Genetic transformation, infection process and qPCR quantification of Verticillium dahliae on smoke-tree Cotinus coggygria

The smoke-tree vascular wilt fungus, Verticillium dahliae, was successfully transformed via PEG-mediated protoplasts using cassettes for eGFP and hygromycin B resistance (hph) gene expression. The transformants were stable after alternative sub-culturing on selective and non-selective medium for 6 months. The transformed isolates did not differ markedly from wild-type strains in terms of growth, morphological characteristics, asexual conidiation and pathogenicity. Fluorescence observations clearly indicated strong expression of the green fluorescent protein in fungal structures. The colonization and penetration of smoke-tree roots by eGFP-tagged V. dahliae was monitored by fluorescence, and the early infection processes were clearly visualized. At 24-hour-post-inoculation (hpi), hyphae were found to be predominantly colonizing the fibers rather than the taproots, and subsequently, hyphae started to grow parallel to the longitudinal axis of the root along the junctions of epidermal cells at 36 hpi. At 48 hpi, the elongated hyphae were interspersed among the root hairs and the first observation of central cylinder colonization occurred at 5 dpi. Elevated amounts of fungal DNA were detectable in stems 6 dpi, in branches 12 dpi, and in leaves 14 dpi. The data indicated that once V. dahliae enters smoke-tree, the pathogen rapidly spreads and increases in planta, and GFP can be used as a vital and stable marker for studying the interaction between V. dahliae and woody plants. Furthermore, the establishment of a PEG-mediated transformation system will contribute to functional genomics studies of smoke-tree Verticillium wilt fungus.