Hygromycin-resistant calli generated by activation ...

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Hygromycin-resistant calli generated by activation and excision of maize Ac/Ds transposable elements in diploid and hexaploid wheat cultured cell lines Shigeo Takumi

Abstract: To investigate the activation and transposition of maize transposable elements in wheat cultured cells, plasmid DNAs containing the maize Ac/Ds elements located between the CaMV 35s promoter and a hygromycin B resistance gene (hph) were introduced into two wheat (Triticum aestivum and Triticum monococcum) cultured cell lines by microprojectile bombardment. In the first experiment, hph was activated by excision of the Ac element, which encodes transposase, in the two wheat cell lines. In the second experiment, the Ds element was excised by a stabilized Ac element, lacking inverted repeats of the Ac element and located on another plasmid, and therefore leading to activation of hph. After selection of bombarded cells by hygromycin B, many resistant calli were recovered in both wheat cell lines. The integration of hph and the Ac transposase gene was confirmed by PCR and genomic Southern analysis. The stable expression of hph and the transposase gene was also assessed by Northern blot and reverse transcriptase PCR analysis, respectively. Moreover, characteristic sequence alterations were found at Ac/Ds excision sites. These findings indicate that the maize Ac/Ds transposable elements are activated and excised by expression of the Ac transposase gene in both diploid and hexaploid wheat cells. Key words: transposable elements, excision site, transgenic wheat callus, particle bombardment, Ac/Ds. RCsumC : Afin d'itudier l'activation et la transposition des kliments transposables du mays Ac/Ds chez des cultures cellulaires du blk, des ADNs plasmidiques contenant les ilkments Ac/Ds insiris entre le promoteur 35s du CaMV et le gene hph (confkrant la risistance a l'hygromycine) ont it6 introduits par bombardement dans deux cultures cellulaires du bli (Triticum aestivum et Triticum monococcum). Dans une premiere expirience, le gene hph a it6 activi chez les deux cultures cellulaires par suite de l'excision de l'iliment Ac, lequel code la transposase. Dans une seconde expirience, l'iliment Ds a it6 excisi sous l'action d'un iliment Ac stabilisk (dkpourvu de siquences terminales ripities inversies et situi sur un autre plasmide) ce qui a entrain6 l'activation du gene hph. Apres silection sur hygromycine B des cellules bombardies, plusieurs cals risistants ont it6 obtenus chez les deux cultures cellulaires. L'intkgration dans le ginome du gene hph et du gene codant la transposase de l'iliment Ac a it6 confirmie par analyse PCR et par hybridation Southern. L'expression stable des genes codant la hph et la transposase a igalement it6 itudiie par l'analyse northern et par RT-PCR, respectivement. De plus, des altkrations nuclkotidiques typiques ont it6 observies aux sites d'excision des iliments Ac/Ds. Ces risultats indiquent que les ilkments transposables du mai's Ac et Ds sont activks et excisis par suite de l'expression du gene de la transposase de Ac chez des cellules de bli diploi'de et hexaploi'de. Mots cle's : klkments transposables, site d'excision, cals de bli transginique, bombardement, Ac/Ds. [Tradui t par la Rkdaction]

Introduction Transposon mutagenesi~is a powerful technique for isolating genes that encode unidentified products in a variety of the use maize and Antirrhinurn transposable ekments has been widely studied in heterologous host plant species lacking well-characterized endogenous elements (Haring et al. 1991). The maize Activator

(Ac) and Dissociation (Ds) elements, first identified and studied genetically by Barbara McClintock (reviewed in Fedoroff 1989), are among the best studied elements in heterologous host plants. These elements have been demonstrated to transpose in various dicotyledonous plants, such as tobacco (Baker et al. 1986), Arabidopsis (Van s l u y s et a1. 19 87), and tomato (Yoder e t a1. 1988). Successful transPoson tagging of the petunia flower color gene, PH6, with the Ac element and of the DRLl locus involved in root and leaf development in Arabidopsis with the twoelement Ac/Ds system has been reported by Chuck et al. (1993) and Bancroft et al. (1993), respectively. In monocotyledonous species, including cereals, the Ac and (or) Ds elements have been introduced into rice protoplasts by

I Corresponding Editor: G. Bellemare. Received February 13, 1996. Accepted September 10, 1996.

