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. 2024 Jun 26;17(6):e14512. doi: 10.1111/1751-7915.14512

An efficient visual screening of gene knockout mutants in the insect pathogenic fungus Beauveria bassiana

Ajing Mao 1, Junyao Wang 1, Shengan Zhu 1, Dan Jin 1, Yanhua Fan 1,✉
PMCID: PMC11201804  PMID: 38923821

Abstract

Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time‐consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana.

Using the red pigment oosporin as a colour vision marker, ectopic transformants obtained through Agrobacterium tumefaciens‐mediated gene transformation predominantly exhibit a red colour with oosporein production. Knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. The correct mutants were obtained by verifying a few white transformants.

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INTRODUCTION

Beauveria bassiana is an entomopathogenic fungus that has been developed as environmentally friendly biopesticides for agricultural and forestry pest control (Meyling & Eilenberg, 2007; Zimmermann, 2007). It is also a model organism for investigating the interactions between pathogens and their hosts (Hajek & St Leger, 1994; Ownley et al., 2004). In addition to infecting insects, B. bassiana is able to grow in plants (endophytes), improving plant growth and enhancing disease resistance against phytopathogens (Ownley et al., 2008; Quesada‐Moraga et al., 2014). Therefore, B. bassiana plays multiple roles in the agricultural ecosystem. Unveiling the functions of specific genes involved in these processes is crucial for understanding fungal interactions with various hosts and improving the control efficiency of fungal agents on plant pests and diseases.

Gene disruption is a highly effective technique for studying gene function, with wide‐ranging applications in molecular biology, genetics, and medical research (St Leger & Wang, 2010). Homologous recombination and genome editing using CRISPR/Cas9 are two vital methods for disrupting fungal genes. The CRISPR/Cas9 system utilizes RNA‐mediated endonuclease to introduce DNA strand gaps at specific target sites in the genome, which can stimulate the host defence mechanisms to repair gaps (Luo et al., 2023). The CRISPR/Cas9 system has high editing efficiency. However, the stable expression of Cas9 protein in mutants also led to the risk of off‐target effect in strains (Cradick et al., 2013). In B. bassiana, most of the genes studied were disrupted by homologous recombination (dos Reis et al., 2004). While the efficiency of obtaining correct mutants is low, screening hundreds or thousands of transformants is often necessary. This process typically involves laborious, high‐cost or time‐consuming tasks, such as preparing genomic DNA, performing PCR, and conducting electrophoresis. To improve the mutant screening efficiency, the green fluorescent protein (GFP) or β‐glucuronidase (GUS) has been introduced into mutant construction as counter‐selective markers (Bae & Knudsen, 2000; Ying & Feng, 2006). In these systems, the GFP or GUS gene is lost upon homologous recombination, distinguishing the mutant from ectopic mutants. These strategies have enhanced the selective efficiency of mutants to a certain extent. However, the utility of GFP or GUS is constrained by the need for specialized instruments or chemicals, and the requirement for labour‐intensive detection procedures.

Beauveria bassiana generates a diverse array of secondary metabolites during its growth and infection of hosts. These compounds could effectively dampen the host's immune response, inhibit the proliferation of competing microorganisms within the host, or directly exhibit toxicity towards insects (Bidochka et al., 2010; Zhang et al., 2020). Oosporein, a red pigment synthesized via the polyketide synthase (PKS) pathway (Opn cluster), exhibits insecticidal, antiviral, and antibacterial activities (Amin et al., 2010; Fan et al., 2017; Feng et al., 2015; Terry et al., 1992). Beauveria bassiana synthesizes oosporein under alkaline conditions (pH > 8) or within fungus‐killed insects. However, no oosporein is produced in conventional artificial media with pH levels below 7.0 (Luo et al., 2015). OpS3 is a Gal4‐like transcription factor located in Opn gene cluster. Overexpressing OpS3 upregulated the expression of oosporein synthetic genes in Opn cluster and led to abundant production of oosporein in B. bassiana.

In this study, a novel screening strategy was developed to easily screen the correct knockout mutants in B. bassiana. By placing a constitutive OpS3 expression cassette outside one of the two homologous arms, most ectopic transformants appear red due to oosporein production. When recombinant events occurred, the OpS3 expression cassette was lost and resulted in white colonies. Therefore, only a small number of white transformants needed to be further verified to identify the knockout mutants. This strategy significantly improves the selective efficiency of mutants and reduces the experimental cost.

