World J Microbiol Biotechnol DOI 10.1007/s11274-014-1610-7

ORIGINAL PAPER

NADH: flavin oxidoreductase/NADH oxidase and ROS regulate microsclerotium development in Nomuraea rileyi Juanjuan Liu • Youping Yin • Zhangyong Song Yan Li • Shasha Jiang • Changwen Shao • Zhongkang Wang

•

Received: 11 October 2013 / Accepted: 19 January 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Based on transcriptome library, an NADH: flavinoxidore ductase/NADH oxidase gene (Nox) was cloned from Nomuraea rileyi. The 1,663-bp full-length cDNA contains an open reading frame of 1,233 bp coding 410 amino acids. The expression level of Nox was upregulated and co-related to the intracellular H2O2 concentration during microsclerotium (MS) initiation. Rotenone inhibition showed that inhibition of Nox could cause a noticeable decrease in the MS yields. Silencing of Nox resulted in the MS yields, H2O2 and virulence decreased by 98.5, 38 and 21.5 %, respectively. On the other hand, MS yields increased by 24.8–61 % when induced by H2O2 or menadione. Furthermore, the reactive oxygen species (ROS) scavenger, ascorbic acid (up to 0.03 g ascorbic acid l-1), completely inhibited the formation of MS. In conclusion, the results obtained suggested that ROS promoted MS development, and that Nox was required for MS differentiation through regulation of intracellular H2O2 concentration. Besides, Nox had a great impact on the virulence in N. rileyi. Keywords Differentiation  Microsclerotium  NADH oxidase  Nomuraea rileyi  Oxidative stress  RNAi

Electronic supplementary material The online version of this article (doi:10.1007/s11274-014-1610-7) contains supplementary material, which is available to authorized users. J. Liu  Y. Yin  Z. Song  Y. Li  S. Jiang  C. Shao  Z. Wang (&) Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China e-mail: [email protected]

Introduction Nomuraea rileyi, a cosmopolitan entomogenous fungus, infects many noctuids and has a potential for development into mycoinsecticide (Thakre et al. 2011). However, its sporulation requires special growth conditions, such as specific carbon source (maltose) and light stimulation, which limit its mass production and commercialization. Nevertheless, in our early research, we successfully induced microsclerotium (MS) formation in N. rileyi in liquid amended medium (AM). The MS exhibited insecticidal activity against Spodoptera litura, and thus can be instead of conidia to develop as a new agent for insect control (Yin et al. 2012). Microsclerotium is a special reproductive structure of compact hyphal aggregates, which has a characteristic of strong environment resistance and pigmentation. Some phytopathogenic (Georgiou et al. 2006; Segmu¨ller et al. 2008) and entomogenous fungi (Jackson and Jaronski 2009; Song et al. 2013) can differentiate to form sclerotia or MS. Due to the sclerotia of phytopathogenic fungi are widespread soil-borne pathogens that cause serious damage to plants (Georgiou et al. 2006). In recent years, many researchers have studied the mechanism of sclerotia development to better control pathogens instead of using traditional toxic fungicides. It has been shown that high oxidative stress could trigger sclerotia differentiation, whereas the reactive oxygen species (ROS) scavengers, such as certain hydroxyl radical scavengers, ascorbic acid and b-carotene, could inhibit sclerotia differentiation (Georgiou et al. 2006). But the mechanism underlying MS development in entomogenous fungi is obscure. NADH oxidases are flavoenzymes with a TIM-barrel fold, and the flavin (FMN/FAD) cofactor acts as their electron mediator (Higuchi et al. 2000). In aerobes and

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facultative anaerobes, such as Escherichia coli and Salmonella typhimurium, NADH oxidases catalyze O2 to generate ROS, including superoxide radical (O2-) and hydrogen peroxide (H2O2). In recent years, researchers have identified as two distinct NADH oxidases that correspond to H2O2-forming oxidase and H2O-forming oxidase (Higuchi et al. 2000; Gao et al. 2012). Among NADH oxidases in plants and animals, they are involved in several diseases because of the formation of ROS (Morre´ 1995; Griendling and Ushio-Fukai 1997; Morre´ and Morre´ 2003). So far, many studies have demonstrated the importance of ROS generated genes-NADPH oxidases in most fungal species including in filamentous fungi (Segmu¨ller et al. 2008; Papapostolou and Georgiou 2010a, b), but there are few reports on the NADH oxidases in filamentous fungi. In the present study, we examined the effects of ROS and NADH: flavin oxidoreductase/NADH oxidase (Nox) gene on N. rileyi MS differentiation, as well as their associations between ROS and Nox in MS formation. The function of Nox gene was investigated by analyzing the Nox expression patterns under different conditions. Furthermore, RNA interference (RNAi) was performed to further explore whether the Nox gene is essential for MS development and virulence in N. rileyi.

