Plant Pathol J > Volume 25(2); 2009 > Article
The Plant Pathology Journal 2009;25(2):179-183.
DOI: https://doi.org/10.5423/PPJ.2009.25.2.179    Published online June 30, 2009.
Protoplast-Mediated Transformation of the Filamentous Fungus Cladosporium phlei: Evidence of Tandem Repeats of the Integrative Transforming Vector
Jung Ae Kim, Jung Mi Kim, Hwan Gyu Kim, Beom Tae Kim, Ki Jun Hwang, Seung Moon Park, Moon Sik Yang, Dae Hyuk Kim
Abstract
To facilitate the genetic manipulation of Cladosporium phlei, a causal agent of leaf spot disease in timothy (Phleum pretense), protoplast-mediated transformation of C. phlei has been developed and the resulting transformants were characterized in this study. Hygromycin B resistance was applied as a dominant selection marker due to the sensitivity of C. phlei to this antibiotic. The transformation efficiency ranged from approximately 20-100 transformants per experiment. Southern blot analysis of stable transformants revealed that transformation occurred by way of stable integration of the vector DNA into the fungal chromosome. PCR analysis and plasmid rescuing of randomly selected transformants suggested that integration of tandem repeat copies of vector DNA was common. In addition, multiple integrations of the transforming vector at different chromosomal sites were also observed. The establishment of a transformation method for C. phlei facilitates strain improvement of this fungus and can be applied as an initial step in the molecular analysis of pigment production in this fungus.
Key Words: Cladosporium phlei, fungal transformation
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