Multifunctional regulation of NADPH oxidase in growth, microsclerotia formation and virulence in Metarhizium rileyi

Microsclerotia (MS), anti-stress structures produced by many filamentous fungi, have been proven to be a great substitute for conidia in the production of insecticides within entomogenous fungi. NADPH oxidase (Nox) is a highly conserved ROS-response protein family that is widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether the formation of MS and pathogenicity in entomogenous fungi is regulated by the Nox inside. In this study, we reported the presence of NADPH oxidase homologs in a great potential biocontrol fungus, Metarhizium rileyi, and further showed multiple biological functions. Three Nox homologous genes in M. rileyi showed high expression throughout the entire process of MS formation. Targeted deletion of MrNoxA, MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, the anti-adversity assay showed that they are indispensable for growth, osmotic pressure and oxidative stress regulation in Metarhizium rileyi. Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria might be unaffected in mutants since there are no striking differences in virulence compared with WT by topical injections. Our results revealed that NADPH oxidase plays important roles in growth regulation, MS formation and pathogenicity in M. rileyi, perhaps in the ROS response and hyphal polarity.


Introduction
Insect pathogenic fungi, also known as entomopathogenic fungi, are one of the largest populations of insect pathogenic microorganism, represented by Beauveria spp.and Metarhizium spp.(Khachatourians et al. 2008;Leclerque et al. 2004).Conidia are the main virulence determinants for fungal pathogenicity to insects; once attached to the cuticle, conidia germinate rapidly to produce appressoria and convert to penetration pegs to penetrate into insects.Then, intrusive hyphal bodies begin to proliferate sharply, compete and consume nutrients with the host, secrete toxins into the insect hemocoel, and eventually result in the death of insects.Soon afterward, hyphostroma inside insects will transform into aerial mycelia to destroy insect the cuticle and produce conidia to enter the next infection cycle (Srisukchayakul 2005).In addition, biological pesticides often share with less impact on plant growth, and even can play a great contributor in the growth, resisting diseases and enhancing immunity of plants (Miranda-Fuentes et al. 2021;Ipsilantis et al. 2012).All these unique features suggest that fungal biological pesticides will be an extensive perspective in agricultural applications.
Metarhizium rileyi (Nomuraea rileyi), an environmentally-friendly entomopathogenic fungus widely distributed worldwide, is a common etiological agent of multiple lepidopteran pests (mainly Lepidoptera: Noctuidae) that causes large-scale infectious diseases in insect populations (Chen. et al. 2012).However, pesticide commercialization of M. rileyi is restricted by its long production period and unstable conidiation compared with Metarhizium spp.and Beauveria spp., which are widely used commercially at present.Hence, it is important to look for effective substitutes for conidia to promote the commercial application of M. rileyi.Microsclerotia (MS), which possess drought tolerance, easy storage, strong resistance and high stability, are a specialized dormant structure formed with intertwined polar hyphae under adverse conditions.Once rehydrated, MS will germinate rapidly to form hyphae and further generate a large number of conidia.Previous studies have made it possible to obtain MS production of M. rileyi in the laboratory.Moreover, liquid fermentation and large-scale production could be realized by optimization of manufacturing circumstances (Song et al. 2014).Comparative transcriptomics revealed that there is a series of highly expressed genes enriched in ROS accumulation and response during MS formation, and MS yield can be improved significantly with exogenous ROS, such as H 2 O 2 (Georgiou et al. 2006;Song et al. 2013).These results indicate that ROS probably play a critical role in the formation of MS in M. rileyi.
It is well known that excessive amounts of toxic reactive oxygen species (e.g.superoxide, H 2 O 2 , and hydroxyl radicals) can be produced in response to various environmental stimuli via membrane-bound NADPH oxidase (Nox) in eukaryotes (Bedard et al. 2007;Lambeth et al. 2004;Takemoto et al. 2007).NADPH oxidase is a typical multienzyme complex that can transfer electrons from NADPH to oxygen substances to generate superoxide and then widely participates in cell proliferation, differentiation, polar growth, apoptosis, immunity, ion transport, signal transduction and even programmed cell death (Foreman et al. 2003;Aguirre et al. 2005;Suzuki et al. 2011).In Aspergillus nidulans, the differentiation of the sexual fruiting body becomes defective and fails to assemble into mature cleistothecium and ascospores in the absence of NoxA (Lara-Ortiz et al. 2003).Similarly, NoxA (Nox1) is also indispensable for the normal generation of reproductive fruiting bodies in Podospora anserina and Neurospora crassa (Aguirre et al. 2005).In Claviceps purpurea, in addition to actively participating in conidia germination and MS formation, NoxA is also involved in the response against antioxidant stress in conjunction with NoxB (Giesbert et al. 2008;Schürmann et al. 2013).In Botrytis cinerea, there is no macroscopic faultiness in mycelial growth, spore production and germination, but restricted fruiting body production is noticeable regardless of the deletion of Nox1 or Nox2.Moreover, the pathogenicity of B. cinerea will be completely lost with the deletion of both Nox1 and Nox2 (Segmuller et al. 2008).Fusarium graminearum, a destructive phytopathogen, will fail to form ascospores and lose its pathogenicity to gramineous crops with deletion of any of these three Nox homologous genes (NoxA, NoxB and NoxC) (Wang et al. 2014;Zhang et al. 2016).Similarly, deformed appressoria and deficient penetration pegs will lead to the loss of pathogenicity in Magnaporthe grisea mutant, regardless of the lack of NoxA, NoxB or NoxR (Egan et al. 2007;Ryder et al. 2013).
A direct correlation between MS production and ROS in M. rileyi has been reported in the early years (Song, et al. 2013).In this study, three homologous genes of NADPH oxidase, NoxA, NoxB and NoxR were identified and functionally characterized in the insect mycopathogen M. rileyi.Disruption of MrNoxR and MrNoxA but not MrNoxB resulted in a dramatic reduction in virulence via inoculation, and destruction of any of these three genes would lead to a decrease in MS yield and impaired morphology, even osmotic pressure and oxidative stress.

