title: Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions.
abstract: Thielaviopsis basicola is a hemibiotrophic root pathogen causing black root rot in a wide range of economically important crops. Our initial attempts to transform T. basicola using standard Agrobacterium tumefaciens-mediated transformation (ATMT) protocols were unsuccessful. Successful transformation required the addition of V8 juice (to induce germination of T. basicola chlamydospores) and higher concentrations of acetosyringone in the co-cultivation medium, and of chlamydospores/endoconidia, A. tumefaciens cells during co-cultivation. With these modifications, two T. basicola strains were successfully transformed with the green (egfp) or red (AsRed) fluorescent protein genes. Chlamydospores/endoconidia transformed with the egfp gene exhibited strong green fluorescence, but their fluorescence became weaker as the germ tubes emerged. Transformants harbouring the AsRed gene displayed strong red fluorescence in both chlamydospores/endoconidia and germ tubes. Fluorescent microscopic observations of an AsRed-labelled strain colonizing roots of transgenic Nicotiana benthamiana plants, which express the actin filaments labelled with EGFP, at 24 hours post inoculation showed varying levels of fungal germination and penetration. At this stage, the infection appeared to be biotrophic with the EGFP-labelled host actin filaments not being visibly degraded, even in host root cells in close contact with the hyphae. This is the first report of ATMT of T. basicola, and the use of an AsRed-labelled strain to directly observe the root infection process.
authors.affiliation: Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece. Electronic address: aliki@aua.gr.
authors.forename: Aliki K
authors.initials: AK
authors.lastname: Tzima
authors.affiliation: Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece.
authors.forename: Epaminondas J
authors.initials: EJ
authors.lastname: Paplomatas
authors.affiliation: Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece.
authors.forename: Charikleia
authors.initials: C
authors.lastname: Schoina
authors.affiliation: Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece.
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Domazakis
authors.affiliation: Department of Plant Pathology & Environmental Microbiology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Seogchan
authors.initials: S
authors.lastname: Kang
authors.affiliation: School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Goodwin
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001203:Ascomycota / Q000235:genetics* / Q000254:growth & development; D003470:Culture Media / Q000737:chemistry; D005453:Fluorescence; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005827:Genetics, Microbial / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018517:Plant Roots / Q000382:microbiology; D014026:Nicotiana / Q000382:microbiology; D014170:Transformation, Genetic
chemical_list: D003470:Culture Media; D008164:Luminescent Proteins
keywords: Agrotransformation; AsRed; Biotrophic infection; Black root rot; egfp