Abstract

The ascomycete fungus Ascochyta rabiei (Pass.) Labr. [teleomorph: Didymella rabiei (Kovacheski) von Arx (= Mycosphaerella rabiei Kovacheski)] causes Ascochyta blight of chickpea and is one of several economically important Ascochyta pathogens infecting legumes worldwide. Despite the importance of these fungi in agriculture, no transformation systems have been developed for them to date. Here we report transformation of A. rabiei using a polyethylene glycol-mediated method with hygromycin B and geneticin resistance genes as selectable markers. Heterologous expression of green and red fluorescent proteins (GFP from jellyfish and DsRed from reef coral) in A. rabiei was attempted through cotransformation with plasmids carrying an antibiotic resistance gene and an engineered fluorescent protein gene. Two kinds of antibiotic resistant transformants were obtained from A. rabiei protoplasts, and transformation frequencies with 3 kinds of plasmid are ranged from 2 to 5 transformants per microgram DNA per 107 protoplasts. Integration of plasmid DNAs into the A. rabiei genome was confirmed by PCR with antibiotic resistance gene-specific primers. Southern blot analysis showed that plasmid integration occurred randomly in the genome. Out of 500 hygromycin B and 100 geneticin resistant transformants, a putative albino mutant and some morphological mutants were screened, and the mutants were confirmed to be derived from A. rabiei using mating type gene primers specific to the fungus developed by our previous research. Analysis of plasmid insertion sites in the mutants is in progress. Establishment of transformation systems in this fungus led to yield marker strains expressing GFP and DsRed genes. Approximately 50% of transformants obtained by cotransformation protocol displayed bright fluorescence when viewed under the fluorescent microscope. GFP expression was detected in conidia and hyphae during development on glass slides and plants. We are currently using these GFP-expressing strains to investigate the infection process of A. rabiei on chickpea plants.

*Department of Plant Pathology, Washington State University