Effect of the M·EcoRII methyltransferase-encoding gene on the phenotype of nicotiana tabacum transgenic cells

Gene, 157 (1995) 283 287 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

283

GENE 08847

Effect of the M.EcoRII methyltransferase-encoding gene on the phenotype of Nicotiana tabacum transgenic cells* (Recombinant DNA; DNA methylation; plant bacterium; agrobacterium; transformation)

Yaroslav I. Buryanov, Natalia S. Zakharchenko, Taras V. Shevchuk and Irene G. Bogdarina Branch of Shem yakin and Ot~chinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Re,4ion 142292, Russia Received by T.H. Bestor: 11 July 1994; Accepted: 23 December 1994; Received at publishers: 13 February 1995

SUMMARY

The EcoRII DNA methyltransferase (M.EcoRII; MTase) modifies a cytosine in the DNA sequence CCWGG which contains a CNG methylation motif characteristic of plant DNA. The gene (ecoRIIM) encoding this MTase has been cloned into the T-DNA of the wild-type Agrobacterium Ti-plasmid pTiC58 downstream from the plant expression nopaline synthase-encoding gene promoter. Nicotiana tabacum cells have been transformed with Agrobacterium tumefaciens harbouring this recombinant Ti-plasmid. The primary transformed tabacco tissue line has given rise to novel stable lines which are morphologically distinctive. Southern hybridization analysis of all transformed tissue lines has shown the presence, in each of them, of ecoRIIM. The tissue studied differed in morphology in callus culture, dependence on phytohormones and the ability to synthesize nopaline.

INTRODUCTION

In higher eukaryotic genomes 5-methylcytosine is the only modified base formed by enzymatic DNA methylation. In animals DNA methylation is involved in the regulation of gene expression, cell differentiation, embryonal development (for reviews, see Cedar, 1988; Bestor, 1990; Razin and Cedar, 1991) and in inheritable epigenetic control of genomic imprinting (Sapienza et al., 1987; Swain Correspondence to: Dr. Ya. I. Buryanov, Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia. Tel. (7-095) 925-2342; Fax (7-095) 923-3602; e-mail: [email protected] *Presented at the Third New England BioLabs Workshop on Biological DNA Modification, Vilnius, Lithuania, 22-28 May 1994. Abbreviations: A., Agrobacterium; Ap, ampicillin; bp, base pair(s); ecoRllM, gene ecoding M.EcoRII; EtdBr, ethidium bromide; kb, kilobase(s) or 1000bp; Km, kanamycin; MS, Murashige-Skoog (1962) (medium); MTase (M-), DNA methyltransferase; N., Nicotiana; Nal, nalidixic acid; nos, gene encoding nopaline synthase; nt, nucleotide(s); R, resistance/resistant; Ti plasmid, plasmid that induces plant tumors; W, A or T; wt, wild type. SSD1 0378-1119(95)00129-8

et al., 1987). Little is known about DNA methylation in plants. Data obtained confirm the correlation of transcription with plant gene undermethylation (Hepburn et al., 1983; Amasino et al., 1984; Hershkovitz et al., 1990) and show the association of DNA methylation with the inactive state of plant transposable elements (Chomet et al., 1987; Fedoroff, 1989). It is possible that the main functions of cytosine methylation in plants and animals are similar, but they can display specific peculiarities. In fact, plant DNA is usually highly methylated (up to 40% of all cytosine residues are modified compared to only 2-10% in mammals (Adams and Burdon, 1985). The difference in the extent of DNA-cytosine methylation between vertebrates and plants can be attributed to site specificity of their MTases. While DNA methylation in animals occurs mainly in CG dinucleotides, in plants it takes place in both CG and CNG sequences where N is any nt (Gruenbaum et al., 1981; Kirnos et al., 1981). It is possible that the CNG type of DNA methylation can play some specific functions in plant cell. The M.EcoRII MTase modifies the internal cytosine in the DNA sequence CCWGG (Buryanov et al., 1978) which con-

284 tains the C N G methylation motif characteristic of plant DNA. We report here the first experimental results of obtaining and analysis of transgenic tobacco cells containing the e c o R I I M gene. Such transgenic cells and plants have been used to study the effect of C N G type of in vivo D N A methylation on the cell cycle and plant development and morphogenesis.

