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

ABSTRACT

Nematode-trapping fungi (NTF) are the majority of carnivorous microbes to capture nematodes through diverse and sophisticated trapping organs derived from hyphae. They can adopt carnivorous lifestyles in addition to saprophytism to obtain extra-nutrition from nematodes. As a special group of fungi, the NTF are not only excellent model organism for studying lifestyle transition of fungi but also natural resources of exploring biological control of nematodes. However, the carnivorous mechanism of NTF remains poorly understood. Nowadays, the omics studies of NTF have provided numerous genes and pathways that are associated with the phenotypes of carnivorous traits, which need molecular tools to verify. Here, we review the development and progress of gene manipulation tools in NTF, including methodology and strategy of transformation, random gene mutagenesis methods and target gene mutagenesis methods. The principle and practical approach for each method was summarized and discussed, and the basic operational flow for each tool was described. This paper offers a clear reference and instruction for researchers who work on NTF as well as other group of fungi.

KEYWORDS:

• nematode-trapping fungi
• transformation method
• restriction enzyme mediated integrations
• homologous recombination
• CRISPR

1. Introduction

Nematode-trapping fungi (NTF) are a kind of fascinating filamentous fungi capable of capturing free-living nematodes by forming sophisticated trapping structures (Yang et al. 2012). NTF live mainly as saprobes in soil. When sensing the presence of living nematodes, they adopt carnivorous lifestyle in addition to saprophytism by developing delicate trapping structures to capture free-living nematodes for extra nutrients. The special lifestyle transition of NTF makes them attractive and appealing to biologists who are interested in fungal ecology and evolution (Yang et al. 2007). They also receive considerable attentions as biocontrol agents against parasitic nematodes of crops and livestock that can cause devastating economic losses. Nowadays, attempt to improve the virulence of NTF against nematode pests are conducting and biological nematicides containing NTF have been commercialized or are under developing (Abawi and Widmer et al. 2000; Dong and Zhang 2006; Khan et al. 2006; Singh et al. 2012; Li et al. 2015).

The species of NTF affiliate in different lineages including Zoopagomycota, Ascomycota and Basidiomycota but more than 90% of the species are Ascomycetes. NTF produce various trapping devices which can be divided into 2-dimensional adhesive traps (adhesive knobs, adhesive columns, non-constricting ring), 3-dimensional adhesive trap (adhesive nets) and mechanical trap (constricting rings). Generally, the process of NTF attacking nematodes includes attraction and trap inducing, adhesion and recognition, penetration and nematode consuming (Dijksterhuis et al. 1994). In order to understand the mechanisms of lifestyle transition, predation process and virulence of NTF, multiple omics studies have been conducted (Table 1), including genomics (Yang et al. 2011; Meerupati et al. 2013; Liu et al. 2014; Youssar et al. 2019; Fan et al. 2021), transcriptomics (Fekete et al. 2008; Andersson et al. 2014; Ramesh et al. 2015; Pandit et al. 2017; Zhang et al. 2020), proteomics (Andersson et al. 2013; Liang et al. 2019) and metabolomics (Wang et al. 2018; Kuo et al. 2020). Those studies have revealed numerous functional genes, gene families, pathways, proteins and metabolites that involve in growth, sporulation, evolution and predatism. Comparative omics studies are also under progress to uncover the common and specific nature of lifestyle transition as well as the mechanism of predatory process in NTF (Pandit et al. 2017; Kuo et al. 2020). Thus, molecular tools are prerequisite to verify those gene functions in NTF.

Table 1. Omics studies in NTF.

Omics studies	Traps	Species	Source
Genomics	AN	Orbilia oligospora ATCC 24927 (=Arthrobotrys oligospora ATCC 24927) (Assembly AOL24927 1.0)	NCBI ID:98790 (Yang et al. 2011)
		Arthrobotrys flagrans (Assembly DF_1.0)	NCBI ID:83355 (Youssar et al. 2019)
		A. oligospora TWF154	(Yang et al. 2020)
	AB	Dactylellina cionopaga (Assembly ASM1218435v1)	NCBI ID:88854 2020
	AK	Dactylellina haptotyla CBS 200.50 (Assembly MHA_v2)	NCBI ID:17457 (Meerupati et al. 2013)
	CR	Drechslerella stenobrocha 248 (Assembly DREv1)	NCBI ID:11565 (Liu et al. 2014)
		Drechslerella dactyloides (Assembly ASM2006472v1)	NCBI ID:106045 (Fan et al. 2021)
Transcriptomics	AN	Arthrobotrys oligospora	(Andersson et al. 2014)
		A. conoides GeneBank No. JX979095	(Ramesh et al. 2015)
		Arthrobotrys conoides	(Pandit et al. 2017)
		Duddingtonia flagrans GenBank No. JX979094	(Pandit et al. 2017)
		Duddingtonia flagrans GeneBank No. SAMN05504105	(Zhang et al. 2020)
	AB	Monacrosporium cionopagum	(Andersson et al. 2014)
	AK	Monacrosporium haptotylum	(Fekete et al. 2008)
	CR	Arthrobotrys dactyloides	(Andersson et al. 2014)
Proteomics	AN	Duddingtonia flagrans	(Liang et al. 2019)
	AK	Monacrosporium haptotylum	(Andersson et al. 2013)
Metabolomics	AN	Arthrobotrys oligospora	(Wang et al. 2018)
		A. oligospora	(Kuo et al. 2020)
		A. thaumasia	(Kuo et al. 2020)
		A. musiformis	(Kuo et al. 2020)

Note: AN represents adhesive nets, AB represents adhesive branches, AK represents adhesive knob, CR represents constricting rings.

