Using an improved site-directed mutagenesis for the creation of constructs to analyse gene function in Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc) is the causal pathogen of Fusarium wilt in banana. Apart from plant resistance, no sustainable solution has been found to control Fusarium wilt. Understanding the genetic basis of pathogenesis in Foc might contribute to developing novel strategies to control Fusarium wilt. We used an improved site-directed mutagenesis to create constructs to analyse gene function in Foc. For the general site-directed mutagenesis, sequencing is the only choice to confirm the mutant clone; however in our protocol, named OE-PCR-S1, we not only improved the efficiency with Dpn I, but also used mismatch cleavage by S1 nuclease to replace sequencing to screen the mutants, which made the protocol more rapid and labour efficient and less expensive. We have used it for Foc, and have successfully deleted three bases from a pathogenic gene, FOW1 (EU795421). The mutant DNA can be inserted into an expression vector and transformed into Foc for functional domain analysis. The overall rate of obtaining the mutant sites was 100%, and the whole mutagenesis process could be completed in less than 2 days, which means that the method is a powerful tool to study pathogenic gene functions and for genetic engineering.