Reduction in solanapyrone phytotoxin production byAscochyta rabieitransformed withAgrobacterium tumefaciens

Abstract

Agrobacterium tumefaciens was used to transform Ascochyta rabiei, the causal agent of chickpea blight. Employing a T-DNA containing a hygromycin resistance gene (hph), 908 transformants were obtained from germinated pycnidiospores on a selective medium containing hygromycin. Transformants were confirmed using PCR and Southern analyses and of four of these that were tested, two had integrated multicopies of the hph gene, one had two copies and one had a single insertion. Transformants were tested for the production of solanapyrone A toxin using a microtitre plate assay. Loss of toxin production by transformants was confirmed by reversed phase high-performance liquid chromatography. Sixteen transformants out of 668 tested produced significantly less solanapyrone A than the wild-type strain.

Agrobacterium-Mediated Transformation of Non-Plant Organisms

“…Indeed, Agrobacterium has been used as a tool for insertional mutagenesis in plants, such as Arabidopsis thaliana, Nicotiana species, and Oryza sativa (Koncz et al, 1989;Koncz et al, 1992;Krysan et al, 1999;Jeon et al, 2000) and more recently in different genera of fungi such as the model eukaryote S. cerevisiae (Bundock et al, 2002), the symbiotic fungus Hebeloma cylindrosporum (Combier et al, 2003), the biocontrol agents Beauveria bassiana (Leclerque et al, 2004) and Coniothyrium minitans (Rogers et al, 2004), the phytopathogens Ascochyta rabiei (Mogensen et al, 2006) and Magnaprthe grisea , and the human pathogens Aspergillus fumigatus (Sugui et al, 2005) and Cryptococcus neoformans (Idnurm et al, 2004).…”

Towards identifying pathogenic determinants of the chickpea pathogen Ascochyta rabiei

“…The hydrolytic enzymes are considered necessary for fungal nutrition and to facilitate spatial spread of fungi (Walton 1994). Ascochyta rabiei was first transformed with the protoplast/PEG protocol with a GUS reporter gene for observing the infection process (Kohler et al 1995), and later transformed with Agrobacterium-mediated transformation (AMT) for studying pathogenicity factors (White and Chen 2006;Morgensen et al 2006). However, little information is currently available about the genetic determinants of pathogenicity of the ascochyta pathogens.…”