Abstract
A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem.
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