Compared with bacteria and yeasts, the development of molecular tools in filamentous fungi is confined due to the cell complexity, such as multicellular morphology, thick chitinous cell wall and multinuclear cell. Great effort to establish multiple molecular tools in NTF has been carried out and the versatile CRISPR system has been established in adhesive net-forming NTF Duddingtonia flagrans (Youssar et al. 2019) and Arthrobotrys oligospora (Chen et al. 2021). Therefore, we summarize the molecular tools that have been established in NTF. This review covers the chassis of genetic manipulation and methodology of transformation, random gene mutagenesis methods and target gene mutagenesis methods, and aims to offer comprehensive understanding and practical strategy for NTF researches.

2. The chassis of genetic manipulation

Genetic manipulation serves the purpose of providing a better understanding of gene function as well as constructing novel mutant. It is achieved by selective breeding traditionally but directly DNA manipulation nowadays. The methods to modify DNA and introduce DNA to the host are two prerequisites. Before that, promoters, selection markers, and fluorescence indicators are fundamental elements needed to be considered for further genetic manipulation.

2.1. Promoters

The choice of promoter is a critical step in initiating gene transcription and optimizing the efficiency and stability of recombinant protein production (Adnan et al. 2022). There are three types of promoters in filamentous fungi, e.g. the RNA polymerase I, II, and III promoters. Among them, the RNA polymerase II promoters contain constitutive and inducible types, and are extensively applied in genetic manipulation. Up to now, the constitutive promoter used in NTF contains heterologous ones and endogenous ones.

Heterologous promoters used in NTF include glyceraldehyde 3-phosphate dehydrogenase (gpdA) promoter, oliC promoter and tryptophan biosynthesis gene (trpC) promoter from A. nidulans, cpc-1 promoter from Neurospora crassa (Tunlid et al. 1999), o2tef promoter from Ustilago maydis (Youssar et al. 2019). Among them, the trpC promoter from A. nidulans was most commonly used in NTF. (Table 2).

Table 2. Promoters used in NTF.

Species	Name	Origin	Gene	Ref
Arthrobotrys oligospora	gpdA	A. nidulans	hph	Tunlid et al. 1999
	trpC	A. nidulans	hph	Tunlid et al. 1999
	cpc-1	N. crassa	hph	Tunlid et al. 1999
Duddingtonia flagrans	trpC	A. nidulans	hph, nat	Youssar et al. 2019
	o2tef	U. maydis	neo	Youssar et al. 2019
	oliC	A. nidulans	GFP, mCherry	Youssar et al. 2019
	tubA	D. flagrans	GFP, mCherry	Youssar et al. 2019
	H2B	D. flagrans	mCherry	Youssar et al. 2019
	sipC	D. flagrans	GFP, mCherry	Wernet et al. 2022
	artA	D. flagrans	H2B, mCherry	Yu et al. 2021
	artB	D. flagrans	H2B, mCherry	Yu et al. 2021
	artC	D. flagrans	H2B, mCherry	Yu et al. 2021
Drechslerella dactyloides	trpC	A. nidulans	hph	Fan et al. 2021

Note: hph, hygromycin-B resistance gene, nat, clonNAT resistance gene, neo, nourseothricin resistance gene, GFP, green fluorescent proteins. artA,

The published cases about endogenous promoters used in NTF were only reported in D. flagrans. The first case is that the native promoters of tubA, sipC and H2B from D. flagrans were used to express fusion fluorescent proteins endogenously to observe the location and expression levels of the target proteins (Youssar et al. 2019). For example, the natural promoter of H2B was amplified together with its coding sequence by PCR using D. flagrans genomic DNA as template, producing a 1.6 kb fragment including the ORF of DFL_000203 (0.6 kb without stop codon), + 1 kb of the 5’ region and a 1 kb fragment 3’ downstream of the locus as reported in Youssar et al.’ s study. Another case is also operated in D. flagrans, the three promoters (artA promoter: 1,407 bp, artB promoter: 1,141 bp, artC promoter: 1,567 bp) were fused with the H2B gene and mCherry respectively to monitor the expression of the artA-C genes in subcellular spatiotemporal dynamics (Yu et al. 2021). The endogenous promoters are preferred choice if researchers want to observe the expression levels of endogenous genes by expressing fusion fluorescent proteins.

Both heterologous promoters and endogenous promoters can be good choices if they drive the expression of target genes efficiently in host cells. By now, there is lack of studies in NTF about the inducible promoters which can be used for temporally adjustable expression of target genes and RNA polymerase III promoters which can be used for fine-tuned expression of small RNAs to regulate cellular conditions. Although the constitutive promoter can meet the demand in most cases, new kinds of promoters should be explored and applied for the NTF studies in future.

2.2. Selection markers

After introducing constructed DNA into host cells, selection markers help select the positive transformants effectively. The commonly used selection markers in filamentous fungi can be divided into the antibiotic resistance markers (e.g. hygromycin-B, benomyl and oligomycin), the auxotrophic markers (e.g. uracil auxotrophic markers and lysine auxotrophic markers), and the visual distinction markers (e.g. Gus and LacZ) (Wang et al. 2017). Among them, the antibiotic resistance makers including hygromycin B and geneticin (G418) have been established in the NTF (Table 3).