3

4

kb

7.7

EXPERIMENTALAND DISCUSSION

6.2 3.4 2.6

(a) Introduction of the ecoRllM gene into the wt Ti plasmid

1.5

The modified B a m H I fragment of the plasmid N3 containing the e c o R I I M gene was cloned into the pLGV2382 cointegrative vector. This vector contains bacterial selectable Ap R and Km R markers, D N A sequence from the HindIII-23 fragment of pTiC58 T - D N A providing homology for recombination with Ti plasmid in A. tumefaciens, and unique cloning sites B a m H I and EcoRI downstream from the nopaline synthase promoter (Herrera-Estrella et al., 1983). The pLGV2382 harbouring the e c o R I I M gene was propagated in E, coli B834 ( d c m - ) cells and transmitted by conjugation to the Nal R A. tumefaciens C58 strain using E. coli carrying the helper plasmids R 6 4 d r d l l and pGJ28 as described by Van Haute et al. (1983). Exconjugants of A. tumefaciens were selected as a single cross-over event between the wt Ti plasmid and pLGV2382 harbouring the e c o R I I M gene. The resulting cointegrative Ti plasmids have had the e c o R I I M gene in addition to the nopaline synthase (nos) and phytohormone genes (tmsl, tins2, tmr) from the T - D N A of the wt Ti plasmid. The structure of the T - D N A region harbouring the e c o R I I M gene is shown in Fig. 1. The e c o R I I M gene product had a normal enzymatic activity as it observed in experiments on A. tumefaciens D N A cleavage with EcoRII and B s t N I (Fig. 2).

Fig. 2. Sensitivity of the .4. tumefaciens DNA to EcoRII and BstNI Total DNA from non-exconjugant and exconjugant cells of A. tumefaciens was digested with EcoRII or BstNI and analyzedby 0.8% agarose gel electrophoresis and staining with 0.5 gg EtdBr/ml. The Styl fragments of ~, DNA were used as size markers. Lanes: l, undigested DNA; 2, non-exconjugant DNA digested with EcoRII; 3, non-exconjugant DNA digested with BstNI; 4, exconjugant DNA digested with EcoRII; 5, exconjugant DNA digested with BstNI.

cointegrative Ti plasmid with the e c o R I I M gene. Three weeks later, tumors were excised and tissue culture was obtained as described by Leemans et al. (1981). Selection of transformants was carried out by culturing cell suspensions in hormone-free MS medium supplemented with cefotaxime (300gg/ml) and sucrose (30mg/ml). The tumor tissues were transferred every 3 weeks. After three transfers, tissues were free of bacteria and were further cultivated on antibiotic-free MS agar medium supplemented with 2 gg naphthalene acetic acid (NAA) and 0.5 gg benzoaminopurine (BAP) per ml. After two months cultivation on this medium spontaneous morphological differentiation of tumor cal-

(b) Transformation and culture of crown gall tumor tissues The stem wound surface of 4-week-old tobacco seedlings was inoculated with A. tumefaciens C58 carrying the

,~

K.___2_ ~

Af -,wA ............ 1.

A

EB

I

7. °.

I

B

i J- k b

E i

Fig. 1. The partial structure of the T-DNA region of the Ti plasmid harbouring the ecoRIIM gene. The BamHI fragment of plasmid N3 carrying the ecoRIIM gene was inserted into BamHI site of the intermediate vector pLGV2382. The resulting plasmid pLGV23ecoRIIM was transferred to A. tumefaciens C58 by conjugation. Agrobacterialexconjugants were selected as a single cross-overhomologyrecombination between pLGV23ecoRIIM and pTiC58 on Ap + Kin-containing medium. Southern analysis of four agrobacterial exconjugants with the ecoRIIM gene as a probe exhibited the only detectable signal in the form of 10.6-kb EcoRI band (data not shown). Regions of the HindIII-23 fragment of pTiC58 are shown as a hatched boxes, Km~ and ApRgenes of the pLGV2382as a open box, the N3 plasmid fragment as a dotted box, pTiC58 sequence as a single line. P indicates the nos promoter. Arrows mark the direction of transcription, jagged lines mark the borders of the ecoRIIM gene. B, BamHI, E, EcoRI.

