Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana.
Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome. The transformation protocol was optimized to generate a library of insertion mutants of Beauveria. Transformation frequencies around 10(-4) and suppression of background growth were achieved. Over 90% of the AIM mutants investigated contained single-copy T-DNA integrations at different chromosomal locations. Integrated T-DNAs were re-isolated from ten transformants by a marker rescue approach. When the sequences flanking these T-DNAs were compared with the corresponding locations of the wild-type genome, truncations of T-DNA borders were found to be common, while none of the sites of integration had suffered deletion or rearrangement. Thus, AIM can be considered a promising tool for insertional mutagenesis studies of entomopathogenic filamentous fungi.
Leclerque
Andreas
A
Zentrum für Molekulare Biologie (ZMBH), Ruprecht-Karls-Universität Heidelberg, Neuenheimer Feld 282, 69120 Heidelberg, Germany. leclerque@hotmail.com
Wan
Hong
H
Abschütz
Anette
A
Chen
Siwei
S
Mitina
Galina V
GV
Zimmermann
Gisbert
G
Schairer
Hans Ulrich
HU
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D005822:Genetic Vectors; D006921:Hygromycin B / Q000494:pharmacology; D006999:Hypocreales / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity*; D007313:Insecta / Q000382:microbiology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*
D017931:DNA Primers; D004269:DNA, Bacterial; D004271:DNA, Fungal; C036483:T-DNA; D006921:Hygromycin B