S. Takumi. Laboratory of Genetic Resources, Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi-machi, Ishikawa 921, Japan.

Genome, 39: 1169-1 175 (1996). Printed in Canada 1 Imprim6 au Canada

Genome, Vol. 39, 1996

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electroporation and their transposition in the rice genome has been demonstrated (Izawa e t al. 199 1 ; Murai e t al. 199 1 ; Shimamoto et al. 1993). The phenotypic assay developed by Baker et al. (1987) relies on the restored expression of a marker gene after excision of a transposable element. Excision of the Ac/Ds elements restores hygromycin resistance by bringing the 3 5 s promoter proximal to a hygromycin B resistance gene (hph). In rice, Izawa et al. (1991) showed that the phenotypic assay using hph a s a selectable marker gene was efficient in detecting Ac transposition, and Shimamoto et al. (1993) successfully used this assay to monitor the transactivation of a D s element. When the Ac/Ds elements are excised, part or all of the 8-bp target duplication remains, and sequence alteration at the excision site is one of the most important characteristics of plant transposable elements. T h e most c o m m o n features of such a site are a deletion of several central nucleotides and the transversion of one or two central nucleotides (Fedoroff 1989). In rice, Izawa et al. (199 l ) , Murai et al. ( I 9 9 l ) , Shimamoto et al. (1993), and Sugimoto e t al. (1994) found the sequence alterations at the excision site of the Ac/Ds elements, as observed in the heterologous systems of dicotyledonous plants (Fedoroff 1989). In wheat species, in which transgenic plants cannot be obtained through electroporated protoplasts, few studies have been reported with regard to transposition of the heterologous transposable elements. In protoplasts of Triticum monococcum, Laufs et al. (1990) transfected the maize Ac/Ds elements by using a wheat dwarf virus (WDV) vector and observed their transposition with the help of viral replication. They also analyzed the excision sites in the W D V vector and observed deletions and transversions. Instead of transfection to protoplasts, the particle bombardment system is widely used as an essential technique for genetic modification in common wheat (Vasil et al. 1992; Weeks e t al. 1993). I have previously demonstrated the production of transgenic wheat calli through particle bombardment (Takumi and Shimada 1995). In this study, I introduced maize Ac/Ds transposable elements into intact wheat cultured cells by particle bombardment. Using the phenotypic assay of Baker et al. (1987) for excision of the Ac/Ds elements from the hygromycin-resistance gene, many transgenic wheat calli were isolated. This is the first demonstration that the Ac/Ds elements can be activated and excised in common wheat cells.

Materials and methods

Plasmids

The plasmids pBI222, pCKR262, pCKR234, and pCKR532 were used. pBI222 includes a hygromycin phosphotransferase gene (hph) that confers hygromycin resistance under the control of the cauliflower mosaic virus (CaMV) 35s promoter. pCKR262 (Fig. 1A) includes the maize Ac autonomous element inserted between the 3 5 s promoter and hph (Izawa et al. 1991). pCKR234 (Fig. 1A) contains the Ds nonautonomous element, which lacks the 1.6-kb HindIII fragment of the Ac element, inserted between the 35s promoter and hph (Shimamoto et al. 1993). pCKR532 (Fig. 1B) contains the Ac transposase coding region under control of the 3 5 s promoter (Shimamoto et al. 1993). This Ac transposase gene lacks the inverted repeats of the

Fig. 1. Structure of the plasmids used in this study. (A) The structure of pCKR234 containing the Ds element and of pCKR262 containing the Ac element derived from maize waxy-m7 allele. The two arrows show the primers for PCR analysis for detecting excision of the Ac/Ds elements. (9) Structure of pCKR532. pCKR532 contains the Ac transposase gene lacking the inverted repeats of the Ac element. The solid bar and arrows show five exons of the ORF in the Ac element and the primers for PCR analysis, respectively. Restriction sites: B, BamHI; H, HindIII; E, EcoRI; P, PstI; and Sal, SalI.