EXPERIMENTAL PROCEDURES

Microbial strains and media

Beauveria bassiana strain CGMCC7.34 (China General Microbiological Culture Collection Center) was routinely cultivated on Sabouraud dextrose agar (SDA, BD, Difco), potato dextrose agar (PDA, BD, Difco), or Czapek Dox agar (CZA, BD, Difco). Escherichia coli DH5α was used for plasmid amplification and was cultured in Luria–Bertani (LB) medium supplemented with Kanamycin (50 μg/mL). Agrobacterium tumefaciens AGL‐1 was utilized for transforming B. bassiana and was maintained on Yeast Mannitol Medium (YEB) supplemented with Kanamycin (50 μg/mL) and Carbenicillin (60 μg/mL).

Constitutive expression of OpS3 in B. bassiana

The constitutive promoter BbgpdA and transcription factor OpS3 were amplified from the genome of B. bassiana using specific primer pairs P BbgpdA ‐F/P BbgpdA ‐R and P OpS3 ‐F/P OpS3 ‐R, respectively (Table S2). The gene fragments of BbgpdA and OpS3 were cloned into the HindIII site of pK2‐Sur/Bar with the ClonExpressTM MultiS one‐step cloning kit (Vazyme, C113‐01). The construct (pK2‐Sur/Bar‐BbgpdA‐OpS3) was verified by sequencing and transformed into B. bassiana mediated by A. tumefaciens AGL‐1 as described (Fang et al., 2004). Transformants were inoculated on CZA, PDA, and SDA media, and oosporein production was observed.

Construction of targeted gene knockout vectors

For construction of the tenS (EJP63694) and NRPS (EJP62835) gene deletion vectors, the sulfonylurea resistance gene (Sur) cassette (~3.6 kb) was utilized to replace a 0.8 kb gene fragment within the tenS gene, and the phosphinothricin acetyltransferase gene (Bar) cassette (~1.6 kb) was used to substitute a 1 kb gene fragment within the NRPS gene. The upstream (L tenS ) and downstream (R tenS ) fragments of the construct were amplified via PCR with primer pairs PLtenS ‐F/PLtenS ‐R and PRtenS ‐F/PRtenS ‐R (Table S2), using B. bassiana genomic DNA as the template, and then cloned into the EcoRI (L tenS ) and XbaI (R tenS ) sites of pK2‐Sur/Bar‐BbgpdA‐OpS3, respectively. The obtained pK2‐L tenS ‐Sur‐R tenS ‐BbgpdA‐OpS3 vector was verified by sequencing (Invitrogen). The same method was used to construct the NRPS gene deletion vector.

Screening and identification of transformants

Fungal transformants grown on CZA with glufosinate or sulfonylurea were picked onto a 48‐well plate with SDA media to observe oosporein production. Compared to random insertion transformants (red colonies), a homologous recombination event resulted in the loss of OpS3 expression cassette and thus no oosporein production (white colonies). Putative homologous recombination transformants are further verified by PCR. DNA preparation for PCR was followed our previous method (Fan et al., 2015). Primers P tenS T1/P tenS T2 or P NRPS T1/ P NRPS T2 were used for the PCR verification.

qRT‐PCR analysis

A 10 μL system was prepared using cDNA as a template with SYBR Premix Ex Taq II master mix (Takara, Shiga, Japan), and a BIO‐RAD CFX96 (BIO‐RAD, Hercules, CA, USA) instrument was utilized to analyse the relative expression levels. The primers are listed in Table S2. The actin was chosen as an internal reference for standardized measurement of gene expression. All quantitative real‐time PCR reactions were repeated with at least three replicates.

Extraction and detection of tenellin

The mycelia and CZA medium cultured in the dark at 26°C for 15 days were collected and extracted in 0.1 M NaOH solution by sonication at 50°C for 30 min, and the extraction was repeated until there was no obvious pigment in the medium. Filtration was used to remove impurities, pH was adjusted to approximately 3.0 with dilute hydrochloric acid, and left at 4°C for 2 h. The supernatant was discarded by centrifugation, and the precipitate was dried completely at 45°C. The precipitate was dissolved in 100% methanol, and the solution was filtered through a 0.22 μm microporous filter membrane.