Biological strains and culture conditions
M. rileyi strain CQNr01 was isolated from the body surface of deceased Spodoptera litura and deposited in the Engineering Research Center for Fungal Insecticides of Chongqing.Wild-type and various Nox mutants were cultured on SMAY medium to harvest conidia or for growth tests.Escherichia coli DH5α was employed for plasmid propagation, and Agrobacterium tumefaciens AGL-1 was used for transformation and mutant construction.Bacterial materials were all cultured in Luria-Bertani media containing appropriate concentrations of antibiotics based on the transferred plasmids as previously reported (Marcet-Houben et al. 2012).Spodoptera litura were artificially reared in our laboratory with a constanttemperature incubator according to previous reports (Wei et al. 2004).

Sequence and genomic manipulation
Based on previous transcriptomics data, three NADPH oxidase homologous genes (MrNoxA, MrNoxB and MrNoxR) were carried out with the M. rileyi genome database (Song et al. 2013).Fungal genomic DNA in M. rileyi was isolated as a template with a genomic extraction kit (AxyPrep Multisource Genomic DNA Miniprep Kit) to clone various fragments.Mutational deletion was performed through homologous recombination by using Agrobacterium-mediated fungal transformation.Based on the complete genomic sequence available, the flanking regions of three homologs were amplified with specific primers (Table 1) designed using the National Center for Biotechnology Information (NCBI) online software Primer-BLAST (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast).Pure fragments were then introduced into pzp-Ptrpc-Hph-Knock to obtain deletion plasmids.The resultant plasmids were eventually transformed into conidia for targeted genedeletion.Transformants were selected on SMAY media supplemented with appropriate antibiotics and verified by conventional PCR and quantitative PCR (primers listed in Table 1).

