pp 1-26 | Cite as

Agrobacterium-Mediated Transformation of Yeast and Fungi

  • Paul J. J. Hooykaas
  • G. Paul H. van Heusden
  • Xiaolei Niu
  • M. Reza Roushan
  • Jalal Soltani
  • Xiaorong Zhang
  • Bert J. van der Zaal
Part of the Current Topics in Microbiology and Immunology book series


Two decades ago, it was discovered that the well-known plant vector Agrobacterium tumefaciens can also transform yeasts and fungi when these microorganisms are co-cultivated on a solid substrate in the presence of a phenolic inducer such as acetosyringone. It is important that the medium has a low pH (5–6) and that the temperature is kept at room temperature (20–25 °C) during co-cultivation. Nowadays, Agrobacterium-mediated transformation (AMT) is the method of choice for the transformation of many fungal species; as the method is simple, the transformation efficiencies are much higher than with other methods, and AMT leads to single-copy integration much more frequently than do other methods. Integration of T-DNA in fungi occurs by non-homologous end-joining (NHEJ), but also targeted integration of the T-DNA by homologous recombination (HR) is possible. In contrast to AMT of plants, which relies on the assistance of a number of translocated virulence (effector) proteins, none of these (VirE2, VirE3, VirD5, VirF) are necessary for AMT of yeast or fungi. This is in line with the idea that some of these proteins help to overcome plant defense. Importantly, it also showed that VirE2 is not necessary for the transport of the T-strand into the nucleus. The yeast Saccharomyces cerevisiae is a fast-growing organism with a relatively simple genome with reduced genetic redundancy. This yeast species has therefore been used to unravel basic molecular processes in eukaryotic cells as well as to elucidate the function of virulence factors of pathogenic microorganisms acting in plants or animals. Translocation of Agrobacterium virulence proteins into yeast was recently visualized in real time by confocal microscopy. In addition, the yeast 2-hybrid system, one of many tools that have been developed for use in this yeast, was used to identify plant and yeast proteins interacting with the translocated Agrobacterium virulence proteins. Dedicated mutant libraries, containing for each gene a mutant with a precise deletion, have been used to unravel the mode of action of some of the Agrobacterium virulence proteins. Yeast deletion mutant collections were also helpful in identifying host factors promoting or inhibiting AMT, including factors involved in T-DNA integration. Thus, the homologous recombination (HR) factor Rad52 was found to be essential for targeted integration of T-DNA by HR in yeast. Proteins mediating double-strand break (DSB) repair by end-joining (Ku70, Ku80, Lig4) turned out to be essential for non-homologous integration. Inactivation of any one of the genes encoding these end-joining factors in other yeasts and fungi was employed to reduce or totally eliminate non-homologous integration and promote efficient targeted integration at the homologous locus by HR. In plants, however, their inactivation did not prevent non-homologous integration, indicating that T-DNA is captured by different DNA repair pathways in plants and fungi.



We acknowledge the contributions of Amke den Dulk-Ras, Alice Beijersbergen, Paul Bundock, Haico van Attikum, Jesus Escudero, Marcel de Groot, Carolien Michielse, Barbara Schrammeijer, Martijn Rolloos, and Suzanne Wolterink to our research regarding AMT of yeast and fungi. We had to restrict the numbers of references. Therefore, we apologize to those whose work related to the topic we did not refer to. The work in our laboratory was supported over the years by grants from the divisions Earth and Life Sciences (ALW) and Chemical Sciences (CW) of the Netherlands Organization of Scientific Research NWO, the organization for Applied Research (STW), and the Royal Netherlands Academy of Arts and Sciences (KNAW).


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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Paul J. J. Hooykaas
    • 1
  • G. Paul H. van Heusden
    • 1
  • Xiaolei Niu
    • 1
  • M. Reza Roushan
    • 1
  • Jalal Soltani
    • 1
  • Xiaorong Zhang
    • 1
  • Bert J. van der Zaal
    • 1
  1. 1.Sylvius Lab, Department of Molecular and Developmental GeneticsInstitute of Biology, Leiden UniversityLeidenThe Netherlands

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