The APSES Gene MrStuA Regulates Sporulation in Metarhizium robertsii

The APSES family is a unique family of transcription factors with a basic helix-loop-helix structure (APSES: Asm1p, Phd1p, Sok2p, Efg1p, and StuAp), which are key regulators of cell development and sporulation-related processes. However, the functions of the APSES family of genes in the entomopathogenic fungus Metarhizium robertsii have not been reported. Here, we report the identification and characterization of the MrStuA gene, a member of the APSES family, in M. robertsii. The selected gene was identified as StuA in M. robertsii (MrStuA) because the gene product contains two conserved sequences, an APSES-type DNA-binding domain and a KilA DNA-binding domain, and has the highest homology with the StuA in the C-II clade of the APSES family. We found that the number of conidia produced by the ΔMrStuA strain was 94.45% lower than that in the wild type. Additionally, in the mutant, the conidia displayed an elongated shape, the sporulation was sparse and the phialide were slender. In addition, transcription levels of two central regulators of asexual development, AbaA and WetA, were significantly reduced in the mutant; furthermore, the transcription levels of other sporulation related genes, such as Mpk, Phi, Med, Aco, Flu, and FlbD, also decreased significantly. We also show that the median lethal time (LT50) of the mutant increased by 19%. This increase corresponded with a slower growth rate and an earlier conidia germination time compared to that of the wild strain. However, the resistance of the mutant to chemicals or physical stressors, such as ultraviolet radiation or heat, was not significantly altered. Our results indicate that in M. robertsii, MrStuA may play a crucial role in regulating sporulation as well as virulence, germination, and vegetative growth. This study improves our understanding of the impact of the transcription factor StuA on sporulation processes in filamentous fungi and provides a basis for further studies aimed at improving sporulation efficiency of these fungi for use as a biocontrol agent.


INTRODUCTION
Transcription factors are an important component of the signal transduction pathway, which regulates the expression of the target gene(s) to further regulate the growth and differentiation of cells (Shelest, 2008). There are several exclusive transcription factor families in fungi, including the TEA/ATTS and APSES families. The transcriptional regulatory proteins in the APSES family belong to the bHLH class of transcription factors. There are five types of proteins in the APSES family, Asm1, Phd1, Sok2, Efg1, and StuA; these proteins play important roles in mycelial growth, sporulation, and pathogenicity (Twumasi-Boateng et al., 2009). In recent years, a large number of APSES family proteins have been identified, especially in filamentous fungi. There are five members identified in most fungi, including Aspergillus fumigatus and A. nidulans; meanwhile, other fungi, such as Magnaporthe oryzae and Cryptococcus neoformans, have fewer members (between two and four) (Nishimura et al., 2009).
A. nidulans StuA was the first transcriptional factor with a basic helix-loop-helix structure found in the APSES family (Ballance et al., 1983). The gene, which has a 3-kb-long promoter that is regulated by the most prominent regulator BrlA, is responsible for the spatial expression of the AbaA transcription factor (Wu and Miller, 1997). This protein plays an important role in the structure and cell morphogenesis during the sexual and asexual phases of reproduction in A. nidulans. However, the transcription factors in the APSES family have different functions in different species. For example, they regulate attachment-mediated infection in M. grisea (Nishimura et al., 2000); yeast-mycelial two-phase transformation in Wangiella dermatitidis; secondary metabolites in A. fumigates (Twumasi-Boateng et al., 2009); bisexual dimorphism, sporulation, and pathogenic development in Ustilago maydis (Garcia-Pedrajas et al., 2010); and conidia development, pathogenicity, and secondary metabolites in Fusarium graminearum (Lysoe et al., 2011).
The STUA protein (65.01 kDa) in A. nidulans contains two conserved fragments: an APSES-type DNA-binding domain and a KILA (N-terminal/APSES-type HTH) DNA-binding domain (Twumasi-Boateng et al., 2009). The two conserved domains regulate the expression of related genes through interactions with the STREs sequence of the promoter region in the AbaA gene, thus impacting the conidiation of strains. For example, a spatial rearrangement of the conidiophores and a small amount of visible conidia are observed in the A. nidulans knockout mutant of StuA; meanwhile, no normal conidiophore structure has been found in a Penicillium oxalicum StuA mutant. The StuA gene has two transcription sites, StuAα and StuAβ. StuAα activates the first intron in StuAβ. Both of the sites have long 5 leaders and mini open reading frames (ORFs) that code regulatory functions of StuA at a translational level.
M. robertsii (formerly known as M. anisopliae) is an important insect pathogen that is commonly used as a biological control agent (Zhou et al., 2012). Spores are the main reproductive organs of fungi as well as the main component of fungal pesticides. The bottleneck limiting the use of M. robertsii as a biopesticide is the low sporulation rate. Thus, a greater understanding of the function of sporulation-related genes will help make the application of strains as fungicides feasible.
There are some existing reports on sporulation-related genes in the genus Metarhizium. The molecular mechanism of the sporulation process is complicated in M. anisopliae and other entomopathogenic fungi (Clarkson and Charnley, 1996). Two sporulation pathways involving many regulatory genes, such as Flu, G-flbs, and FadA, co-regulate sporulation in M. robertsii; this process is similar to that of A. nidulans. The Cag8 gene, which is homologous with the FlbA gene in A. nidulans, is required for sporulation. This requirement has been confirmed by showing that a knockout strain cannot produce conidia (Timberlake, 1980). Using RNA interference (RNAi), a microcirculation sporulation gene Pky was found to produce more varied shapes of conidia (Xu et al., 2011).
The mechanisms of sporulation in A. nidulans and Neurospora crassa have been well studied (Park and Yu, 2012). Blasting the sequences of StuA and other sporulation related genes from A. nidulans, such as BrlA, AbaA, WetA, FlbA, FluG, AcoB, FlbC, FlbD, MedA, PhiA, SteC, MpkA, FadA, HymA, and Sak A, show that homologous genes also exist in M. robertsii. Thus, it is speculated that the molecular mechanism of sporulation in M. robertsii may be similar to that of A. nidulans. However, the functions of the APSES family of proteins in the entomopathogenic fungi M. robertsii have not been reported. We generated a MrStuA knockout mutant ( MrStuA) and found that StuA may play a crucial regulatory role in sporulation, virulence, germination, and vegetative growth in M. robertsii. This study deepened our understanding of the impact of the StuA-like transcription factor on sporulation in filamentous fungi.