Tenellin was detected by a reverse‐phased C18 column (ZORBAX C18, 2.1 × 150 mm, 1.8 μm, Shimadzu SIL‐20A). The detector was PDA with a wavelength of 210/254 nm. Methanol‐H2O was used as the mobile phase, and the detection procedure was as follows: 5% methanol at 0–5 min, 5%–100% methanol at 5–30 min, and 100% methanol at 30–35 min.

RESULTS

Knockout vector construction for visualized screening

OpS3 is a GAL4‐type transcription factor located in oosporein synthetic gene cluster and positively regulated the expression of oosporein synthetic genes (Figure 1A). The constitutive promoter BbgpdA and transcription factor OpS3 fragments were ligated to pK2‐Sur/Bar through HindIII site (Figure 1B). The OpS3 overexpression strains were obtained by A. tumefaciens‐mediated fungal transformation (Figure 1C). The OpS3 OE strains produced large amounts of red oosporein on SDA (Figure 1D). We hypothesized that it would be possible to use this red pigment as a negative selective marker to easily screen gene deletion mutants by ruling out T‐DNA random insertion mutants. For gene deletion, there are two types of transformants, including ectopic mutants obtained by T‐DNA random insertion, and deletion mutants resulting from homologous recombination. When T‐DNA insertion occurs, both the resistance gene and the transcription factor OpS3 expression cassette are integrated into the genome of B. bassiana. In this case, the mutants can grow in a medium containing antibiotic and produce red oosporein because of the expression of OpS3 (Figure 2A). When homologous recombination occurs, the target gene that needs to be knocked out is replaced by the resistance genes, and therefore the OpS3 expression cassette is lost. Thus, no oosporein is produced in homologous recombinant mutants, which can be distinguished from the random insert transformants that produce oosporein (Figure 2B).

FIGURE 1.

FIGURE 1

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Expression of OpS3 gene in Beauveria bassiana led to oosporeion production. (A) Conserved domain in OpS3 was predicted by SMART. GAL4, GAL4‐like Zn(II)2Cys6 binuclear cluster DNA‐binding domain. Fungal trans, fungal‐specific transcription factor domain. TR, transmembrane region. (B) Construction of OpS3 overexpression vector. (C) Molecular identification of the BbgpdA::OpS3 strains. M, Maker 5000. N, Negative control; P, positive control. Lane 1 to 4，four transformants were randomly selected for verification, and PCR was performed with primer P BbgpdA ‐F/P OpS3 ‐R. A fragment of ~3.7 kb was amplified in correct transformants and positive control. (D) Plasmids pK2‐Sur and pK2‐Sur‐BbgpdA‐OpS3 were transferred into B. bassiana. Most of the transformants produce red pigment in SDA when OpS3 expressed.

FIGURE 2.

FIGURE 2

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Schematic of gene knockout using oosporein as a visual selective marker. (A) Homologous arms of target gene are cloned into the EcoRI and XbaI sites of pK2‐Bar/Sur vector. Two types of transformants are obtained after transformation, including random T‐DNA insertion and homologous recombination. (B) The phenotype of transformants on a 24‐well plate. The T‐DNA insertion transformants appear red as the expression of OpS3 and oosporein production. However, the homologous recombination mutant is white due to the loss of OpS3 expression cassette.

Effects of culture media on oosporein production

The effects of culture media on oosporein production were determined by inoculating OpS3 OE transformants on CZA, PDA, and SDA. It was observed that only a very few BbgpdA::OpS3 transformants produced weak red pigments in CZA medium (Figure 3A). However, on PDA or SDA media, all transformants exhibited obvious oosporein production, indicating that culture media are an important factor affecting oosporein production (Figure 3B–D).

FIGURE 3.

FIGURE 3

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The efficiency of oosporein production of BbgpdA::OpS3 strains on different culture media. (A–C) The transformants were inoculated on CZA, PDA, and SDA media, respectively. (D) The rate of oosporein production in fungal colonies on different culture media.