I

I

I1

I11 IV

H

**

ORF Primer A+B Primer C+D

Ac element and cannot transpose by itself. These Ac/Ds transposable elements were originally derived from the entire Ac element of the maize waxy-m7 allele (Klosgen et al. 1986). These plasmids were amplified in liquid cultures of Escherichia coli, isolated by alkaline lysis, and purified twice by CsCl ethidium bromide density centrifugation (Maniatis et al. 1982).

Plant materials, particle bombardment, and selection of transformants Two wheat cultured cell lines were used in this study. One is Mono-3 from einkorn wheat (T. monococcum) and the other is HY- 1 from Japanese common wheat (Triticum aestivum cv. Haruyutaka). These calli were induced from immature embryos and grew vigorously on Linsmaier-Skoog (LS) medium (Linsmaier and Skoog 1965) containing 30 g - ~ psucrose, ' 2 mg.L-' 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.25% (w/v) Gelrite. These two cell lines were subcultured into fresh medium every 3 weeks. Approximately 1 mL (fresh packed cell volume) callus was spread onto a petri dish containing solid LS medium supplemented with 2 mg.L-' 2,4-D. After 10 days of incubation, these calli were bombarded. The conditions of particle borrtbardment have been described previously (Takumi et al. 1994). After bombardment, the calli were transferred to a selection medium composed of LS medium with 2 mg.LP' 2,4-D and 50 mg-Lp' hygromycin B. After selection for about 1 month, the hygromycin-resistant calli were recovered.

Analysis of transgenic calli Genomic DNAs isolated from both wheat calli were used in the DNA analysis as described previously (Takumi and Shimada 1995). The primers 5'-CCACTGACGTAAGGGAT-3' and 5'-ACCAATGCGGAGCATATACG-3' were designed and

Takumi

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Fig. 2. Phenotypic assay by production of hygromycin-resistant calli through particle bombardment in wheat cultured cell line HY-1. (a) Nontransformant as a negative control. When only pCKR234 was introduced, no hygromycin-resistant calli appeared. (b) Hygromycin-resistant calli generated after pBI222 was introduced. (c) Hygromycinresistant calli generated after pCKR262 was introduced. ( d ) Hygromycin-resistant calli generated after pCKR234 was cotransformed with pCKR532.

synthesized based on the sequences of the 35s promoter and hph for detection of hph integration and Ac/Ds excision (Fig. 1A). These primers were expected to produce a 1-kb product after PCR amplification of pCKR262 and pCKR234, following excision of the Ac/Ds elements. Two sets of primers for detecting the integration and expression of the Ac transposase gene were designed and synthesized based on the sequence of a portion of the Ac transposase coding region that is deleted in the Ds element of pCKR234 (Fig. I ) . One set is 5'-GCGATTGTTCTTGCTGTA-3' and 5'-GCGAGGATCTGTTTT-3' and the other set is 5'-ATTTGATGTTGAGGGATGC-3' and 5'-TTTGGAGCTGAAGGACTAC-3'. These primer sets were expected to produce a 177-bp product within the second exon of the Ac transposase gene and a 542-bp product containing an intron between the second and third exons after PCR amplification, respectively. Fifty nanograms of template DNA and 10 nM of each primer were mixed with 2 p L of 10X Taq DNA polymerase buffer, 100 p M (final concentration) of a dNTP mixture (equimolar dATP, dCTP, dGTP, and dTTP), and 1 U Taq DNA polymerase (Pharmacia) in a total volume of 20 pL. Thirty cycles of PCR were performed in a programmed temperature control system (PC-700, Astec). A single cycle consisted of the following steps: denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and DNA synthesis at 72°C for 1 min.