Fungal Culture
The wild-type (WT) strain M. robertsii ARSEF23 (ATCC No. MYA-3075) was obtained from the Anhui Provincial Key Laboratory of Microbial Pest Control (RCEF5501). To obtain conidia, the WT strain, a MrStuA knockout strain ( MrStuA), and a complemented strain (C') were grown on potato dextrose agar (PDA; 200 g of potato, 20 g of dextrose, 20 g of agar and 1,000 ml of water) at 25 • C for 7-8 days. Subsequently, conidial suspensions (0.05% v/v Tween 80) were obtained by filtering the cultures through sterile non-woven fabrics to remove mycelia. The conidial suspensions were inoculated onto PDA medium overlain with sterile cellophane paper using spreaders and incubated at 25 • C. Colony phenotype assays were performed on solid PDA, 1/4 Sabouraud dextrose agar with yeast extract (1/4 SDAY; 10 g of glucose, 2.5 g of yeast extract powder, 2.5 g of peptone, 20 g of agar, and 1,000 ml of water) and SDAY.
The host E. coli Trans 10 was purchased from the TransGen Biotech Corporation (China). The plasmid pDHt-bar/ben and Agrobacterium tumefaciens AGL-1 were donated by Chengshu Wang (Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences).

Peptide Domain and Homology Analysis
The amino acid sequences of APSES proteins were retrieved from NCBI 1 . The conserved domains were predicted using InterProScan 5.0. We identified the protein structure of the ARSEF 23 APSES transcription factor (XP_007819177) ( Figure 1A) and found that this protein has high homology with the APSES family of proteins from a diverse range of fungi.

Multiple Alignment of the APSES-Type DNA-Binding Domain and Construction of a Phylogenetic Tree
The conserved amino acid sequences of the APSES domains (IPR003163) were aligned using Clustal W parameters (Supplementary Figure S1). MEGA version 5.1 was used to construct a neighbor-joining (NJ) tree with bootstrap analysis using 1,000 replicates. In total, 58 APSES domains from different fungi were identified for phylogenetic analysis. The neighborjoining tree was submitted to TreeBase ( Figure 1B) (Study Accession URL 2 ).  Table S1) were designed to amplify the MrStuA fragment. The PCR amplification product was purified, cloned, and sequenced.