Materials and methods Fungal culture, treatment with factors and MS collection Nomuraea rileyi CQNr01 strain was obtained from the School of Life Science, Chongqing University, China. The strain was grown on Sabouraud’s Maltose Agar Yeast media (SMAY) (Thakre et al. 2011). For liquid culture, the conidia were harvested from sporulated SMAY plates, suspended in sterile deionized water with 0.05 % Tween 80, and then inoculated into 100 ml of AM (Song et al. 2013) to obtain a final conidial concentration of 1 9 106 conidia ml-1. All cultures were incubated in an incubator shaker at 28 °C and 250 rpm. MS morphology was observed by a light microscope (Motic B5 Professional Series). Digital images were captured with a Canon EOS350D camera. We evaluated the effects of ROS on growth and differentiation of MS in N. rileyi by employing the single factor test. The ROS modulators used in this study were prepared fresh as sterile aqueous stock solutions: 10 M H2O2, 10 mM menadione and 20 mM rotenone [Sigma; dissolved in 0.05 % dimethyl sulfoxide (DMSO)]. H2O2 was added to the AM with conidial suspension at the final concentrations of 10-11, 10-9, 10-7, 10-5 and 10-3 M, respectively; menadione was administered at 0.01, 1 and

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100 lM concentrations, respectively; and the exogenous ascorbic acid was administered at final concentrations of 0.001, 0.01 and 0.03 g ascorbic acid l-1, respectively. The strain grown under untreated conditions was used as the control. Rotenone concentration was controlled at 40 and 100 lM. Meanwhile, 0.05 % DMSO was used as the negative control and the untreated sample was employed as the blank control. All the treatments were cultured for 3 days under conditions described above. The effects of different factors on the growth and differentiation of MS were evaluated by analyzing biomass accumulation and MS yields. For biomass evaluation, the liquid fermentation products were collected by vacuumfiltering in a Buchner funnel to remove the culture solution, and MS yields were measured as described by Jackson and Jaronski (2009). The resulting MS mixtures were vacuumfiltered and washed three times with sterile water for RNA extraction. Rapid amplification of cDNA end and genomic structure analysis of Nox The total RNA was extracted with TRIzol and subjected to first-stand cDNA synthesis according to the manufacturer’s instructions (Invitrogen). The gene-specific primers for 50 and 30 -rapid amplification of cDNA end (RACE) were designed based on the expressed sequence tag (EST) sequence of Nox from the transcriptome library (Supplementary Table 1). The 50 - and 30 -RACE were performed according to the manufacturer’s recommendation (Clontech). For the 50 -RACE, the first round of PCR amplification was carried out with SMART II oligonucleotide and reverse primer Nox-R1, and subsequently, the SMART II oligonucleotide and Nox-R2 primers were used in the nested PCR. For the 30 -RACE, the PCR product was amplified with 30 -CDS and forward primer Nox-F1 in the first-round PCR, and then, with the primers 30 -CDS and Nox-F2 in the nested PCR. To confirm the assembled cDNA sequence from overlapping PCR products, the entire coding regions of the Nox gene were amplified from the cDNA with primers Nox-FG and Nox-RG, and the genomic DNA was also used as a template to obtain the genomic structure of Nox. Short interfering RNA mixture preparations To synthesize long dsRNA, the PCR templates for Nox and enhanced green fluorescent protein gene (eGFP) were amplified from the cDNA sequence of Nox and plasmid C: Bar-eGFP with primer pairs (Si-F1, Si-R1; eGFP-F, eGFPR), containing T7 promoters in the 50 -end (Supplementary Table 1). The PCR products obtained were purified by employing a gel extraction kit (Sangon) and used as a

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template to synthesize long dsRNA with a MEGAscript High Yield Transcription Kit (Ambion), followed by generation of heterogeneous mixtures of short interfering RNAs (siRNA) from dsRNA by ShortCut RNase III (New England Biolabs) digestion.

Table 1 Effects of exogenous oxidative stress producing agents (H2O2, menadione) and ROS scavenger (ascorbic acid) on N. rileyi MS differentiation Reagents