Amplified DNAs were analyzed by ethidium bromide staining after 2% agarose gel electrophoresis at 50 V. PCR products were transferred to nylon membrane (Hybond N', Amersham) after electrophoresis. The identity of the major fragments was established by Southern hybridization using as a probe the cDNA of hph and the HindIII fragment of the Ac open reading frame (ORF) with the ECL (enhanced chemiluminescence) system (Amersham). Moreover, total DNAs were digested by either EcoRI or HindIII for genomic Southern analysis. The methods of electrophoresis, Southern blotting, hybridization probes, and autoradiography were the same as with 32~-labelled those reported by Liu et al. (1990). Total RNA was isolated from wheat calli by using ISOGEN (Nippongene, Japan). The expression of hph and the Ac transposase gene was determined by Northern blot analysis (Maniatis et al. 1982) with a labelled probe consisting of the hph coding region and by reverse transcriptase (RT) PCR (First Strand cDNA Synthesis Kit, Amersham, U.K.), respectively. Determination of sequences produced by excision of the Ac/Ds elements was analyzed as previously described by Izawa et al. 199 1. PCR products containing the excision site were cloned to pUC 119 and sequenced by dyedeoxy terminator cycle sequencing using an ABI Model 373 DNA Sequencer (Applied Biosystems, U.S.A.).

Genome, Vol. 39, 1996

Table 1. Summary of transformation experiments.

HY- 1

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Plasmid Experiment 1 pCKR262 pBI222 Nontransformant Experiment 2 pCKR234 + pCKR532 pCKR 234 pBI222 Nontransformant

No. of plates bombarded

Mono-3 No. of resistant calli

No. of plates bombarded

No. of resistant calli

7 9 2

23 6 12 2

Fig. 3. Molecular analyses of hph in hygromycin-resistant calli. (A) PCR amplification of hph from total DNA isolated from hygromycin-resistant calli. The ethidium bromide staining pattern after agarose gel electrophoresis. (B) Results of Southern hybridization with hph as a probe after transfer of the DNA shown on the agarose gel of A. (C) Northern blot analysis of the hph transcripts in total RNA isolated from hygromycin-resistant calli. Each lane was loaded with 1 0 pg of total RNA. Lane: P, pBI222 as a positive control; N l and N2, pCKR262 and pCKR234, respectively, as negative controls; 1, nontransformed callus; 2, hygromycin-resistant callus produced by introducing pBI222; 3, hygromycinresistant callus produced by introducing pCKR262; 4 and 5, hygromycin-resistant calli produced by introducing both pCKR234 and pCKR532.

Results Transformed wheat cultured cell lines To monitor Ac/Ds transposition in wheat by phenotypic assay (Baker et al. 1987), I introduced the plasmids used in rice (Izawa et al. 1991; Shimamoto et al. 1993) into two cultured cell lines of wheat, 7: monococcum and 7: aestivum, through particle bombardment, and hygromycin-resistant calli were recovered. In the two cultured cell lines, Mono-3 and HY- 1, no hygromycin-resistant calli were recovered when no plasmid was introduced ( a negative control; Fig. 2a), but many hygromycin-resistant calli were recovered when pBI222 was introduced (a positive control; Fig. 26). In the first experiment, pCKR262 was introduced into Mono-3 and HY-1 by particle bombardment, and hygromycin-resistant calli appeared in calli of both wheat lines (Fig. 2c). In the second experiment, pCKR234 was

introduced into both Mono-3 and HY-1. No hygromycinresistant calli were recovered when pCKR234 was introduced alone, but many hygromycin-resistant calli were generated in both wheat cultured cell lines when pCKR234 was cotransformed with pCKR532 (Fig. 2d). These findings indicated that excision of the Ac/Ds elements occurred only in the presence of the Ac transposase gene. Table 1 shows a summary of the transformation experiments. Integration of hph into the wheat genome and excision of the Ac/Ds elements were confirmed by both PCR amplification analysis and genomic Southern hybridization analysis in the hygromycin-resistant calli of both Mono-3 and HY-1. PCR amplification of total DNAs from all hygromycinresistant calli yielded the expected fragment (Fig. 3A), and the identity of the observed fragment was determined by Southern hybridization with labelled hph cDNA (Fig. 3B). The PCR fragment obtained after excision of