Table 3. Selection markers used in NTF.

Species	Name	Concentration	Reference
Arthrobotrys oligospora	hygromycin-B	100 µg/mL	Andersson et al. 2014
	geneticin (G418)	200 µg/mL	Andersson et al. 2014
	clonNAT resistance	350 µg/mL	Andersson et al. 2014
Duddingtonia flagrans	hygromycin-B	100 μg/mL	Youssar et al. 2019
	nourseothricin	60 μg/mL	Youssar et al. 2019
	geneticin (G418)	120 μg/mL	Youssar et al. 2019
Monacrosporium haptotylum	hygromycin-B	200 μg/mL	Zhen et al. 2018
Drechslerella dactyloides	hygromycin-B	100 μg/mL	Fan et al. 2021
	geneticin (G418)	200 μg/mL	Fan et al. 2021

The principle of hygromycin-B as selection marker is to inhibit protein synthesis by interfering with 70S ribosome translocation and inducing misreading of mRNA templates (Borovinskaya et al. 2008). Likewise, the geneticin (G418) also inhibits protein synthesis through binding with 80S ribosome (Prokhorova et al. 2017). Optimal concentration of antibiotics is important for transformant screening. Low concentrations lead to high false positive rate and labour-consuming process, while high concentration results in reagent waste and longer selection process. The optimal concentration of antibiotics is not only species-dependent, but also strain-dependent. For example, hygromycin-B concentration was 200 µg/ml for A. oligospora ATCC 24927 (Zhen et al. 2018), while 100 μg/mL for A. oligospora TWF154 (Yang et al. 2020). Thus, optimal antibiotics concentrations should be determined when new strain is applied. To do such experiment, the professional way is to transfer hypha fragments and/or spores (not fungus culture discs) without substrate or medium by sterilized toothpicks to the agar plate (24 well tissue culture plate is recommended) containing a series of concentrations of the antibiotics.

Gus from E. coli, a visible report marker, was also applied in A. oligospora under the control of gpdA promoter (Tunlid et al. 1999). Adding the substrate X-Gluc (5-bromo-4-chloro-3-indyol-β-glucuronide) at a concentration of 50 μg/mL into the selection medium, the positive transformants show blue appearance on agar plate (Tunlid et al. 1999). The difference of visible appearance between the wide type and transformants significantly shortened the selection process. However, further genetic confirmation should be conducted.

Some fungal strains have selection difficulties using antibiotic resistance markers because loss of inhibition after continuous culture or the extremely high resistance to the antibiotics. The auxotrophic selection markers can be an alternative. However, it seems that the two kinds of commonly used auxotrophic markers are inapplicable in NTF since urea metabolism and amino acid metabolism are essential for their lifestyle transition (Dijksterhuis et al. 1994; Wang et al. 2014). New auxotrophic markers that are capable of selecting transformants effectively without interfering the predation process and lifestyle transition in NTF should be explored in the future. Besides, new and various selection markers also provide possible choice for multiple gene deletions in NTF.

2.3. Fluorescence indicators

To illustrate a protein function, monitoring its subcellular spatiotemporal localization is required. The fluorescence indicators provide powerful tools to link to the target genes which can directly exhibit the location of target proteins during the biological process under a fluorescence microscope.

Fluorescence indicators including GFP (green fluorescent protein), RFP (red fluorescent protein), YFP (yellow fluorescent protein) and mCherry have been widely applied in filamentous fungi. Table 2 provides information about fluorescence indicators used in NTF. The first report to apply the fluorescent proteins in NTF is for adhesive network-forming D. flagrans and genes encoding GFP and mCherry were tested with gpdA promoter from A. nidulans (Youssar et al. 2019). Although genes encoding GFP and mCherry have been extensively applied in N. crassa, A. nidulans, U. maydis, C. albicans, and Botrytis cinerea, only that applied in B. cinerea was successfully expressed in D. flagrans (Youssar et al. 2019). Both mCherry and GFP genes were successfully fused to the target gene histone H2B at C-terminus to visualize nuclei in living cells (Youssar et al. 2019) and the mCherry gene was tagged with the target gene TubA at N-terminus in D. flagrans (Wernet et al. 2022). Thus, either C or N terminus of target genes can be tagged with the fluorescence indicators to effectively monitor the location and expression levels of target proteins. Obviously, application of fluorescence indicators to visualize the expression of target proteins in vivo without disrupting cells is a good choice since there was no fluorescence background detected in NTF so far.

3. Transformation methods

Transformation method helps deliver or introduce foreign nucleic acid or proteins into hosts. Several transformation methods have been applied in filamentous fungi, including protoplast-mediated transformation, Agrobacterium tumefaciens-mediated transformation (ATMT), electroporation, biolistic bombardment, and shock wave-mediated transformation (Li et al. 2017). Up to now, only protoplast transformation method and ATMT method have been established in NTF.

3.1. Polyethylene glycol (PEG) mediated protoplast transformation method

PEG mediated protoplast transformation method can deliver exogenous nucleic acids into protoplast cells due to the change of cell membrane permeability with help of PEG (De Filippis et al. 2000). Protoplast transformation method was first applied in Saccharomyces cerevisiae (Hutchison et al. 1967), then expanded rapidly to other filamentous fungi, such as Neurospora crassa (Case et al. 1979) and A. nidulans (Tilburn et al. 1983). The protoplast transformation with CaCl₂/PEG treatment was first developed in A. oligospora in 1999 (Tunlid et al. 1999), then gradually established in other hematophagous fungus including Monacrosporium sphaeroides (Jin et al. 2005) and Monacrosporium haptotylum (Zhen et al. 2018). The brief procedure of PEG-mediated protoplast transformation for NTF is summarized as following. (Figure 1).