Phenotypic assays
Mature conidia were harvested to measure the conidial yield as described previously (Lin et al. 2019).Briefly, conidia (cultured for 14 days) from tested strains were prepared as a 10 6 conidia mL −1 suspension with 0.05% Tween-80, and then 100 μL was coated onto SMAY plates (9-cm diameter) followed by incubation at 25 °C with light 16 h/dark 8 h to produce fresh conidia.On day 14, three culture plugs from each strain were taken using a 1 cm diameter borer, and then placed into 50 mL centrifuge tubes with 10 mL of precooled sterilized pure 0.05% Tween-80.The supernatant was then diluted 10 times with the same solvent to count the conidia number by using a hemocytometer.
To check the difference in percentage germination, 50 μL conidial suspensions (10 6 conidia mL −1 ) of the tested strains were inoculated onto SMAY media under optimum conditions.Starting from 12 h postinoculation, germinated conidia were counted every 4 h.Three random fields of view were counted under a microscope (Nikon Co. Ltd., Japan), and the number of germinated conidia of 200 conidia in each view was recorded.The average number in these fields of view was recorded as the germination frequency.
The colony morphologies of the tested strains on SMAY media were observed, and morphological development was recorded using a digital camera (60mm Macro lens, Canon Inc., Tokyo, Japan) and a light microscope.In addition, the capacities for dimorphic transition of the tested strains were assessed as previously described (Li et al. 2016).In short, conidial suspension was prepared as noted above and 3 μL was pipetted onto SMAY plates and incubated at 25 °C.The colony diameter was measured with cross methods every day until the 10th day.SMAY plates supplemented with 0.5 M NaCl, 0.5 M KCl, 1 mM CaCl 2 , 10 mM CaCl 2 , 20 mM CaCl 2 , 0.5 M glycerin, 1 M sorbitol, 100 μg Congo red and 0.01% SDS were used to test abiotic stress tolerance.Colony morphologies were recorded at continuous time points using a digital camera as mentioned above.

ROS sensitivity and histochemical detection of ROS production
ROS, including superoxide, H 2 O 2 , and hydroxyl radicals are produced via a membrane-bound NADPH oxidase (Nox) to perform multiple regulatory functions among species (Marino et al. 2012;Mithofer et al. 1997).Thus, antioxidant defenses and accumulation of ROS production in mutational impairment of NADPH oxidase were tested in M. rileyi.SMAY plates supplemented with 0, 10 -4 , 10 -2 , and 1 mM H 2 O 2 were used to test the antioxidant capacity of the tested strains.Superoxide anion (O 2 − ) is one of the major components of ROS, and its production in fungal hyphae was measured by staining with nitroblue tetrazolium (NBT).Briefly, conidia were dropped onto SMAY plates and incubated at 25 °C for 4 ~ 5 d until the plates were covered with white hyphae.Appropriate 0.2% NBT solution (pH 7.5) staining solution was dropped onto the surface of the whole colony, and then plates were incubated for 2 h.The colony was washed twice with sterilized pure water, and the staining reaction was stopped with 70% ethanol.Color changes are observed under the microscope.NBT reacts with O 2 − to form a dark blue insoluble formazan compound, and the depth of color indicates the relative content of O 2 − .

Microsclerotium differentiation
Microsclerotia (MS) are effective infective and spreading structures produced by multiple pathogenic fungi in nature (Wang et al. 2011).Conidial suspensions (10 8 conidia/mL) prepared with test strains were inoculated into liquid AM medium followed by shaking to culture for 6 days before MS yields were quantified and determined.In addition, morphologies were observed and recorded using a digital camera and microscope.

Bioassays
Third instar larvae of Spodoptera litura were used to determine fungal pathogenicity.Conidia from tested strains were used for topical infection by dipping the larvae into the conidial suspension (1 × 107 conidia mL −1 ) prepared with 0.05% Tween 80 for 90 s or by directly injecting 5 μL conidial suspension (5 × 10 5 conidia mL −1 ) into the hemocoel of each larva.Thirty larvae were tested in each group, and the experiment was repeated three times.Pure cotton seed oil alone was brushed on the surface of the insects, and 0.05% Tween 80 was injected into the hemocoel as a control.
Mortality was recorded at continuous time points, and LT 50 was established with analysis of the data.