Construction of a
A MrStuA deletion vector, pDHt-bar-StuA-Del, was constructed by inserting two flanking sequences of MrStuA into the left and right sides of bar (a kanamycin resistance gene) in the pDHt-bar vector using the primers MrStuAupF+MrStuAupR (Supplementary Table S1). A 1,008 bp upstream flanking sequence of MrStuA was amplified from M. roberstii genomic DNA and was inserted into the EcoR I-Pst I sites of the pDHtbar vector to generate a plasmid, pDHt-bar-StuA-upstream. Subsequently, a 1,285 bp downstream flanking sequence of MrStuA was amplified from M. roberstii genomic DNA using the primers MrStuAdownF+MrStuAdownR (Supplementary  Table S1). This sequence was inserted into the XbaI site of the pDHt-bar-StuA-upstream vector to generate the pDHt-bar-StuA-Del plasmid. Finally, a 3,267 bp fragment containing the StuAupstream-bar-StuA-downstream cassette was obtained through PCR amplification with the MrStuAupF+MrStuAdownR primers from the pDHt-bar-StuA-Del vector. The resulting PCR product was purified and used for A. tumefaciens-mediated transformation. The MrStuA strain was constructed using Agrobacterium-mediated fungal transformation (ATMT) and inserted by homologous recombination as previously described (Duan et al., 2009).
All recombinant strains were confirmed by PCR and reverse transcription-PCR (RT-PCR). PCR analysis indicated that a 1,050-bp fragment corresponding to the partial MrStuA gene was present in the WT and C' strains, but not in the MrStuA strain. A partial bar gene fragment (814-bp) was present in the MrStuA and C' strains, and a partial Ben gene fragment (328-bp) was present in the C' strain.

Construction of the Complement Plasmid for MrStuA
The MrStuA complement plasmid MrStuA::MrStuA was constructed using the backbone of the pDHt-ben vector. The full-length MrStuA (3,528 bp including the promoter region) was amplified from the genomic DNA of the WT strain using cpMrStuAupF+cpMrStuAupR primers (Supplementary  Table S1). This sequence was subsequently cloned into the Spe I-Xba I site of pDHt-ben to create the pDHt-ben-MrStuA::MrStuA plasmid. Transformation of pDHt-ben-MrStuA::MrStuA with the full length of MrStuA was performed as previously described using carb (50 µg/mL) and kan (50 µg/mL) as selective agents. Transformants were co-cultured with the MrStuA strain. A portion (1,050 bp) of the MrStuA sequence was amplified using MrStuAF+MrStuAR primers (Supplementary Table S1) to obtain the C' strain.

Morphological Observations at the Microscopic Level
The WT, MrStuA, and C' strains were grown on PDA medium at 25 • C for 3 days to determine hyphal growth and conidiation. Fresh mycelia and conidia were taken from the edges of colonies of each strain and stained with lactophenol cotton blue. Micromorphology was examined using an Olympus optical microscope (BX51, Olympus Co, Tokyo, Japan) and measured using the Measurement Tool program in the DP2-BSW software.

Sporulation Capacity Tests
Fresh conidia from the WT, MrStuA, and C' strains were collected after being cultured at 25 • C for 8 days. The conidia were suspended, and 30 µl of each suspension was inoculated onto solid PDA (35 mm disposable plastic petri dish) for incubation at 25 • C for 12 days (Liu et al., 2013;Xiong et al., 2013). The quantity of conidia in each plate was counted using a hemacytometer, after which the number of conidia per unit area was calculated. There were three replicates for each strain and all assays were repeated in triplicate.

Color, Morphology, and Growth of Colonies
The WT, MrStuA, and C' strains were grown on PDA, SDAY, and 1/4SDAY plates, respectively, at 25 • C for 7-8 days. Conidial suspensions (10 7 conidia/mL) were prepared for each strain to inoculate a new plate (1 µl of suspension/plate). These plates were cultured at 25 • C for 12 days. Each strain was inoculated on 3-5 plates. All assays were repeated in triplicate. The morphology and growth status of the colonies were observed and measured every 48 h starting on the fourth day of culture. The diameter of each colony was measured by the cross method, and all assays were repeated in triplicate.

Changes in Germination Patterns
Conidia were harvested and suspended in Tween 80 solution (0.05% v/v). Then, 30 µL of the filtered suspension (10 6 conidia/mL) was resuspended in 30 µL of GM (0.2% trehalose, 0.25% peptone, and 0.25% yeast extract powder) liquid medium and incubated at 25 • C for 16-22 h. Germination was observed at 400× magnification after the conidia were placed on the medium. All assays were repeated in triplicate.

