precision genetic modifications: a new era in molecular biology and crop improvement. Planta, http://dx.doi.org/10.1007/s00425-014-2029-y. Fischer, R., Hain, R., 1995. Chapter 29: tobacco protoplast transformation and use for functional analysis of newly isolated genes and gene constructs. Methods Cell Biol. 50, 401–410, http://dx.doi.org/10.1016/S0091-679X(08)61046-8. Foulger, D., Jones, M.G.K., 1986. Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars. Plant Cell Rep. 5, 72–76, http://dx.doi.org/10.1007/BF00269723.

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Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts.

Potato is the third largest food crop in the world, however, the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depre...
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