Transformation of SR1 tobacco and JTE-h apple rootstock with the EPS-depolymerase gene from an Erwinia amylovora phage

S. Süle, E. Kiss, W. S. Kim, K. Geider

Research output: Chapter in Book/Report/Conference proceedingConference contribution

3 Citations (Scopus)

Abstract

The gene of an Erwinia amylovora bacteriophage encoding an extracellular polysaccharide depolymerase (Dpo), which specifically degrades the surface polysaccharides of the fire blight pathogen, was cloned into pUC-vectors including His-tagged fusions. These insertions were transferred into plasmid pBinAR and named pBinAR-dpo. pBinAR-dpo was transformed into Agrobacterium tumefaciens strain LBA 4404 and were used to transform SR1 tobacco and JTE-H apple rootstock leaves. The putative transformant shoots appearing were analysed by PCR for the presence of nptII gene and the 35S promoter. Plants which were positive in PCR analysis, were further analysed by Western blots. Antiserum against recombinant EPS depolymerase was prepared in order to detect expression of the dpo gene. Several transformed tobacco and apple plants were positive for the presence of EPS-depolymerase. The tobacco clone EPS-dpo 6 that expressed the protein at a high level was chosen for the production of seeds and the establishment of a transgenic Nicotiana tabacum line. Seeds of the transgenic plants were germinated on 1/2 MS medium containing 100 mg kanamycin per litre. Out of 100 seeds 62 exhibited normal roots and leaf phenotypes. Several problems were encountered in transformation of the JTE-H apple rootstock. Out of 432 infected leaves, 24 transformed cell lines were obtained. Incorporation of the 35S promoter, the nptII and dpo genes were confirmed by PCR analysis. These clones are currently under investigation for expression of the EPS-depolymerase and their resistance to E. amylovora. Preliminary data with five transformed shoots showed that four out five plants were resistant to the E.amylovora infection. All of the five control plants were sensitive.

Original languageEnglish
Title of host publicationActa Horticulturae
Pages407-409
Number of pages3
Volume590
Publication statusPublished - 2002

Publication series

NameActa Horticulturae
Volume590
ISSN (Print)05677572

Fingerprint

Erwinia amylovora
bacteriophages
rootstocks
tobacco
apples
polysaccharides
genes
promoter regions
clones
leaves
shoots
seed crop production
kanamycin
seeds
Agrobacterium radiobacter
Nicotiana tabacum
antiserum
transgenic plants
plasmids
Western blotting

ASJC Scopus subject areas

  • Horticulture

Cite this

Süle, S., Kiss, E., Kim, W. S., & Geider, K. (2002). Transformation of SR1 tobacco and JTE-h apple rootstock with the EPS-depolymerase gene from an Erwinia amylovora phage. In Acta Horticulturae (Vol. 590, pp. 407-409). (Acta Horticulturae; Vol. 590).

Transformation of SR1 tobacco and JTE-h apple rootstock with the EPS-depolymerase gene from an Erwinia amylovora phage. / Süle, S.; Kiss, E.; Kim, W. S.; Geider, K.

Acta Horticulturae. Vol. 590 2002. p. 407-409 (Acta Horticulturae; Vol. 590).

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Süle, S, Kiss, E, Kim, WS & Geider, K 2002, Transformation of SR1 tobacco and JTE-h apple rootstock with the EPS-depolymerase gene from an Erwinia amylovora phage. in Acta Horticulturae. vol. 590, Acta Horticulturae, vol. 590, pp. 407-409.
Süle S, Kiss E, Kim WS, Geider K. Transformation of SR1 tobacco and JTE-h apple rootstock with the EPS-depolymerase gene from an Erwinia amylovora phage. In Acta Horticulturae. Vol. 590. 2002. p. 407-409. (Acta Horticulturae).
Süle, S. ; Kiss, E. ; Kim, W. S. ; Geider, K. / Transformation of SR1 tobacco and JTE-h apple rootstock with the EPS-depolymerase gene from an Erwinia amylovora phage. Acta Horticulturae. Vol. 590 2002. pp. 407-409 (Acta Horticulturae).
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abstract = "The gene of an Erwinia amylovora bacteriophage encoding an extracellular polysaccharide depolymerase (Dpo), which specifically degrades the surface polysaccharides of the fire blight pathogen, was cloned into pUC-vectors including His-tagged fusions. These insertions were transferred into plasmid pBinAR and named pBinAR-dpo. pBinAR-dpo was transformed into Agrobacterium tumefaciens strain LBA 4404 and were used to transform SR1 tobacco and JTE-H apple rootstock leaves. The putative transformant shoots appearing were analysed by PCR for the presence of nptII gene and the 35S promoter. Plants which were positive in PCR analysis, were further analysed by Western blots. Antiserum against recombinant EPS depolymerase was prepared in order to detect expression of the dpo gene. Several transformed tobacco and apple plants were positive for the presence of EPS-depolymerase. The tobacco clone EPS-dpo 6 that expressed the protein at a high level was chosen for the production of seeds and the establishment of a transgenic Nicotiana tabacum line. Seeds of the transgenic plants were germinated on 1/2 MS medium containing 100 mg kanamycin per litre. Out of 100 seeds 62 exhibited normal roots and leaf phenotypes. Several problems were encountered in transformation of the JTE-H apple rootstock. Out of 432 infected leaves, 24 transformed cell lines were obtained. Incorporation of the 35S promoter, the nptII and dpo genes were confirmed by PCR analysis. These clones are currently under investigation for expression of the EPS-depolymerase and their resistance to E. amylovora. Preliminary data with five transformed shoots showed that four out five plants were resistant to the E.amylovora infection. All of the five control plants were sensitive.",
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