Chemical Stress Challenges
To examine chemical stress tolerance, the mycelium of the WT, MrStuA, and C' strains were cut off from YEB medium by perforations (Chen et al., 2015a). The mycelium of each strain were grown on solid PDA and PDA supplemented with 2 µg/mL sodium dodecyl sulfate (SDS), 2 µg/mL Congo red (CR), 1 mol/L NaCl, 2 µg/mL carbendazim, 2 µg/mL menadione, and 2 mmol/L H 2 O 2 . Then, the mycelium were incubated at 25 • C for 10 days. Morphological changes in the colonies were observed and measured. All assays were repeated in triplicate.

UV Radiation and Heat Shock Assay
Thirty microliters of conidial suspensions (10 6 conidia/mL) were placed on a glass slide and dried on a sterile table to eliminate moisture. The UV light wavelength of the hybrid furnace was set to UV 254 nm, Time1, Energy2. Germination liquid (GM) was added after UV irradiation, and samples were cultured at 25 • C for 16-24 h. At the same time, the filtered suspensions were transferred to Eppendorf tubes and treated in a 42 • C water bath for 1 h. Aliquots of the suspensions (30 µL) were inoculated onto PDA medium (35 mm disposable plastic petri dish) and cultured at 25 • C for 16-24 h for examination of conidium germination. The data were recorded every 2 h. The inhibition rates were then analyzed (Rangel et al., 2006;Chen et al., 2015b).

Virulence Assays
To investigate the effects of MrStuA on fungal virulence, insect bioassays were conducted using wax moths. Conidia harvested from cultures grown on PDA medium were applied topically by immersing the wax moths for 1 min in an aqueous suspension (10 7 conidia/mL). Each treatment had three replicates with 15 insects each, and mortality was recorded every 12 h to calculate the median lethal time (LT 50 ).

Real-Time Quantitative Reverse Transcription PCR
Hyphal and conidia were harvested from cultures grown on solid PDA for 60 h (initial period of conidia formation). Total RNA was extracted from the samples using the Trizol reagent (Invitrogen, Foster City, CA, United States). cDNA was synthesized from 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (TaKaRa, Dalian, China) according to the manufacturer's instructions. This cDNA was used as the template for qRT-PCR with specific primer pairs (Supplementary Table S2). The qRT-PCR was performed using the SYBR Green (TAKARA) detection method (CFX96TM Real-Time System, Bio-Rad Laboratories, Hercules, CA, United States). All PCR reactions were performed in triplicate. The 2-CT method (Livak and Schmittgen, 2001) was used to calculate relative expression levels, and the glyceraldehyde 3-phosphate dehydrogenase gene (primer pair gpdF/gpdR) was used as an internal control for each sample. The data are expressed as the mean ± standard deviation (SD) for three independent experiments.

Summary of the Gene StuA in Metarhizium robertsii
An analysis of the M. robertsii genome database revealed that the putative StuA transcription factor gene consists of 2,074 nucleotides with two introns and three exons (427 bp, 94 bp, and 1,336 bp) and is predicted to encode a protein with 618 amino acids. The protein contains two conserved sequences, the APSEStype DNA-binding domain (IPR003163, 93rd-208th amino acid) and the KILA, N-terminal/APSES-type HTH, DNA-binding domain (IPR018004, 135th-207th amino acid, Figure 1A). The APSES core domain in fungi contains 73 amino acid residues consisting of six beta-folded and two pairs of alpha helices. The amino acid sequence from M. robertsii putative StuA had 65%, 43%, and 43% homologies with those of F. graminearum, A. nidulans, and P. chrysogenum, respectively.
Based on the phylogenetic tree (Figure 1B), the selected APSES family proteins were divided into four clades (A, B, C, and D), each of which has differently conserved amino acid sequences. The M. robertsii putative StuA transcription factor gene (XP_007819177) is in the C clade, which also includes the StuA genes from F. graminearum, A. nidulans, P. chrysogenum, and Beauveria bassiana. Within the clade, the M. robertsii gene is most closely related to the homologous genes from F. graminearum and F. verticillioides and is most distinct from that of B. bassiana. Based on the conserved domains, homology with other genes, and similarity within the phylogenetic tree, the gene was identified as the StuA transcription factor and was named MrStuA.