Transcriptional analysis
Fungal samples were collected from SAMY plates from days 0 to 7 for RNA extraction, and then reverse transcribed to cDNA to quantify the transcript abundance of specific genes associated with fungal development.In addition, RNA from MS was also collected to quantify the expression level of genes within MS formation.Total RNA was isolated with TRIzol regents, and cDNA was synthesized by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA).Quantitative reverse transcription-PCR (RT-qPCR) was performed using SYBR Green (Invitrogen).All primers used for RT-qPCR were designed by using the National Center for Biotechnology Information (NCBI) online software Primer-BLAST (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast/ index.cgi?LINK_ LOC= Blast Home) (Table 1), and the Tub gene (GenBank: AF399718.1)from M. rileyi was used as an internal control to normalize gene expression.

Results and discussion
Nox homolog in M. rileyi Three orthologous Nox genes in M. rileyi were identified based onpreviously published transcriptome data (Song et al. 2013), and were all cloned by using specific primers with genomic DNA as the template. .Moreover, the transcriptional profile of three genes during MS production was investigated (Fig. 1A and  B).The expression levels of three genes were observably increased at 36 h.Until 84 h, there was a sudden increase in the expression level of MrNoxB and MrNoxR, reaching the peak value.The expression of MrNoxA basically maintained a state of continuous increase during the formation process, reaching the highest value at 120 h, the mature stage of MS production.These results revealed that three Nox homologs coordinate to play a crucial role in the whole process of MS formation in M. rileyi.

Three homologous genes are required for the right track of growth and morphological building in M. rileyi
Conidia from different strains were harvested to observe the morphologies under a microscope.The results showed that the conidia of △MrNoxR were swollen compared with those of the wild type, which was not observed in △MrNoxA and △MrNoxB Vol:.(1234567890) (Fig. 2A).Moreover, germination experiments were conducted on SMAY medium to investigate the contribution of Nox homologs to germination in M. rileyi.MrNoxA might not participate in the germination procedure but may be involved in the regulation of conidia production yield in M. rileyi.Moreover, the germination rate was decreased in the absence of MrNoxB, but accelerated in △MrNoxR (Fig. 2B).Additionally, reduced conidial yield was observed in △MrNoxR but not in △MrNoxB (Fig. 2C).
Biological characteristics of mycelial growth and morphologies were investigated by inoculating conidia inside the SMY liquid medium and SMAY solid medium.Hyphal differentiation was completed at 3 days whether in both SMY and SMAY (Fig. 3A  and B).Compared with the wild type, the colonies of △MrNoxA and △MrNoxB were maintained in the yeast state for a longer time, while △MrNoxR entered and maintained the yeast state for only a short time.On the fourth day, polarized growth appeared in all strains (Fig. 3B and C).Furthermore, the spreading rate of colonies of mutants was measured on SMAY solid medium by cross-bonding methods.As the results showed, there was no obvious difference in the colony size of △MrNoxA or △MrNoxB compared with the wild type during the whole growing period, but an enlarged size was found in △MrNoxR from the 7th day compared with the wild type (Fig. 3D).These phenomena indicated that MrNoxR may be involved in the polar growth process, perhaps through negative regulation.Hyphal polarized growth is a typical feature in numerous filamentous fungi that is involved in various basic processes of the life cycle, such as colony extension, nutrition acquisition, vegetative and reproductive growth, and infection structure formation (Malagnac et al. 2004).Moreover, growing evidence shows that ROS produced by NADPH oxidase are mainly located in the tip of mycelium to control apical dominance (Rolke et al. 2008;Jiang et al. 2014).