Concentration

Control

0

Collection of blastospores and fungal transformation The conidial suspension was inoculated in 100 ml of SMY medium (SMAY with no agar) and incubated in an incubator shaker at 28 °C and 250 rpm for 2–3 days for blastospores formation. The blastospores were collected for siRNA transformation and the blastospore-based transformation system was modified according to the Beauveria bassiana protocol (Ying and Feng 2006). As described by Ying and Feng, for the transformation, the reagents, including 240 ll of 50 % PEG 4000, 36 ll of l M LiCl, 200 or 500 nM siRNA mixtures and 35 ll of 1 M dithiothreitol were sequentially added to a tube containing 1 9 108 blastospores ml-1 suspension. As a negative control, 500 nM eGFP siRNA was used, and as a wildtype, the untreated sample was employed. Subsequently, the transformed blastospore suspension was inoculated into 40 ml of AM and incubated for 3 days under the conditions described above. Real-time quantitative PCR (RT-qPCR) analysis and intracellular H2O2 assay For the analysis of the transcriptional levels of the Nox gene, the total RNA was isolated and treated with DNase I (Takara) and then converted into cDNA by using RevertAid First Strand cDNA Synthesis Kit (Thermo scientific). RT-qPCR was carried out with the specific primer pairs L1 and R1 of Nox using the iCycler system (Bio-Rad) (Supplementary Table 2). As internal controls, b-tubulin (TUB) and translation elongation factor (TEF) genes were used. The relative quantification of the target gene expression was evaluated using the 2-DDCt method (Vandesompele et al. 2002). All the primers used in this study are listed in Supplementary Tables 1 and 2. The intracellular H2O2 concentration in the fungal tissue (mycelia/MS with the weight of vacuum filtration) was measured by using the hydrogen peroxide assay kit (Beyotime Biotechnology). Virulence assays The virulence of the controls and RNAi mutant were tested by inoculating the strains into 3rd-instar cabbage caterpillar larvae. Aliquots of 5 ll MS suspensions in cottonseed oil (107 MS ml-1) of the controls and RNAi mutant were inoculated into 30 larvae of cabbage caterpillar per

10.5 ± 0.5 -11

10 H2O2

M

3.63 ± 0.11

12.0 ± 1.0

3.8 ± 0.06

12.5 ± 0.5

3.91 ± 0.19

10-7 M

21.5 ± 0.5**

5.85 ± 0.07**

10-5 M

15.5 ± 1.5**

4.53 ± 0.15**

10-3 M

3.5 ± 0.5**

0.2 ± 0.16**

1 lM 100 lM

12.33 ± 0.8

3.35 ± 0.11

9.5 ± 0.5

4.19 ± 0.15*

8.5 ± 0.5

0.28 ± 0.08**

12.33 ± 0.9

0.37 ± 0.03**

0.01 g l-1

9 ± 0.6

0.17 ± 0.04**

-1

10.67 ± 1.1

0.001 g l-1 Ascorbic acid

MS yields (MS ml-1) 9 103

10-9 M

0.01 lM Menadione

Biomass (mg ml-1)

0.03 g l

0**

The results are the mean ± SD * P \ 0.05; ** P \ 0.01, when compared with the control

treatment. Larval mortalities were recorded everyday till 15th day. The lethal time values for 50 % mortality (LT50) were estimated with the infectivity of N. rileyi. Statistical analysis All experiments were repeated three times with three replicates for each experiment. The data obtained were analyzed by one-way ANOVA, followed by Duncan’s Multiple Range test with the SPSS 16.0 and GraphPad Prism 5 program. The results were given as the mean ± standard deviation (SD).

Results Role of oxidative stress in N. rileyi MS differentiation In the present study, exogenous oxidative stress producing agents (H2O2, menadione) and ROS scavenger (ascorbic acid) were added to AM for N. rileyi growth to explore the effects of ROS on MS differentiation. The results obtained showed that the biomass and MS yields of N. rileyi under high-oxidative stress were significantly increased. Noticeably, higher MS yields were obtained following treatments with 10-7 and 10-5 M H2O2, with yields increased by 61 and 24.8 %, respectively. However, a higher dose of H2O2 (10-3 M) significantly decreased the biomass and MS yields (Table 1). Addition of 1 lM menadione to AM significantly increased the MS yields by 27.8 %, but did not produce

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formation under low-oxidative stress, with MS yields 88.6, 94.8 and 100 % reduction at 0.001, 0.01 and 0.03 g ascorbic acid l-1, respectively (Table 1). Furthermore, there was no puce pigment, but only hyphae or mycelial pellets in the AM when treated with 0.03 g ascorbic acid l-1 (data not shown).

Importance of H2O2 generation by N. rileyi in MS development

Fig. 1 Profile of intracellular H2O2 concentration during MS development. The results are the mean ± SD. *P \ 0.05, **P \ 0.01, when compared with the results observed at 2 days

any change in the biomass. Furthermore, when compared with the control, the MS produced were smaller and more uniform (data not shown). On the other hand, a higher dose (100 lM) of menadione significantly decreased the MS yields without affecting the biomass. However, ascorbic acid was disadvantageous to MS formation. It resulted in a dose-dependent decrease in MS

Fig. 2 Phylogenetic tree was performed with the Nox of N. rileyi homologues from different species by MEGA 4.0 (NJ) program: Metarhizium anisopliae (GenBank accession No. EFZ03372.1), Metarhizium acridum (EFY85092.1), Glomerella graminicola (EFQ2 9664.1), Beauveria bassiana (EJP64292.1), Cordyceps militaris (EGX91727.1), Botryotinia fuckeliana (CCD34521.1), Aspergillus niger (XP001395504.2), Aspergillus fumigatus (EDP54368.1),