MrStuA Is Required for Sporulation
There were numerous morphological differences between the colonies of the MrStuA strain and those of the WT and C' strains. The MrStuA strain formed white colonies with only a few conidia; meanwhile, the WT and C' strains produced dark green colonies with many green conidia (Figure 2A). On PDA medium, the MrStuA strain yielded 94.45% fewer conidia than did the WT strain, and the mutant was nearly totally unable to produce conidia ( Figure 2B). Microscopic observations showed that the conidium shape, sporulation structure, and phialides were elongated, sparser, and more slender, respectively, in the mutant compared to those in the wild type. In the wild-type and C' strains, the conidia column contained many parallel conidial chains; however, this feature was not present in the mutant. A length-width ratio was calculated for conidia to better determine the variety in conidia shape. This ratio revealed that the MrStuA strain (ratio = 3.64 ± 0.36) had conidia that were significantly more slender (40%) than those of the WT strain (ratio = 2.6 ± 0.36) (P < 0.01) ( Figure 2D).
After 16 h, the germination rate of the MrStuA strain was more than 90%; meanwhile, the germination rates of the WT and FIGURE 2 | Changes in conidia capacity, conidium shape, conidiophore structure, and germination time between the wild type and mutant strains. (A) Morphology of the conidia, hypha and conidiophore structures, and colonies for the WT, MrStuA, and C' strains. (B) Conidia numbers per growth area (cm 2 ) for the WT, MrStuA, and C' strains. (C) Germination rate of conidia for the WT, MrStuA, and C' strains. (D) The aspect ratio of the conidia for the WT, MrStuA, and C' strains. Student's t-test: * p < 0.05, * * p < 0.01. C' strains were less than 50%. However, there were no differences in germination rate between strains at 24 h ( Figure 2C). Thus, it appears that germination occurs sooner in the mutant strain than that in the WT or C' strains.
Deletion of MrStuA led to changes in sporulation capability, conidia shape, conidiophore structure, and germination time; thus, it appears that MrStuA is required for sporulation.

Expression of Sporulation Related Genes in MrStuA
We monitored the mRNA levels of genes involved in conidiogenesis in M. robertsii by qRT-PCR, including the FlbA, BrlA, FluG, AcoB, AbaA, FlbC, FlbD, MedA, PhiA, WetA, SteC, MpkA, FadA, HymA, and SakA genes. The Brl A, Aba A, and Wet A genes are pivotal to the central regulatory pathway of asexual sporulation in A. nidulans. The transcription levels of two of these pivotal sporulation-related genes, AbaA and WetA, were significantly downregulated in MrStuA compared to those in the WT and C' strains (p < 0.01) (Figure 3). In addition, the transcription levels of other sporulation related genes, including MpkA, PhiA, MedA, AcoB, FluG, and FlbD, were also downregulated. Meanwhile, the expression levels of other genes, such as FlbA, BrlA, FlbC, SteC, FadA, HymA, and SakA, were not altered in the MrStuA mutant. Collectively, the results indicate that the MrStuA gene regulates two key transcription factors, AbaA and WetA, that in turn impact a series of downstream sporulation-related genes at the molecular level. Therefore, MrStuA is necessary for normal sporulation in M. robertsii.

Effect of MrStuA Function on Other Phenotypic Traits
Morphological observations showed differences between MrStuA colonies and those of the WT and C' strains. On solid PDA, SDAY, and 1/4SDAY medium, the MrStuA mutant produced white colonies, while the WT and C' strains produced dark green colonies ( Figure 4A). The radial growth of MrStuA was significantly reduced compared to that of the WT and C' strains (p < 0.05) under all growth conditions ( Figure 4B).
In the UV treated group, the relative germination rates of the WT, MrStuA, and C' strains at 24 h were 62.55%, 63.38%, and 66.06%, respectively (Supplementary Figure S4). Under the 42 • C water-bath treatment condition, the relative germination rates of the WT, MrStuA, and C' strains at 24 h were 76.77%, 81.95%, and 81.8%, respectively (Supplementary Figure S5). There were no significant differences in the tolerance of the germination of conidia to UV or heat treatments. Furthermore, there were no differences in the growth diameters of the WT, MrStuA, and C' strains on all chemical stress medium (Supplementary Figures S2, S3).
The MrStuA strain exhibited reduced virulence compared to WT and C' strains based on infection bioassays performed on the greater wax moth (Galleria mellonella). The MrStuA strain (LT 50 = 8.07 ± 0.68 d) took a longer time (19%) to kill the wax moths than the WT strain did (LT 50 = 6.78 ± 0.77 d) (P < 0.05) ( Figure 4C).
There were also differences in sporulation of the WT, MrStuA, and C' strains on host cadavers. The dead greater wax moths infected with the three M. robertsii strains were incubated at 25 • C at 100% relative humidity for a week. Those killed by the WT and C' strains produced numerous green spores; however, there were only white mycelia and a few conidia on the surface of host cadavers killed by the MrStuA strain ( Figure 4C).
Our results indicate that MrStuA may be involved in virulence and vegetative growth but has no impact on resistance to heat, UV, or chemical stress.