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Takumi

the Ac/Ds elements was slightly longer than that obtained with pB1222 in both Mono-3 and HY-1. This is due to the presence of 60 bp of waxy sequence flanking the Ac/Ds elements. Moreover, total DNA digested with HindIII from hygromycin-resistant callus was used for genomic Southern analysis with 32~-labelledhph cDNA. The integration of hph was also confirmed by Southern blot pattern (Fig. 4A). To confirm the expression of hph in transgenic wheat calli, total RNAs were isolated from the calli of both Mono-3 and HY-I and analyzed by Northern hybridization with the labelled probe of the hph coding region. All transgenic calli showed hph expression (Fig. 3C). This indicated that hph, having become adjacent to the 35s promoter following excision of the Ac/Ds elements, was precisely transcribed. Integration and expression of Ac transposase The integration and expression of the Ac transposase gene, which is essential for excising the Ac/Ds elements, were detected by genomic Southern analysis and PCR analysis. First, PCR fragments located within the HindIII fragment of the Ac ORF were amplified by using total DNAs isolated from the hygromycin-resistant calli of both Mono-3 and HY-1. In most transgenic calli, the expected bands were amplified and the integration of the Ac transposase gene was confirmed by the amplification of two different sequences (Figs. 5A and 5B). No bands were seen in transgenic calli in which pB1222 was introduced as a positive control. In some of the transgenic calli, the expected bands were not seen. Total DNAs from transgenic wheat calli were digested with EcoRI and used for genomic Southern analysis with the 32 P-labelled second exon of the Ac element. The integration of the Ac/Ds elements was also confirmed by Southern blot (Fig. 4B). Second, the expression of the integrated Ac transposase gene was analyzed by RT-PCR using total RNAs isolated from transgenic calli of both wheats. Two sets of primers were designed and synthesized according to sequences in the HindIII fragment of the Ac element (Fig. 1B). In one primer set, both primers are located in the second exon of the Ac transposase gene. In the other set, the 5' primer is located in the second exon, while the 3' primer is located in the third exon. Therefore, the RT-PCR fragments generated by the former set of primers were of the expected length (Fig. 5C), but the fragments generated by the latter set of primers were 72-bp shorter (Kunze et al. 1987) than those of the positive control, for which the template is pCKR532 (Fig. 5D). These findings indicated that the Ac transposase gene was expressed and that the mRNA was exactly processed in both wheat cells. Analysis of the Ac/Ds excision sites To characterize the sequence alteration at the excision site in transgenic wheat calli, the DNA fragments containing the Ac/Ds excision sites were cloned and sequenced. From three independently recovered hygromycin-resistant calli, three different excision sequences were determined (Table 2). These excision sequences contained deletions of three or five nucleotides that occurred at the junction of the two direct 8-bp repeats. These findings indicated that the excision of Ac/Ds in wheat cells depended on the function of maize Ac transposase.

Fig. 4. Autoradiograms of Southern blots of total DNAs from transgenic wheat calli. Each lane contains 20 kg of total DNA. The enzyme-probe combinations used are HindIII with hph cDNA (A) and EcoRI with the second exon of the A c element (B). Lane: N, nontransformed callus as a negative control; 1, transgenic callus produced by introducing pBI222; 2, transgenic callus after excision of the Ac element of pCKR262; 3, transgenic callus generated after pCKR234 was cotransformed with pCKR532.

Discussion Recently a wheat genetic transformation system using the particle gun apparatus has been established (Vasil et al. 1992; Weeks et al. 1993). In this study, the maize transposable element was introduced into wheat cells by particle bombardment. First, transgenic wheat calli were derived from bombarded suspension cultures, as reported by Vasil et al. (1991). Our previous work demonstrated that transgenic calli can be obtained without suspension culture, but with a very low frequency of transformation (Takumi and Shimada 1995). Therefore, in this study, hygromycinresistant transgenic calli were generated via suspension culture for 10 days in the beginning of the selection step to increase the transformation frequency. To demonstrate the somatic activation and excision of the Ac/Ds elements in wheat cells, two cultured cell lines were used, but they are not regenerable calli. When activation and transposition of the maize Ac/Ds transposable elements are demonstrated in germ cells of wheat, transgenic plants that have integrated the Ac/Ds elements must be produced through particle bombardment of the scutellar tissues. Excision of the Ac/Ds elements in cereals other than maize has been reported previously for rice (Izawa et al. 1991; Murai et al. 1991; Shimamoto et al. 1993; Sugimoto et al. 1994). In diploid wheat, T. monococcum, the Ac/Ds elements have only been excised in protoplasts transfected with vectors based on the geminivirus WDV (Laufs et al. 1990). Laufs et al. (1990) demonstrated that the combination of the transposable element Ac with the autonomously

