Targeted Gene Knockout and Complementation in Valsa mali

VmLaeA and the core enzyme genes of cluster PKS7, PKS11, NRPS14, PKS16, PKS23, PKS31, NRPS/PKS33, and PKS39 were selected to construct deletion mutants. The gene knockout cassettes were assembled by double-joint PCR with upstream and downstream flanking sequences of the target gene and neo gene as selective marker (Yu et al., 2004 (link); Supplementary Figure 8). The gene knockout cassettes were then transformed into protoplasts of V. mali mediated by polyethylene glycol (Gao et al., 2011 ). Putative deletion mutants filtered by selection with geneticin were confirmed by PCR and further confirmed by Southern blot hybridization using the DIG DNA Labeling and Detection Kit II (Roche, Mannheim, Germany). Gene complementation was conducted by cloning the target genes into plasmid PDL2 by the yeast gap repair approach and then transforming the recombined plasmids into the respective target gene deletion mutant (Bruno et al., 2004 (link); Zhou et al., 2012 (link)). The complemented mutants were finally confirmed by PCR. All primers used in gene deletion and complementation are listed in Supplementary Table 11.