DISCUSSION
Proteins in the APSES family are regulatory transcription factors that exist ubiquitously in fungi. The functions of these proteins are related to sporulation, development, and the tolerance to unfavorable conditions (Doedt et al., 2004;Nishimura et al., 2009). StuA is a common member of Clade C-II of the APSES family; the function of StuA varies across different fungi. For example, StuA regulates sexual/asexual sporulation in A. nidulans, appressorium-mediated infection in M. oryzae (Doedt et al., 2004), two-phase conversion in W. dermatitidis (Wang and Szaniszlo, 2007), and secondary metabolites in A. fumigatus (Grosse et al., 2008). We found that in M. robertsii, MrStuA may play a crucial regulatory role in sporulation as well as minor roles in regulating virulence, germination, and vegetative growth.
The M. robertsii APSES transcription factor (EFZ01759) was identified as a C-II clade transcription factor based on a phylogenetic tree of APSES family gene data from different fungi. Some APSES family proteins are divided into the A-I, A-II, B, C-I, C-II, and D clades; at the same time, other APSES family genes, including XP_007819413, XP_007818991, XP_007821139, and EFY97406 from M. robertsii, belong to the A-I, A-II, B, and D clades, respectively. Thus, all five APSES protein family members are present in M. robertsii, demonstrating the richness and diversity of transcription factors of this family within M. robertsii. These APSES family proteins may play important roles in regulating the growth, differentiation, and infection processes in entomopathogenic fungi, especially for sporulation-related processes.
We found that the conidial yield from the MrstuA mutant was 94.45% lower than the yield from the control strains. This observation is consistent with results from A. nidulans and P. oxalicum, as well as those from other fungi (Park and Yu, 2012). Microscopy revealed that the morphology of the conidia and sporulation structures of the mutant strains were significantly altered compared with those of the WT strains in some fungi (Mirabito et al., 1989;Prade and Timberlake, 1993). We found that in the M. robertsii mutant, the conidia became elongated, the sporulation became sparse, and the phialide became slender. However, the knockout of this gene in A. nidulans resulted in shortened conidiophores, reduced conidiophores, missing metulae and phialide, and a "stunted" phenotype. It has also been reported that there were no conidiophore structures when StuA was deleted in P. oxalicum. In addition, transcription levels of two central regulators of asexual development, AbaA and WetA, were significantly reduced in the MrstuA mutant. These results indicate that MrStuA plays a regulatory role in some key genes, which in turn impact sporulation ability, conidia morphology, and sporulation structure. Thus, the gene MrStuA is required for sporulation.
The mutant strain has a significantly early germination time indicating that the StuA gene may be involved in regulating the germination process in M. robertsii. In A. nidulans, it has been reported that the StuA protein may regulate the temporal expression of genes (Adams et al., 1998). Thus, we speculate that this gene can impact the temporal expression of related genes.
The virulence of the mutant was significantly decreased, and the MrstuA strain only produced a few conidia on the surface of host cadavers. The sporulation ability on host cadavers is also an important factor when determining insect virulence. In M. acridum, virulence decreased when a sporulation-related gene was knocked out; this effect is mainly due to the increased immunity of the host insect or the impact on its ability to form appressoria (Zhang et al., 2017). Thus, we conclude that the MrStuA gene is related to the virulence of M. robertsii.
In summary, the knock-out of the MrStuA gene led to significant changes in M. robertsii sporulation at both a phenotypic and molecular level; the deletion impacted virulence, germination, and vegetative growth. Thus, the MrStuA gene is involved in both infection and vegetative growth and is required for the sporulation process. It remains unclear how MrStuA regulates downstream genes, and further studies should focus on sporulation regulation pathways.

AUTHOR CONTRIBUTIONS
BH and ZW conceived and designed the study and edited the manuscript. WY wrote the manuscript, conducted the experiments, and analyzed the data. HW, QS, and LQ did a part of the experiments. BH supervised the project. All authors read and approved the final manuscript.