Highly efficient transformation system for Malassezia furfur and Malassezia pachydermatis using Agrobacterium tumefaciens-mediated transformation.
Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe, a highly efficient Agrobacterium-mediated genetic transformation system for Malassezia furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in M. furfur and M. pachydermatis by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. Optimal temperature and co-cultivation time for transformation were 5 and 7days at 19°C and 24°C, respectively. Transformation efficiency was 0.75-1.5% for M. furfur and 0.6-7.5% for M. pachydermatis. Integration of the hpt resistance cassette and gfp was verified using PCR and fluorescence microscopy, respectively. The T-DNA was mitotically stable in approximately 80% of the transformants after 10 times sub-culturing in the absence of hygromycin. Improving transformation protocols contribute to study the biology and pathophysiology of Malassezia.
['Celis|A M|AM|', 'Vos|A M|AM|', 'Triana|S|S|', 'Medina|C A|CA|', 'Escobar|N|N|', 'Restrepo|S|S|', 'Wösten|H A B|HA|', 'de Cock|H|H|']
D000364:Agaricus D016960:Agrobacterium tumefaciens D018920:Coculture Techniques D003455:Cryptococcus neoformans D004269:DNA, Bacterial D003881:Dermatomycoses D005822:Genetic Vectors D049452:Green Fluorescent Proteins D006801:Humans D008290:Malassezia D008856:Microscopy, Fluorescence D017853:Phosphotransferases (Alcohol Group Acceptor) D016133:Polymerase Chain Reaction D014170:Transformation, Genetic
Transformation Agrobacterium Malassezia spp. T-DNA Hygromycin GFP