Abstract
The present study presents a novel approach combining a tetracycline-inducible system (Tet-On) and CRISPR-Cas9 techniques to investigate the function of two essential genes in Phytophthora sojae. We constructed a donor vector in which the reverse tetracycline transactivator (rtTA) is driven by an oomycete promoter. Additionally, it contains a fused TetR binding site and the minimum oomycete promoter, as well as 1000-bp homologous arms of the promoter upstream and downstream sequences. The promoter of the target gene was replaced with a tetracycline-responsive promoter (P(tet)) using a CRISPR-Cas9 system. In the native transformants, the target gene was induced by the administration of tetracycline and repressed in its absence. Using the Tet-On/CRISPR-Cas9 system, we obtained inducible transformants of PsAF5 and PsCesA3. The phenotype of PsAF5 inducible transformants without doxycycline was consistent with that of ΔPsAF5 transformants, specifically characterised by an increase in oospore production and heightened sensitivity to H(2)O(2). PsCesA3 inducible transformants could not grow in the absence of doxycycline, which means PsCesA3 is an essential protein for P. sojae. In conclusion, the Tet-On/CRISPR-Cas9 system represents an effective approach for investigating crucial genes in P. sojae.