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Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer

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An Erratum to this article was published on 15 May 2004

Abstract

Two transformation systems, based on the use of CaCl2/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS + marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl2/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl2/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.

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Acknowledgements

The authors would like to thank Prof. Masamichi Takagi for providing us with the plasmids pRNU54 and pLPGUS21. This research was supported in part by a grant from Orsan to TNO Nutrition.

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Author notes

  1. K. Salim

    Present address: , 24 Résidence le Bois du Roi, 91940, Les Ulis, France

  2. P. Ragas

    Present address: Molecular Microbial Ecology Group, BioCentrum-DTU, Technical University of Denmark, 2800, Lyngby, Denmark

  3. B. Kudla

    Present address: , 52 Rue de Gometz, 91970, Les Moulieres, France

  4. B. Jarry

    Present address: Tate and Lyle/Amylum N.V., Burchtstraat 10, 9300, Aalst, Belgium

Authors and Affiliations

  1. Fungal Genetics Group, Clusius Laboratory, Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands

    C. B. Michielse, A. F. J. Ram & C. A. M. J. J. van den Hondel

  2. Orsan, 46 Route de Nesle, 80190, Mesnil Saint Nicaise, France

    K. Salim, B. Kudla & B. Jarry

  3. Department of Microbiology, TNO Nutrition, Utrechtseweg 48, 3700 AJ, Zeist, The Netherlands

    C. B. Michielse, P. Ragas, A. F. J. Ram, P. J. Punt & C. A. M. J. J. van den Hondel

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An erratum to this article can be found at http://dx.doi.org/10.1007/s00438-004-1020-x

Communicated by C. P. Hollenberg

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Michielse, C.B., Salim, K., Ragas, P. et al. Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer. Mol Genet Genomics 271, 499–510 (2004). https://doi.org/10.1007/s00438-004-1003-y

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