Figure 1. Basic steps of PEG-mediated transformation method.

Procedure

Spores/hypha culture and collection

Fungus is incubated on agar plates for conidia production (normally 1 week – 4 weeks) depending on the conidiation ability of different fungi. Conidia and hypha fragments are washed from agar plates.

Hypha harvest and enzyme digestion

Circa 10⁸ conidia were then incubated in Gamborg’s B-5 basal medium (Sigma) in an Eppendorf tube on a rotary shaker (130 r/min –180 r/min) at 24 °C – 28 °C for 36 h – 48 h. The fresh mycelia are harvested on membrane filters (hydrophilic Durapore filters, Millipore) and washed with MM solution, then suspended into enzymes solution (0.5 mL – 10 mL) containing 5 mg/mL snailase, 5 mg/mL cellulase or 15 mg/mL of Glucanex for cell wall digestion to release protoplasts on shaker at 28 °C for 3 h – 12 h.

Filtration and counting

The protoplasts are passed through a plug of glass wool fitted in a funnel to remove undigested mycelium, then collected by centrifugation at 6,000 r/min for 10 min at 4 °C. After washed with 500 μL KTC or MTC buffer, the protoplasts are resuspended in 100 μL KTC and used for transformation immediately.

Mix with DNA

Circa 10⁷ protoplasts (ca. 100 μL – 500 μL) are mixed with 10 µg of vector DNA and incubated on ice for 30 min.

Regeneration

Equal or double volumes of PTC are added and mixed gently. The mixture is incubated at 28 °C for 10 min – 60 min for the regeneration of protoplasts.

Selection

200 µL – 300 µL of the mixture is then added to 10 mL OCM medium (2% agar), and poured into Petri dishes. The plates are incubated overnight at room temperature, after which they were overlaid with 10 mL OCM medium (1% agar) containing antibiotics to select for the growth of transformants.

Reagents and media

MM medium

20 mmol/L 2-(N-morpholino) ethanol-sulfonic acid (MES), pH 5.8, 1 mol/L MgSO₄.

KTC buffer

1.2 mol/L KCl, 10 mmol/L Tris–HCl, pH 7.5, 50 mmol/L CaCl₂.

MTC medium

10 mmol/L Tris-HCl, pH 7.5, 10 mmol/LCaCl₂, 1 mol/L MgSO₄.

PTC buffer

10 mmol/L Tris–HCl, pH 7.5, 50 mmol/L CaC1₂, 50% w/v PEG6000 (10 mmol/L CaC1₂, 20% w/v PEG4000 was used in another version).

OCM medium (/L)

1 g yeast extract, 1 g mycological peptone; 1 g casein hydrolysate, 10 g glucose, 273.8 g sucrose, 0.5 g KCl, 0.5 g MgSO₄•7H₂O, 1.5 g KH₂PO₄, 0.8 mg MnSO₄•4H₂O, 0.8 mg FeCl₂•4H₂O, 8.0 mg ZnSO₄•7H₂O, 0.8 mg Na₂MoO₄•2H₂O, 0.4 mg CuSO₄•5H₂O, 0.04 mg sodium tetraborate, 0.10 mg nicotinic acid, 0.05 mg thiamine, 0.25 mg pyridoxine, 0.40 mg myoinositol, 0.40 mg p-aminobenzoic acid, 0.20 mg calcium pantothenate, 0.10 mg riboflavin, 1.40 mg choline chloride, 0.01 mg biotin. The pH was set to 6.5.

Note: Normally, protoplasts should be placed on ice and the transformation process should be carried out at low temperature. Protoplast transformation method is common and effective for filamentous fungi; however, the process of protoplast transformation method involves many critical reagents. Enzymes used for digesting cell walls and regeneration conditions are needed to be optimized.

3.2. Agrobacterium tumefaciens mediated transformation (ATMT)

Foreign DNA was delivered into the genome of host cells through the binary plasmid of A. tumefaciens in ATMT. Naturally, A. tumefaciens can infect the root of injured plants through the tumor-inducing plasmid (Ti plasmid). (Van Larebeke et al. 1974) Furtherly, scientists optimize the Ti plasmid to two plasmids, one plasmid conserving the tumor-inducing ability, the other plasmid carrying the T-DNA insertion fragment between the left and right borders. (Gordon et al. 2014) Like PEG-mediated protoplast transformation, the ATMT method was also first applied in S. cerevisiae (Bundock et al. 1995), and then in many other filamentous fungi (de Groot et al. 1998; Gouka et al. 1999; Campoy et al. 2003). Nowadays, three NTF species e.g. Arthrobotrys conoides, A. oligospora (Nourani et al. 2018) and Drechslerella dactyloides (Fan et al. 2021) were reported to be successfully established with ATMT. The brief procedure of ATMT for NTF is summarized as follows. (Figure 2).

Figure 2. Basic steps of PEG-mediated transformation method.

Procedure

NTF preparation

Conidia or hyphae are inoculated on agar plates or in liquid medium and incubated for 2 weeks –3 weeks. The conidia are washed from agar plates and then resuspended in an Eppendorf tube. The mycelia in liquid culture are harvested through membrane filters (Miracloth) and washed with sterile water, then homogenized in 1.5 mL centrifuge tube with tissue grinder.