Nox contributes to stress tolerance
To examine the role of Nox homologs in M. rileyi in diverse chemical stress environments, the growth variation of both WT and Nox mutant strains was evaluated on SMAY medium that was enriched with a range of chemical agents, such as NaCl, KCl, CaCl 2 , CR, SDS, glycerol, sorbitol, H 2 O 2 and menadione (Fig. 4A).The findings indicate that the growth state of the △MrNoxB strain remains constant in comparison to the wild type when exposed to any stress.Conversely, the △MrNoxA and △MrNoxB mutants exhibited heightened sensitivity toward multiple stressors such as NaCl, KCl, CaCl 2 , glycerol, and sorbitol, as well as oxidative stresses of varying concentrations of H 2 O 2 (Fig. 4B).Furthermore, it has been observed that the yield of conidia is reduced in both △MrNoxA and △MrNoxB strains under conditions of multiple stresses.
Perhaps the absence of Nox would result in a significant decrease in intracellular ROS that in turn interfered with the transport of metal ions among these two mutants.Generally, K + is the key factor related to numerous metabolic processes, and an appropriate concentration of K + and Na + can maintain cell growth in fungi (Page et al. 2006).There have been reports demonstrating that the MAPK pathway is related to the oxidative stress response, including the Hog1 pathway, which is indispensable for cells to maintain internal and external osmotic pressure balance in a hypertonic environment (Nevoigt et al. 1997).These findings indicated that MrNoxR could probably regulate the stress response by interacting with factors in the MAPK pathway via ROS in M. rileyi.

Superoxide (O 2 −
) anion is one of the most principal ROS species that can react with NBT to form a dark blue insoluble formazan compound.Thus, it is often used as a qualitative assay to detect the presence and relative quantity of superoxide anions in biological samples (Kumar et al. 2014).Here, the hyphal body of M. rileyi was harvested from SMAY medium to measure the ROS content.As the results showed, blue formazan precipitation was noticeably accumulated in the hyphae of △MrNoxB compared with wild type, while there was an extremely small

Nox is involved in the formation of MS in M. rileyi
The evolution of MS in AM medium was observed with macroscopically and microscopically.After incubation with conidia for 2.5 d, a brown precipitate was generated in the medium.The sediment pigment was slightly deeper in △MrNoxB but shallower in △MrNoxA than in the wild type.Moreover, there was no formation of reddish brown pigment in △MrNoxR.Until 5 d, MS production accumulated extensively in AM medium incubated with wild type.By comparison, there was a reduced yield in MS production accompanied by different sizes in △MrNoxA and △MrNoxB, while the proper crystallization of MS was seriously decimated in △MrNoxR.Instead, there was only a small amount of MS with flabby and short peripheral polar hyphae and the formation of reddish brown pigment was completely invisible in △MrNoxR (Fig. 5A).MS was originally a dormant structure formed in fungi under diversified stresses, and the biological characteristics are bound to be affected by the sharp change in differentiation.Pigment (melanin) is considered to be related to the long-term survival of MS in Verticillium dahliae (Klimes et al. 2008), so it may be speculated that the stability and tolerance of white MS would be reduced in △MrNoxR.
Furthermore, MS production was harvested to quantify the expression of related genes with special primers (Table 1).According to the RT-qPCR results, there was an infinitesimal expression change Moreover, MrNoxA was highly expressed, while MrNoxB was maintained at a low level of expression in △MrNoxR (Fig. 5B).In general, the expression of MrNoxB and MrNoxR subunits was unaffected by the lack of MrNoxA, but with the deletion of MrNoxB or MrNoxR, the expression levels of the other two genes were significantly affected.It could be deduced from the above evidence that the pigment deposition of MS may be regulated by MrNoxA, and the size and morphology of MS was mainly regulated by MrNoxB, while MrNoxR is the most critical participant in the whole formational process.These inferences may need to be further confirmed by constructing double or triple knockout mutants.
Pks is one of the most cardinal genes interrelated to pigment synthesis, such as melanin (Takano et al. 1997).The correlation between Pks expression and the accumulation of brown pigment in the △MrNoxR mutant was analyzed during the formation of MS.It was observed that Pks maintained a high level of expression in the MS of △MrNoxR from the very early stage to the mature period, indicating that pigment can be produced in △MrNoxR, which might be white pigment deposition (Fig. 5C).

