Genetic Manipulation of P. fici

The plasmids and primers are listed in Table S1 and Table S2, respectively. PCR amplifications were executed in the T100TM Thermal cycler (Bio-Rad, Hercules, CA, USA). TransStart-FastPfu DNA polymerase as a High-Fidelity DNA polymerase (TransGene Biotech, Beijing, China) was used to amplify the gene fragments. PCR screenings for transformants were performed by using 2×Taq Mix kit (Tiangen Biotech, Beijing, China). PCR reaction and thermal profiles were referred to the manufacturer´s instructions. The restriction enzymes used in this work were obtained in New England Biolabs (New England Biolabs Inc. (NEB), Ipswich, MA, USA). To generate the deletion cassette, we used Fusion PCR strategy as described previously [51 (link)]. Briefly, G418 was amplified from the pAG1-H3-G418, and around 1.1 kb of fragments upstream and downstream of the gene PfptaA were amplified from P. fici genomic DNA using the designed primers. The three PCR fragments were ligated into the T-vector p-Blunt, and then were amplified for transformation in P. fici strains.