A. tumefaciens cultivation

The A. tumefaciens is inoculated on agar plates of YEP containing selection pressure and incubated at 28 °C for 24 h. The individual colony is transferred to 15 mL – 50 mL centrifuge tube or flask with minimal medium (MM) containing selection pressure and incubated at 28 °C for 24 h – 48 h under constant shaking at 100 r/min.

A. tumefaciens induction

One ml of the above culture mixture is transferred to induction medium (IM) containing selection pressure and acetosyringone and kept at 28 °C on shaker (120 r/min) for 3 h – 6 h to reach OD₆₀₀ to 0.2 – 0.4.

Co-cultivation

The collected conidia or homogenized mycelia of fungus and the A. tumefaciens culture in the IM are mixed. The mixture is inoculated and smeared with sterilized glass balls on nitrocellulose membrane pre-placed on IM plates for co-cultivation at 26 °C.

Selection

The nitrocellulose membrane with co-cultures of fungus and A. tumefaciens is transferred onto PDA plate containing selection pressures and co-cultured at 26 °C for two weeks. Putative transformants (colonies) are transferred to fresh selective agar plates for further PCR verification and phenotype assay.

Reagents and media

YEP medium

Yeast extract peptone media (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

MM medium

10 mmol/L K₂HPO₄, 10 mmol/L KH₂PO₄, 2.5 mmol/L NaCl, 2 mmol/L MgSO₄ · 7H₂O, 0.7 mmol/L CaCl₂, 9 μmol/L FeSO₄ · 7H₂O, 4 mmol/L (NH4)₂SO₄, 10 mmol/L glucose, pH 7.0.

IM medium

MM containing 0.5% (wt/vol) glycerol, 200 μmol/L acetosyringone, 40 mmol/L 2-(N-morpholino)-ethanesulfonic acid (MES), pH 5.3.

PDA

Potato dextrose agar (Difco, Detroit, Mich).

Mitotic stability test

A piece of mycelial from the colony edges of transformants are serially transferred five times at one-week intervals on PDA plate with hygromycin-B. Then the colony diameters are measured at the day 4 and 10 respectively.

Note: The ATMT is a stable transformation method and can transform diverse samples, such as spores and mycelial fragments. Since the T-DNA fragment is randomly integrate into the genome of the host cells, the ATMT is also used as a random gene mutagenesis method. According to previous studies, ectopic integration of single-copy T-DNA into non-coding region is usually observed. Factors influencing the ATMT efficiency include the type of fungal material (spores, hypha or germlings) and A. tumefaciens strains, the concentration of acetosyringone, the ratio of fungi to A. tumeaciens and the co-cultivation conditions. The A. tumefaciens strains and binary plasmids used in NTF were summarized in Table 4. Other influence factors need to be considered and optimized when ATMT efficiency is not high.

Table 4. Agrobacterium tumefaciens strains and binary plasmids used in NTF.

Name	Reference
A. tumefaciens strains
LBA4404	Nourani et al. 2018
AGL1	Nourani et al. 2018; Fan et al. 2021
Binary plasmid
pCAMBIA1304	Nourani et al. 2018
pAg1-H3	Fan et al. 2021

4. Random gene mutagenesis methods

Random gene mutagenesis offers a good choice to screen genes related to certain phenotypic properties. The mutants are generated randomly by insertion a foreign DNA or by the movement of mobile genetic elements. The commonly used methods for random gene mutagenesis in filamentous fungi include ATMT, restriction enzyme-mediated integration (REMI) and transposon-arrayed gene knockout (TAGKO) (Wang et al. 2017). The REMI was first established in S. cerevisiae (Schiestl et al. 1991) and also successfully applied in NTF (Tunlid et al. 1999). The REMI method originates from protoplast transformation, but it increases transformation frequency by delivering enzymes that can cut genomic DNA to provide integration site for the linear DNA fragment that is digested with the same enzymes.

The REMI method for random gene mutation applied in NTF was established in A. oligospora in 1999 (Tunlid et al. 1999) and 13 transformants were achieved and 7 of them were single copy integrated. Subsequently, this method was applied in M. sphaeroides and five mitotic stable mutants were obtained, three of which exhibited integration at a single location (Jin et al. 2005). Recently, this method has been applied in A. oligospora to identify novel genes related to trap formation, conidiation and virulence, 37 random-insertional mutants were screened (Jiang et al. 2017). Here, the brief procedures of REMI for NTF were summarized as follows.

Procedure

Protoplast preparation

Protoplast suspension can be prepared following the previous description and ca. 1 × 10⁷ – 10⁸ protoplasts/mL can be generated and used.

Enzyme digestion of plasmid

Recombinant plasmid such as pBChygro containing selection marker gene (hph) and restriction enzyme (including XbaI, KpnI, SmaI, XmaI, SacI, SalI and HindIII) sites is digested with the selected enzymes (HindIII). The enzyme digestion system is prepared according to the manufacturers’ instructions.

Protoplast transformation

The rate of endonuclease-induced damage to protoplasts is determined for suitable concentration (normally 30 U). For transformation, 100 μL protoplasts (ca. 8.0 × 10⁷) is mixed with the restriction enzyme (HindIII), then10 μg of the above linear vector DNA is added into the mixture. Incubate the whole transformation mixture for 30 min on ice. Then 250 μL of PTC are added and mixed gently.

