In Sun Hwang, and Il-Pyung Ahn

The Plant Pathology Journal 2016;32:173-181; Published online June 1, 2016

Abstract

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

See: http://www.ppjonline.org/journal/view.html?uid=2103&&vmd=Full

Figure 2: Phenotypic characterization and fumonisin analysis in the fcc1 mutant strains compared with the wild-type (WT). (A) Phenotypical analysis of the WT and the fcc1 mutants in PDB and on PDA for 5 and 7 days. (B) The effect on fumonisin production by Fusarium cyclin C1 (FCC1) disruption in vitro and in vivo. Error bars are represent as ± standard deviation. Statistical significance was analyzed by Student’s t-test. Asterisks indicate significant differences (^**P < 0.01).