Deletion of tenS and NRPS

To assess the efficiency of the visual screening system, the tenellin synthetase gene (tenS) and a nonribosomal peptide synthetase gene (NRPS) were chosen to construct mutants. A fragment within the tenS gene (~ 0.8 kb) was selected as the deletion region (Figure 4A). After transformation, the obtained transformants were plated onto SDA medium. Most transformants exhibited red pigment production on 48‐well plates; however, two white transformants, lacking pigment production, were obtained and verified using primers P tenS T1 and P tenS T2 (Figure 4B). One of the transformants was confirmed to be correct by regular PCR and qRT‐PCR analyses (Figure 4C,D). Compared with wild‐type strain, the tenS mutant did not produce yellow pigment (tenellin) on nitrogen‐deficient medium (Figure 4E,F). These results indicate that tenS had been successfully deleted.

FIGURE 4.

FIGURE 4

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Construction and identification of tenS knockout mutants. (A) The schematic diagram for tenS deletion by homologous recombination with visual screening marker OpS3. (B) Mutants were selected by different colours. Mutants that did not produce red pigment could be the tenS knockout mutants and were circled in blue. (C) PCR verification of ΔtenS mutants with primer pairs P tenS T1/P tenS T2. (D) qRT‐PCR analysis of tenS expression in WT and ΔtenS strains. (E) The phenotype of ΔtenS. Conidial suspension (1 μL, 106 conidia/mL) was inoculated on nitrogen‐deficient medium and cultured at 26°C for 5 daya. (F) Detection of tenellin by HPLC. The green arrow indicates the peak of tenellin.

Nonribosomal peptide synthetases (NRPSs) are a class of cytosolic enzymes that synthesize various bioactive secondary metabolites such as antibiotics and siderophores, and are prevalent in both prokaryotes and eukaryotes (Suring et al., 2023). We constructed the knockout vector of an NRPS with Bar resistance gene (Figure 5A). Four white colonies were tested by PCR screening, and two mutants exhibited the predicted DNA fragments (Figure 5B,C). The qRT‐PCR results showed that NPRS expression was undetectable in both knockout mutants (Figure 5D).

FIGURE 5.

FIGURE 5

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Construction and identification of NRPS knockout mutants. (A) Schematic diagram of homologous replacement of NRPS by the Bar gene cassette. (B) Mutants were selected by different colours. Mutants that did not produce red pigment could be the NRPS knockout mutants and were circled in blue. (C) PCR verification of ΔNRPS mutants with primer pairs P NRPS T1/P NRPS T2. (D) qRT‐PCR analysis of NRPS expression in WT and ΔNRPS strains.

Knockout efficiency comparison

The traditional method, which lacks the OpS3 expression cassette, was also utilized for the deletion of tenS and NRPS. Genomic DNA templates from 288 transformants of each gene were prepared and utilized for PCR validation. As a result, three deletion mutants of tenS and two deletion mutants of NRPS were successfully obtained. The efficiencies of the traditional method for obtaining tenS and NRPS knockouts were 1% and 0.7%, respectively, which was significantly lower compared to the designed visual screening system that achieved a positive efficiency exceeding 50% (Table S1).

DISCUSSION

Gene disruption is a crucial technique for elucidating gene functions in entomopathogenic fungi. However, the low efficiency and labour‐intensive nature of gene deletion often hinder gene function studies. Homologous recombination‐mediated gene knockout represents one of the most efficient techniques for investigating gene function. However, the rate of homologous recombination is generally low, frequently falling below 2%, which presents a significant obstacle in obtaining the desired mutants. In this study, we have devised a visual screening system for gene deletion in B. bassiana by introducing the transcription factor OpS3 overexpressing cassette into the gene deletion vector. This alteration enables straightforward discrimination between gene deletion mutants, which lack oosporein production and display a white phenotype, and random insertion transformants, which exhibit oosporein production and appear red. Compared to the traditional methods that required screening several hundreds of colonies or more, the improved method only requires PCR testing of an average of 10 colonies for each gene. Since oosporein is a red pigment, the presence or absence of oosporein production can be visually assessed without the requirement for specialized equipment or chemicals. The visual screening method improved the gene knockout efficiency to 50%–75%, and reduced the workload of DNA preparation and PCR amplification, greatly saving the experimental cost and time.