Transformants selection

After incubation at 28 ^oC for 20 min – 30 min, 100 μL of the protoplast mixture is added to 10 mL of PDSSA medium containing 100 μg/mL ~ 400 μg/mL of hygromycin-B (previously kept at 48 °C), then poured onto Petri dishes. The plates are incubated at 28 ^oC for transformants growth.

PDSSA medium

PDA supplying with 0.6 mol/L sucrose, 0.3 g/L yeast extract, 0.3 g/L tryptone, 0.3 g/L peptone.

Note: The transformation frequency of the REMI is affected by the concentration of restriction enzyme and PEG. The highest transformation frequency of 175 transformants/μg of DNA was obtained (the concentration of PEG3350 was 20%) when 30 units HindIII per transformation reaction was used (Xu et al. 2005). There was no obvious difference of transformation frequency when PEG3350, PEG4000 or PEG6000 were applied (Xu et al. 2005). Another advantage of REMI is that the disrupted genes can be obtained by plasmid rescue in E. coli, which is more efficient for subsequent function studies.

Except molecular tools, gene mutagenesis can be induced by physical mutagens or chemical treatment. The ethyl methane sulfonate and UV as the mutagens were applied in NTF to identify potential genes and pathways involved in trap morphogenesis and predation in A. oligospora, and 15 mutants with strong defects in trap morphogenesis were identified from 5,552 randomly mutagenized A. oligospora mutants (Huang et al. 2021). Although the process of screening is labor-consuming and tedious, physical or chemical mutagens are easily to apply and screen multiple potential genes for certain physiological features without conducting genetic manipulation.

5. Target gene mutagenesis methods

5.1. Homologous recombination (HR)

HR is a common strategy for deleting target genes in genetic manipulation. The principle is based on the HR pathway of DNA double strands breaks repairing (DSBR) by delivering foreign DNA sequences flanked by homologous fragments (Jasin and Haber 2016). A number of target genes in NTF have been knocked out through HR approach. The homologous sequence length used in NTF ranges from 1.3k bps to 2.5k bps, but the most frequently used length is around 2k bps (Table 5).

Table 5. Gene deletion in NTF through HR.

