Tools and basic procedures of gene manipulation in nematode-trapping fungi

**Background:** Nematode-trapping fungi (NTF) are carnivorous microbes that capture nematodes using specialized trapping structures derived from hyphae. They can switch between saprophytic and carnivorous lifestyles, making them valuable model organisms for studying fungal lifestyle transitions and potential biocontrol agents against parasitic nematodes. Despite numerous omics studies revealing genes and pathways associated with carnivorous traits, molecular tools to verify these gene functions have been limited. This review aims to provide a comprehensive reference for gene manipulation tools in NTF.

**Methods:** The authors conducted a narrative review of published literature on gene manipulation tools in NTF, covering transformation methods, random mutagenesis, and targeted gene editing. They summarized protocols for PEG-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation (ATMT), including detailed reagent compositions and step-by-step procedures. Random mutagenesis methods covered include restriction enzyme-mediated integration (REMI) and chemical/UV mutagenesis. Targeted methods include homologous recombination (HR) and CRISPR/Cas9 ribonucleoprotein (RNP) approaches.

**Key Results:** The review catalogs promoters used in NTF, including heterologous promoters (gpdA, trpC, oliC from A. nidulans; cpc-1 from N. crassa; o2tef from U. maydis) and endogenous promoters (tubA, sipC, H2B, artA-C from D. flagrans). Selection markers include hygromycin-B (100-200 µg/ml), geneticin/G418 (120-200 µg/ml), clonNAT (350 µg/ml), and nourseothricin (60 µg/ml), with species- and strain-dependent optimal concentrations. Fluorescence indicators GFP and mCherry have been successfully expressed in D. flagrans. For transformation, PEG-mediated protoplast transformation was first established in A. oligospora in 1999, while ATMT has been established in three NTF species. REMI achieved 175 transformants/µg DNA under optimized conditions. Homologous recombination has been used to delete over 60 genes across multiple NTF species, with homologous arm lengths typically around 2 kb. The HR rate in A. oligospora was reported as extremely low (~3%), but deletion of ku70 improved this rate without phenotypic effects. CRISPR/Cas9 RNP was first applied in D. flagrans in 2019, successfully inserting the hph resistance gene at the cleavage site in 3 of 4 transformants. RNA interference has been established in knob-forming Dactylellina haptotyla, where knockdown of DhFIG_2 significantly decreased conidiation and knob formation.

**Clinical Implications:** NTF are being developed as biological control agents against parasitic nematodes that cause devastating economic losses in crops and livestock. The molecular tools described in this review are essential for verifying gene functions related to trap formation, virulence, and lifestyle transition, which could lead to improved NTF strains for biocontrol applications. Biological nematicides containing NTF have been commercialized or are under development. The review identifies gaps including the lack of inducible promoters, auxotrophic markers, transposon-arrayed gene knockout, plasmid-form CRISPR, and broader RNAi application in NTF.