Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori.
Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is Agrobacterium-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of Aspergillus awamori. This protocol has been used to determine the function of Agrobacterium virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate A. awamori gene knockout transformants. This protocol in not only applicable to A. awamori, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced Agrobacterium, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200-250 transformants per 1 x 10(6) conidiospores. Using this protocol, transformants can be obtained within 10-12 d.
Michielse
Caroline B
CB
Department of Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
Hooykaas
Paul J J
PJ
van den Hondel
Cees A M J J
CA
Ram
Arthur F J
AF
D001230:Aspergillus / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D007612:Kanamycin; D012231:Rhizobium / Q000378:metabolism*; D014170:Transformation, Genetic / Q000502:physiology*
D004271:DNA, Fungal; D005819:Genetic Markers; D007612:Kanamycin