Zn(II)2Cys6 transcription factor genes encode transcription regulators that manage the infection potential and production of
secondary metabolites such as toxins in fungi. In this study, a gene named rolP that encodes a putative Zn(II)2Cys6 transcription factor
regulating leucinostatin production in the filamentous fungus Paecilomyces lilacinus was characterized by a gene knockout approach. The
deduced proprotein consists of 705 amino acids and is highly homologous to the Zn(II)2Cys6 transcription factor of the entomopathogenic
fungus Metarhizium brunneum. Predictive analysis of the secondary structure of the proportion showed that it has two domains. Paecilomyces
lilacinus can produce nematotoxins leucinostatins A and B. Deletion of rolP from the P. lilacinus wild-type strain Pl36-1 leads to the absence
of leucinostatin A, while a large increase in the leucinostatin A level was achieved in the rolP overexpression strain Ov-Pl36-1. During the
process of nematode infection, rolP showed high expression levels at 48–72 h and peaked at 48 h. Bioassay tests confirmed the requirement of
rolP for leucinostatin production. However, the root-knot second-stage juvenile-toxicity was minimized to 35.3% in ΔrolP; toxicity was 97.2%
with Ov-Pl36-1 compared with 68.4% with the wild type strain Pl36-1. Interestingly, neither knockout nor overexpression of the rolP gene
affected the growth or sporulation of P. lilacinus. Additionally, fungal nutrition and acidic media might stimulate the rolP activity of the wild
type strain against nematodes. These findings suggested that the rolP gene is required for the induction and production of leucinostatins and is
considered a leucinostatin regulatory gene in P. lilacinus.