Nox contributes significantly to the virulence of M. rileyi
To assess insect virulence, conidia of M. rileyi were harvested, and a biological test was carried out with third instar larvae of Spodoptera litura by means of topical infection and intrahemocoel injection.The half lethal time compared to wild type was used as the criterion of virulence.Intrahemocoel injection bioassays revealed that the LT 50 values in △MrNoxA, △MrNoxB and △MrNoxR were 10.23 d, 9.55 d and 9.19 d respectively, which were delayed by 1.99 d, 1.31 d and 0.95 d compared with the wild type (8.24 d) (Fig. 6A and B).The findings indicated that the virulence was reduced as the worst with the loss of MrNoxA.Topical injection bioassays showed that the LT 50 values in △MrNoxA, △MrNoxB and △MrNoxR were 11.04 d, 10.17 d and 12.29 d respectively, which were delayed by 1.93 d, 1.06 d and 3.18 d compared with that in the wild type (9.11 d) (Fig. 6C and D).Moreover, compared with the morphological changes of muscardine cadavers infected with wild type, hyphae and conidia coating the whole body in the △MrNoxA and △MrNoxR strains took more time (Fig. 6E).According to previous research results, branch and shorter hyphae may cause obstacles in the differentiation of invasive hyphae in the process of infection and reduce virulence.These results suggest that all three Nox homologs are essential for the complete virulence of M. rileyi against Spodoptera litura.

Conclusions
Reactive oxygen species (ROS) produced by various eukaryotes and prokaryotes via aerobic respiration were considered harmful metabolic byproducts for a long time, as excessive reactive oxygen species lead to oxidative stress toward DNA, proteins, lipids and other cellular components.However, increasing evidence has also implicated ROS in numerous normal cellular responses, such as proliferation, differentiation, resistance and signal transduction (Georgiou 1997;Georgiou et al. 2006).Like many bacteria that form bacterial spores to spend the adverse environment, fungi, especially filamentous fungi, will also produce dormant structures such as sclerotia and microsclerotia (Georgiou 1997).As an insecticidal fungus with great application prospects, pesticide commercialization of M. rileyi is restricted by its long production period and unstable conidiation.Therefore, MS would be a better alternative production.MS production of M. rileyi was induced by Song under laboratory conditions, and ROS were determined to be a vital inducible factor in the process (Song et al. 2013).NADPH oxidase is the major regulator of ROS production in many species, and functional annotation is well defined in animals, plants and filamentous plant pathogenic fungi but rarely reported in insect pathogenic fungi.
In this study, three Nox homologous genes were identified and characterized in the entomopathogenic fungus M. rileyi.Furthermore, the roles of these three genes in the process of MS formation, virulence, growth and stress tolerance were further measured.In the growth assay, compared with the wild type, there was more time spent growing from conidia to hyphae in the △MrNoxA and △MrNoxB strains, while germination and transformation (conidia to hyphae) were significantly accelerated in △MrNoxR.Conidia size was significantly expanded, but yield was reduced in the absence of MrNoxR.Under chemical pressure, the sensitivity of △MrNoxA and △MrNoxR significantly increased.There were almost no conidia but tight hyphae collected from SMAY medium supplemented with glycerol and sorbitol in △MrNoxR.
Environmental factors such as temperature, light and pH are certified as regulators during MS formation in numerous fungi (Georgiou 1997;Song et al. 2013;Liu et al. 2014).In this study, it was found that MS morphologies were changed among all three Nox mutants.The change in size and pigment deposition in MS was visible with the absence of the two catalytic subunits (MrNoxAp or MrNoxBp), while the MS cannot normally format with the deletion of MrNoxR in M. riley.Moreover, a major discovery was that the reddish brown pigment could not be seen in △MrNoxR.During the formation of MS, the expression levels of the MrNoxB and MrNoxR genes in △MrNoxA were impervious, while the expression of the other two Nox related genes was significantly reduced with the deletion of MrNoxB or MrNoxR.
Conidia of M. rileyi first germinate to turn into hyphal bodys, which are morphologically comparable to most yeast in the hemolymph post infection, and then hyphal bodies proliferate by means of heteroschism and budding.After occupying the hemolymph, the hyphal body will than transform into slender invasive mycelium, penetrate the insect body wall, and eventually kill host insects.Due to the lack of receptors recognized by the host immune system on the cell surface, yeast like hyphal bodies can escape host immune defenses and proliferate rapidly in the blood cavity.However, there is no infection ability of the hyphal body, and prolongation of the yeast phase is not conducive to the pathogenicity of entomogenous fungi (Srisukchayakul et al. 2005;Egan et al. 2007;Ryder et al. 2013).A virulence assay showed that △MrNoxA was severely reduced in virulence.Host insects can be destroyed post topical inoculation, indicating that the ability to form appressoria and penetrate the cuticle might not be destroyed completely.There was no significant change in half lethal time with deletion of MrNoxB, indicating that this gene may not play a primary role in pathogenicity.Topical inoculation and intrahemocoel injection bioassays both revealed that the △MrNoxR strain was severely reduced in virulence compared with the wild type, which may be involved in the defects of appressorium formation and penetration peg.
In summary, our results demonstrated that the NADPH oxidase gene family is indispensable for fungal virulence and physiological function in M. rileyi, such as conidial yield, stress resistance, virulence and the important phenotypes associated with fungal biocontrol potential.Moreover, NADPH oxidase is involved in ROS metabolism as the crucial factor related to MS production in entomogenous fungi.