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Genome, Vol. 39, 1996 Fig. 5. Molecular analyses of the Ac element and the Ac transposase gene in transgenic wheat calli. (A, B) PCR amplification of the Ac element from total DNA isolated from transgenic wheat calli. One primer set (primers A + B) amplifies a 177-bp fragment (A) and the other set (primers C + D) produces a 542-bp fragment (B). (C, D) RT-PCR analysis of the Ac transposase transcript using two primer sets. One set (primers A + B) recognizes the second exon (C) and the other set (primers C + D) amplifies a 470-bp spliced transcript from the second and third exons (D). Lane: P, pCKR532 as a positive control; N, nontransformed callus as a negative control; 1, transgenic callus produced by introducing pBI222; 2 and 3, transgenic calli after excision of the Ac element of pCKR262; 4-6, transgenic calli generated after pCKR234 was cotransformed with pCKR532.

Table 2. DNA sequences from waxy-m7 at the point of the Ac/Ds excision in three transgenic wheat calli and at the Ac/Ds original site. Excised transposon Transgenic calli Clone 9 (HY- 1 ) Clone 67 (Mono-3) Clone 141 (HY- 1) Ac/Ds at the original site

Altered DNA sequence

Ac Ds Ds

replicating genome of WDV leads to the rapid and genuine excision of the Ac element. In this study, somatic activation and excision of the Ac/Ds elements not only in T. monococcum but also in 7: aestivum was clearly demonstrated by using transgenic wheat callus without a replicative gene expression vector. In the first experiment, many hygromycinresistant calli were recovered when an autonomous Ac element, inserted between the CaMV 35s promoter and hph, was introduced into wheat cells. In these resistant calli,

hph and the Ac element were integrated into the wheat genome and expressed in wheat cells. Moreover, the characteristic sequence alteration at the Ac excision site was found. These findings clearly demonstrated that the maize autonomous Ac element was activated and excised in wheat cells, as has been observed in rice (Izawa et al. 1991; Murai et al. 1991). In the second experiment, many hygromycin-resistant calli were also generated when a nonautonomous Ds element was cotransformed together

Takumi

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with the Ac transposase gene. The phenotypic assay and molecular characterizations, including sequence analysis of the excision site, clearly indicated that the nonautonomous Ds element was activated and excised by expression of the Ac transposase gene in wheat cells, as has been observed in rice (Shimamoto et al. 1993; Sugimoto et al. 1994). The trans-activation of the nonautonomous element suggests that the two-element system, which is superior to the transposon tagging strategy using only an autonomous element (Haring et al. 1991), can be developed in wheat. The CaMV 3 5 s promoter is not always an efficient promoter in wheat cells (Takumi et al. 1994), but the expression of hph and the Ac transposase gene was sufficient to give hygromycin resistance and to excise the Ac/Ds elements in transgenic calli. Both hygromycin-resistant calli that stably expressed the Ac transposase gene and hygromycin-resistant calli that did not stably express the Ac transposase gene were present in this study. As Shimamoto et al. (1993) has previously reported, the Ac transposase gene may have been transiently expressed and the Ac/Ds elements activated when these plasmids were transformed into wheat cultured cells. This study clearly demonstrates that the maize Ac/Ds transposable elements are activated by the expression of the Ac transposase g e n e in wheat cells. Agriculturally important genes may be isolated by the transposon tagging system in wheat, but many problems, such as the t m n s activation of a nonautonomous element in crosses of transgenic wheat plants and the re-integration of the element into the wheat genome, remain.

Acknowledgements

This work was partly supported by a Sasakawa Scientific Research Grant from T h e Japan Science Society. I a m grateful to Dr. K. Shimamoto for his valuable suggestions and for providing the plasmids pCKR262, pCKR234, and pCKR532. I thank Dr. Ishii for the gift of the T. monococcum cell line. I also thank Dr. K. Yamaguchi, Dr. K. Murai, and Mrs. R. Murai for their kind help in this work.

References

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