Species	Gene	Gene function	DNA length (bp)	Reference
Arthrobotrys oligospora	AoBck1	Cell wall integrity	5’: 1911 3’: 2047	Xie et al. 2021
	AoMkk1		5’: 1835 3’: 2141	Xie et al. 2021
	AoATG5	Autophagy	5’: 1991 3’: 1989	Zhou et al. 2021a
	Aolatg4		5’: 2150 3’: 2098	Zhou et al. 2020
	Aoatg8		5’: 2029 3’: 364	Chen et al. 2013
	Aoatg1		5’: 2046 3’: 1938	Zhou et al. 2021b
	Aoatg2		5’: 2106 3’: 2229	Zhou et al. 2021b
	AoSlt2	MAPK pathway	5’: 2123 3’: 2009	Zhen et al. 2018
	AoIme2	Inducer of meiosis	5’: 1866 3’: 2076	Xie et al. 2020
	AoCaMK A	Calcium/calmodulin-dependent protein kinases	5’: 2014 3’: 1890	Zhen et al. 2019
	AoCaMK B		5’: 2146 3’: 1962	Zhen et al. 2019
	AoCaMKKC1		5’: 1601 3’: 2041	Zhen et al. 2019
	AoCaMKKC2		5’: 1816 3’: 1792	Zhen et al. 2019
	AoCaMKD		5’: 1865 3’: 1977	Zhen et al. 2019
	AoGPB1	G-protein signalling	5’: 2000 3’: 1000	Bai et al. 2021
	AoRic8		5’: 1842 3’: 2094	Bai et al. 2021
	AoFlbA		/	Ma et al. 2021
	AoRgsA		/	Ma et al. 2021
	AoRgsB		/	Ma et al. 2021
	AoRgsB2-1		/	Ma et al. 2021
	AoRgsB2-2		/	Ma et al. 2021
	AoRgsB2-3		/	Ma et al. 2021
	AoRgsC		/	Ma et al. 2021
	AoMad1	Adhesin protein	/	Liang et al. 2015
	AoPalH	pH sensor	5’:2002 3’:2153	Li et al. 2019
	AoNoxA	NADPH oxidase	/	Li et al. 2017b
	AoFig1	Low-affinity calcium uptake system proteins	5’:1350 3’:1323	Zhang et al. 2019b
	AoFig2		5’:1364 3’:1342
	AoRab-2	Rab GTPase	5’:1895 3’:1975	Yang et al. 2018
	AoRab-7A		5’:2087 3’:1937	Yang et al. 2018
	AoCrn1	Actin-associated protein	/	Zhang et al. 2017
	AoHex1	Woronin body component	5’:1971 3’:1719	Liang et al. 2017
	AoMls	Malate synthase	5’:1833 3’:1719	Zhao et al. 2014
	AoGph1	Glycogen phosphorylase	/	Wu et al. 2016
	AovosA	Transcription factors	5’:2081 3’:1913	Zhang et al. 2019a
	AoVelB		5’:2122 3’:2048	Zhang et al. 2019a
	AoStuA		5’:1586 3’:1876	Xie et al. 2019
	AoQde2	MicroRNA-processing protein	/	Ji et al. 2020
	AoRas2	Ras GTPases	5’:1914 3’:1910	Yang et al. 2021
	AoRas3		5’:1721 3’:1994	Yang et al. 2021
	AoRheb		5’:2023 3’:2038	Yang et al. 2021
	AoGlo3	Arf-GAPs	/	Ma et al. 2021
	AoRho2	Rho GTPases	/	Yang et al. 2022a
	AoRac
	AoCdc42
	AoYBP-1	YAP- binding protein 1	5’:2000 3’:2000	Yang et al. 2021
	AoSTE7	MAPK pathway	1.5k −2k	Ma et al. 2020
	AoFUS3			Ma et al. 2020
	AoSte12			Ma et al. 2020
	AoGlo3	Pleiotropic regulator	/	Ma et al. 2020
	AOL-s00215g277	Cupin-like family gene	5’:2000 3’:2000	Chen et al. 2020
	AOL-s00215g279	Dehydrogenase	5’:2000 3’:2000	Chen et al. 2020
	AOL_s00215g281	Amidohydrolase	5’:1970 3’:2021	Xu et al. 2016
	AOL_s00215g282	Cytochrome P450 monooxygenase	5’:1974 3’:1777	Xu et al. 2016
	AOL_s00079g287	PKS III-1	/	Wang et al. 2018
	AOL_s00079g828	PKS I-1	/	Wang et al. 2018
	AOL_s00079g496	PKS I-2	/	Wang et al. 2018
	AOL_s00079g926	PKS I-4	/	Wang et al. 2018
	AOL_s00079g283	PKS I-3	5’: 2114 3’: 2117	Xu et al. 2015
	AOL_s00215g280	Cytochrome P450 monooxygenase	5’: 2064 3’: 1812	Song et al. 2017
	AOL_s00215g278	P450 gene	/	Teng et al. 2020
	AOL_s00215g273	Acetylation of SECs	/	Teng et al. 2020
	AoniaD	Nitrate assimilation pathway	5’: 1948 3’: 1873	Liang et al. 2016
	AoniiA		5’: 2067 3’: 1803	Liang et al. 2016
	AonrtB		5’: 1755 3’: 1797	Liang et al. 2016
	AonirA		5’: 1901 3’: 1787	Liang et al. 2016
	AoPEX1	Peroxisome biogenesis	5’: 2124 3’: 2384	Liu et al. 2022
	AoPEX6		5’: 1807 3’: 1810	Liu et al. 2022
	Aossk1	Response regulator	5’: 2153 3’: 2081	Jiang et al. 2022
	AoUBR1	N-end rule pathway	5’: 1545 3’: 1356	Zhang et al. 2021
	Aog207	F-box-domain-encoding gene	5’: 2000 3’: 2000	Peng et al. 2022
	AOL_s00054g276	Lectin gene	5’: 2500 3’: 2500	Si et al. 2022
	AopdeH	cAMP phosphodiesterases	5’: 2179 3’: 2082	Ma et al. 2022
	AopdeL		5’: 2528 3’: 1897	Ma et al. 2022
	AoGcs1	Arf-GAP proteins	5’: 2050 3’: 2561	Yang et al. 2022b
	AoGts1		5’: 2057 3’: 2038	Yang et al. 2022b
	Aoplc2	Phospholipase C	5’: 1198 3’: 2140	Xie et al. 2022
	YBP1	YAP-binding protein 1	5’: 2000 3’: 2000	Huang et al. 2021
Monacrosporium haptotylum	MhSlt2	MAPK pathway	5’: 1971 3’: 1905	Zhen et al. 2018
Drechslerella dactyloides	DdaSTE12	MAPK pathway	5’: 2598 3’: 2576	Fan et al. 2021
Duddingtonia flagrans	artA	Polyketide synthase gene cluster	5’: 856 3’: 1039	Yu et al. 2021
	artB		5’: 1430 3’: 931	Yu et al. 2021
	artC		5’: 1141 3’: 1067	Yu et al. 2021
	artD		5’: 1311 3’: 912	Yu et al. 2021
	SipC	STRIPAK component	5’: 1000 3’: 1000	Wernet et al. 2022

Note: hygromycin (hph) phosphotransferase, NAT1, nourseothricin acetyltransferase gene, G418, geneticin, MAPK, Mitogen-activated protein kinases, SECs, Sesquiterpenyl epoxy-cyclohexenoids.

Usually, expression cassette of the gene sequence encoding selection marker locates between the forward and downstream homologous fragments. In a special case, the author not only insert the selection marker expression cassette between the homologous fragments, but also smartly altered the start codon ATG to a stop codon TAG in the forward fragment sequence using Gene Editor mutagenesis kit (Balogh et al. 2003). Total 15 transformants were screened through PCR assay from two hundred mutants and 5 mutants were identified to contain the target integrated fragments by Southern blot analysis (Balogh et al. 2003). This strategy provides double insurance for target gene knockout.

HR strategy is a good choice for target gene deletion, but the deletion rate is depending on the HR rate of the host. In mammalian cells, non-homologous end joining (NHEJ) pathway is dominant over the HR-pathway when DNA double strands breaks happen (Jasin and Haber 2016). Thus, in order to improve the HR rate to further increase the gene deletion efficiency, researchers usually delete genes essential in NHEJ pathway, e.g. ku70 and ku80. It is reported that the HR rate in A. oligospora was extremely low (~3%) (Kuo et al. 2020). A NHEJ deficient A. oligospora mutant generated by deleting ku70 showed no phenotypic difference from the wide type but improved HR rate (Kuo et al. 2020). The HR strategy is still the first choice for gene manipulation in NTF although it is difficult to delete multiple genes.