Fig. 1
Fig. 1 Expression level of NADPH oxidase homolog during the development of microsclerotia in M. rileyi.A The morphologies of MS. a, the germination period of conidia (36 h); b, elongation of hyphae (48 h); c, The initial period of the formation of MS (60 h); d, considerable formation of polarity

Fig. 4
Fig. 4 Stress tolerances in M. rileyi.A The morphology of colony growth on SMAY media supplemented with the stress agents.Pictures were taken on the 14th day, and bar = 1 cm.B

Fig. 5
Fig. 5 The formation of MS in M. rileyi.A The morphology of MS.B Transcriptional levels of NADPH oxidase genes during the formation of MS.C Expression analysis of pigment

Fig. 6
Fig. 6 Virulence tests.A Insect survival rate post intrahemolymph injection.B LT 50 for injection assay; C Insect survival rate post topical infection; D LT 50 for topical infection assay.E Muscardine cadavers of Spodoptera litura larva infected by

Table 1
Primers used in this study The MrNoxA gene (sequence ID: OAA37686.1)shared a 1674-bp ORF fragment that encoded a predicted protein of 557 amino acids, and the MrNoxB gene (sequence ID: OAA50529.1)presented a 3195bp ORF fragment containing four exons, which encoded a predicted protein of 603 amino acids.Both catalytic subunits MrNoxAp and MrNoxBp hold the typical domains of Nox-like orthologs in numerous filamentous fungi, such as the FNR_blinding homeodomain and NAD_blinding homeodomain, which are presumed to be involved in the electron transfer of the respiration chain by binding with FAD or NADP.The MrNoxR gene (sequence ID: OAA41034.1)contains an open reading frame (ORF) of 1953 bp (with 402 bp introns) encoding a putative 516 amino acid protein with a calculated molecular weight of 126.6 kDa.The regulatory subunit NoxRp contains a TPRS domain at the N-terminus, which is involved in binding to the RacA protein, and a phox + bem1 domain (PB1) at the C-terminus, which is the main binding domain of small G protein family members to the Nox complex.Phylogenetic analysis of NADPH oxidase and related orthologs of different filamentous fungi suggested that all NADPH oxidase subunits in M. rileyi are closely related to NADPH oxidase in Metarhizium spp.