Visual selective markers have been extensively studied for their application in transformation and screening processes across a variety of species, including both microorganisms and plants (Kortstee et al., 2011; Ying & Feng, 2006). Fluorescent proteins such as GFP and RFP are frequently used as selective markers. However, their detection often necessitates the use of laser equipment for colour visualization (Gao et al., 2005; Ikawa et al., 1995; Jin et al., 2008). The absence of MET15 in Candida albicans leads to brown colony formation on Pb2+ containing medium, while the reintroduction of MET15 restores the white colony phenotype, suggesting its potential as a selective marker in C. albicans (Viaene et al., 2000). In certain plants, the transcription factor MYB10 stimulates anthocyanin synthesis and has been engineered as a visual selective marker in the transformation process (Kortstee et al., 2011). In B. bassiana, oosporein production is governed by multiple factors, including OpS3 within the PKS gene cluster, Bbsmr1 and Bbmsn2 (Fan et al., 2017; Feng et al., 2015; Luo et al., 2015). Among these transcriptional factors, OpS3 acts as a positive regulator, controlling the expression of genes involved in oosporein synthesis. For gene deletion experiments, employing an OpS3 expression cassette may not result in any unintended consequences, as this cassette is lost in the homologous mutants.

The oosporein synthetic gene cluster is found in various B. bassiana strains, including B. bassiana ARSEF 2860 and B. bassiana D1‐5, and the visual screening strategy works well in another B. bassiana strain (ARSEF 2860). Additionally, oosporein production is not exclusive to B. bassiana but has also been observed in other fungi such as Oospora, Acremonium, Chaetomium, and Epicoccum. However, direct application of this method to these fungi is not feasible due to the lack of knowledge concerning the specific genes responsible for efficiently regulating oosporein production. Fungi have the capacity to generate a variety of secondary metabolites in different hues during their growth and development. Typically, positive regulators control the synthesis of these secondary metabolites, and their overexpression can enhance the biosynthesis of secondary metabolites in fungi. For instance, Trichoderma reesei secretes a yellow pigment primarily regulated by the transcription factor YPR1, whose overexpression triggers the formation of the yellow pigment (Zhang et al., 2020). In Fusarium fujikuroi, deletion of the GAL4‐like transcription factor bik5, which is involved in the biosynthesis of the red pigment bikaverin, resulted in significantly reduced pigment production compared to the wild type (Wiemann et al., 2009). Although the visual screening of gene knockout mutants has potential applications in these fungi, the prerequisite is to identify a specific gene which is able to positively regulate the synthesis of coloured substance.

In conclusion, using oosporein production as a negative selective marker in B. bassiana provides a convenient tool for screening gene knockout mutants. This simplifies the investigation of gene functions. This strategy shows that visual selection of mutants can be done by overexpressing transcription factors. Furthermore, based on the pigment production capabilities of different fungi, using visual markers related to pigment production could be extended to other fungal species.

AUTHOR CONTRIBUTIONS

Ajing Mao: Writing – original draft; writing – review and editing; investigation; data curation; formal analysis; validation; visualization. Junyao Wang: Data curation; formal analysis; validation; visualization; writing – original draft. Shengan Zhu: Investigation; writing – review and editing. Dan Jin: Investigation; writing – review and editing. Yanhua Fan: Conceptualization; funding acquisition; resources; project administration; supervision; writing – review and editing.

FUNDING INFORMATION

This work was supported by the National Natural Science Foundation of China (32170197, 32300168) and the Technology Innovation and Application Development Project of Chongqing (No. CSTB2023TIAD‐KPX0045).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no known competing financial interests or personal relationships to influence the work reported in this paper.

Supporting information

Table S1.–S2.

MBT2-17-e14512-s001.docx (13.5KB, docx)

Mao, A. , Wang, J. , Zhu, S. , Jin, D. & Fan, Y. (2024) An efficient visual screening of gene knockout mutants in the insect pathogenic fungus Beauveria bassiana . Microbial Biotechnology, 17, e14512. Available from: 10.1111/1751-7915.14512

Ajing Mao and Junyao Wang are co‐first authors and contributed equally to this work.

DATA AVAILABILITY STATEMENT

The data that supports the findings of this study are available in the supplementary material of this article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1.–S2.

MBT2-17-e14512-s001.docx (13.5KB, docx)

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article.

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