5.2. CRISPR technique

The CRISPR is abbreviation of the clustered regularly interspaced short palindromic repeats. The type II CRISPR/CRISPR-associated gene (Cas) system is the most used genome-editing tool currently (Lanigan et al. 2020). The principle to disrupt target genes through CRISPR technique is using RNA-guided nuclease to nick DNA double strand breaks at target gene composed of a 20 bp sequence matching the guide RNA (gRNA) and an adjacent downstream 5’-NGG nucleotide sequence (protospacer-adjacent motif (PAM) (Nødvig et al. 2015). The CRISPR technique is originated from an immune defence system for foreign DNA invasion in bacteria and archaebacteria. In filamentous fungi, it was first established in Trichoderma reesei (Liu et al. 2015).

CRISPR technique was applied in NTF since 2019 in Duddingtonia flagrans (Youssar et al. 2019). After attempt was failed with the plasmid-based approach, the Cas9 ribonucleoproteins (RNPs) was applied with tested simultaneous transformation of the linear resistance gene hph and the repair template. Total four transformants were obtained and the resistance gene hph was successfully inserted into the cleavage site of three of the four transformants. Then, the RNP form of CRISPR system was also applied in A. oligospora (Chen et al. 2021). The basic procedures for RNP form of CRISPR in NTF are as follows.

Procedure

sgRNA design

The sgRNA was designed using online website (http://crispr.mit.edu). To avoid off-target cleavage, the uniqueness of the protospacer region and the protospacer adjacent motif (PAM) sequence should be analyzed in the host genome using BLASTN.

sgRNA synthesis

The sgRNA for the target gene mutation can be synthesized using the EnGen sgRNA Synthesis Kit (New England Biolabs, Frankfurt).

Ribonucleoprotein formation

RNP formation is carried out at 37 °C for 20 min with 4 μL sgRNA, 10 μL Cas9 (200 nmol/L), 5 μL 10x Cas9 Nuclease Reaction Buffer and DEPC-treated water in 50 μL total volume.

Linear resistance gene amplification

The linear resistance gene is amplified by PCR, subsequently purified using FastGene Gel/PCR Extraction kit (Nippon Genetics) and lastly about 2 μg purified PCR-product is added to the transformation-mix.

Protoplast preparation

The protoplast of NTF is prepared as described above (Xu et al. 2005).

Protoplast transformation

100 μL protoplasts (ca. 5 × 10⁶) are mixed with 5 μg – 8 μg of DNA and incubated for 2 min on ice. Then 1 mL of PTC is added and incubated at room temperature for 20 min. 10 mL PDSSA medium is added to the transformation mix and poured onto PDA plates supplemented with corresponding antibiotics and the plates incubate at 28 °C for 4 – 7 days.

The mentioned reagents and media in the procedure are covered in previous description (Youssar et al. 2019).

CRISPR technology is a powerful and versatile tool of gene editing and can manipulate multiple genes at the same time. However, off target effect and cell damage effect in CRISPR limit its application to some extent (Haapaniemi et al. 2018; Ihry et al. 2018; Manghwar et al. 2020). RNP and plasmid are the two kinds of strategies for delivering the CRISPR system. RNP strategy extremely depends on the protoplast transformation method, while the plasmid strategy is still not established in NTF yet. The sgRNA array construction strategies and Cas proteins choice are two factors to be considered. Currently, CRISPR is only used for target gene mutagenesis in NTF and extensive application of CRISPR technique should be exploited in future.

When gene deletion is vital to host cells, RNA interference (RNAi) technology is a pivotal alternative to investigate the function of target gene. Instead of changing DNA sequence to eliminate or impair the function of target genes, RNAi repress the transcription of target genes by triggering gene-silencing mechanism in the host to degrade the target mRNA. Although it has been successfully applied in many filamentous fungi (Zheng et al. 1998; Ngiam et al. 2000; Kadotani et al. 2003; Janus et al. 2007), but still not be extensively applied in NTF. RNAi approach is just established for a knob-forming NTF in our lab. Knockdown of the transcription of DhFIG_2, a component of NTF-specific low-affinity calcium uptake system in knob-forming Dactylellina haptotyla by hairpin RNA forming binary plasmid together with ATMT significantly decreased its conidiation and knob formation (Zhao et al. 2023). RNAi will offer a good genetic manipulation option in NTF when target genes are indispensable.

6. Summaries and perspectives

Nematode-trapping fungi are the important enemies to regulate nematode dynamics in nature. As fascinating creatures with dual lifestyles of saprophytism and carnivorism and sophisticated trapping devices, NTF is attracting researchers to study the mystery of their lifestyle, trapping mechanisms and morphogenesis. Genetic manipulation is an essential and basic tool for NTF studies. Here we summarized the current development and application of gene manipulation tools and basic procedures in NTF, which should provide crucial methodological reference to promote the NTF studies. However, other molecular tools should be established in NTF in the near future, such as inducible promoters, auxotrophic markers, the transposon-arrayed gene knockout, the plasmid form CRISPR, as well as the RNA interference strategies. We believe those tools are under establishing by mycologists who work on NTF.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

The research is supported by the National Natural Science Foundation of China (31770065, 32200009), the National Key Research and Development Program (2018YFD0201202).