285 luses took place. The primary transformed tobacco tissue line gave rise to several novel stable morphological lines (Fig. 4). At the same time, the tumorous calluses obtained by agrobacterium infection with the same wt Ti plasmid but without MTase gene, were unchanged.

TABLE I

(c) Phenotype and some properties of the different transformed tobacco tissue lines The primary transformed tobacco crown gall tumors demonstrated the typical properties: nopaline synthesis, phytohormone-independent growth, presence of the nopaline synthase gene and genes of phytohormone region of the agrobacterial T-DNA (data not shown). In addition they contained also the e c o R I I M gene (Fig. 3). In spite of the novel tobacco tissue lines contained the same genes, they differed in morphological and biochemical properties (Fig. 4, Table I). The results indicate that the phenotypical changes of the transformed tobacco tumor tissues are caused by the effect of the e c o R I I M gene integration into plant genome. There are three possible explanations of the e c o R l l M

'White'

l

2

3

4

5

kb -23.1

-9.4 --6.5

--4.~

Fig. 3. Southern analysis of tobacco DNA. High-molecular-weight DNA was isolated from tobacco tissue cultures as described (Draper and Scott, 1988). 10lag of plant DNA was digested to completion with BamHI, electrophoresed on a 0.8% agarose gel and transferred to the Zeta-Probe membrane (Bio-Rad) in 10×SSC buffer (1.5M NaCI/0.15 M Naa'citrate pH 7.6). Southern blot was hybridized with the 32P-labelled ecoRllM gene probe prepared by random-primer labeling of 4.2-kb BamHI fragment of pLGV23ecoRIIM to a specific activity of approx. 109 crop/lag of DNA. Hybridization was performed under standard conditions (Maniatis et al., 1982). The HindIII fragments of ;~ DNA were used as size markers. Lanes: 1, DNA from "yellow" callus; 2, DNA from teratoma; 3, DNA from "white" (compact) callus; 4, DNA from initial tumor callus; 5, DNA from untrasformed tobacco callus. Tissue names correspond to the different morphology types shown in Fig. 4.

Properties of transgenic tobacco tissue culture lines containing the ecoRllM gene Tissue culture linea

Nopaline synthesisb

Phytohormone dependence

Initial

+

-

(compact) 'Yellow' Teratoma

+ -

+ -

a Tissue names correspond to the different morphology types shown in Fig. 4. b Nopaline synthase activity was detected as described by Otten and Schilperoort (1978).

gene integration effect. First, the effect could be caused by "in Agrobacterium" Ti plasmid methylation (including T-DNA) by the M.EcoRII MTase. Second, the effect could be caused by "in plant cell" plant genome DNA methylation (including integrated T-DNA) by the M.EcoRII MTase. Third, the effect could be caused by the possible homologous recombination between the ecoRIIM gene incorporated into tobacco genome and the plant own MTase-encoding gene. It is known that the animal and plant MTases display significant homology with the bacterial DNA cytosine MTases (Bestor et al., 1988; Lauster et al., 1989; Finnegan and Dennis, 1993; Scheidt et al., 1994), especially with the M.EcoRII MTase (Som et al., 1987). The homologous recombination could be responsible for disruption of the plant and e c o R I I M MTase genes (Li et al., 1992) and for DNA rearrangements. We are attempting to test possible explanations listed above.

(d) Conclusions Integration of the ecoRIIM gene into the tobacco genome causes the phenotypic changes of tissue culture. The possible explanations of the mechanism of this effect are discussed.

ACKNOWLEDGEMENTS

The research described in this publication was made possible in part by grant N94-04-12212-a from Russian Foundation of Fundamental Research and RMTOOO from the International Science Foundation. We thank Dr. N.I. Strizhov for helping in the construction of the Agrobacterium with the e c o R I I M gene and Dr. I.V. Orlova for the establishing of the tobacco tissue cultures.

286

Fig. 4. Different morphology of transgenic tobacco tissue culture lines containing the ecoRIIM gene. Callus tissues were named according to their morphology (colour and density). (A) Initial tumor tissue; (B) "white" (compact) culture; (C) "yellow" culture; (D) teratoma.

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