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The MS exhibited a characteristic morphology in each developmental stage (Supplementary Fig. 1). The stages of MS development were identified as follows: undifferentiated (UD, 2 days), MS initiation (MI, 3 days), MS development (MD, 4 days) and MS maturation (MM, 5 days). The Profile of intracellular H2O2 concentration was measured in MS and correlated with its developmental growth stages. The H2O2 level was not constant throughout developmental stages: it was extremely low in the UD stage, and then suddenly peaked in the MI stage, which was about threefold higher than that observed in the UD stage. During the later stages of MD and MM, the intracellular H2O2 concentration decreased (Fig. 1). These results indicated that oxidative stress may promote MS differentiation.

Mycobacterium smegmatis (YP007291051.1), Rhodococcus erythropolis (WP003945637.1), Segniliparus rotundus (YP003660231.1), Bacillus sp. (WP006835248.1), gamma proteobacterium (YP003811351.1). The numbers at the branches represent bootstrap values after 1,000 replications. The scale bar indicates 0.1 amino acid substitutions per site

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Gene cloning and bioinformatics analysis of Nox The Nox gene was isolated from the transcriptome library of N. rileyi and cloned by 50 - and 30 -RACE (GenBank Accession No. KF425266). The 1,663-bp full-length cDNA contains 41 bp of 50 -untranslated region (UTR) and 389 bp of 30 -UTR. An open reading frame (ORF) of 1,233 bp encodes 410 amino acids. Analysis of the Nox protein reveals its predicted molecular mass as 44.81 kDa and isoelectric point as 7.12. In addition, the Nox protein contains old yellow enzyme (OYE)-related FMN binding domain (15–356 amino acids), which belongs to the TIM phosphate binding superfamily. The Nox genomic sequence of 1,788 bp consists of three exons interrupted by two introns, corresponding to the cDNA sequence. In order to examine the phylogenetic relationship of Nox with other species, a phylogenetic tree of the Nox protein was constructed with the MEGA 4.0 program, which showed the presence of two clades in the phylogenetic tree, including aerobic fungi and bacteria. The Nox of N. rileyi, along with those of other aerobic fungi, was found to belong to the Nox-1 clade, and it had a closest genetic relationship with that of Metarhizium spp. (Fig. 2).

Increase in the transcription level of Nox during MS development The transcription level of N. rileyi Nox during MS development was analyzed by RT-qPCR. The results demonstrated that the mRNA level of Nox expressed in all developmental stages, but dramatically increased during MI and MD stages, exhibiting 5.6- and 3.1-fold higher than that observed during UD stage. During the later stage of maturity, the transcription level of Nox was low (Fig. 3). Effect of inhibition of Nox on MS formation To study the participation of Nox in the process of MS development, we examined the effects of rotenone, an inhibitor of NADH oxidase, impairing ROS production by inhibiting the electron transfer in respiratory chain. With the inhibition of rotenone, MS formation remarkably decreased. The MS yields were apparently inhibited by 96 or 97.8 % at a concentration of 40 or 100 lM, respectively. On the other hand, there was little impact on the MS yields following DMSO treatment (negative control) (Table 2). Transcription responses to rotenone inhibition The transcription level of Nox was examined under various conditions of rotenone inhibition. As shown in Fig. 4, transcription reduction was obvious in the Nox expression under a 100 lM rotenone inhibition condition. The Nox mRNA expression approximately decreased by 50 % following the addition of 100 lM rotenone, whereas DMSO or even 40 lM rotenone had no significant effect on Nox expression. Nox-RNAi efficiency

Fig. 3 RT-qPCR analysis of Nox expression during MS development. TUB and TEF genes were used as the internal controls. The results are the mean relative expression ± SD. *P \ 0.05, **P \ 0.01, when compared with the results observed at 2 days

To investigate genetically the role of NADH oxidase in MS development, gene-specific siRNA for Nox and eGFP was employed to disrupt the Nox expression in vitro. The efficiency of RNAi in the transformants was analyzed by RTqPCR. In comparison with the controls, increasing the siRNA concentration enhanced the specific silencing effects. The Nox transcription showed a significant reduction of 57 %, following the treatment with 500 nM siRNA,

Table 2 Effect of rotenone on N. rileyi MS yields Rotenone (lM) -1

3

MS yields (MS ml ) 9 10

0

0.05 % DMSO

5

40

3.58 ± 0.38

3.22 ± 0.04

0.9 ± 0.14**

0.13 ± 0.03**

The results are the mean ± SD * P \ 0.05; ** P \ 0.01, when compared with the control

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Fig. 4 RT-qPCR analysis of the characteristic of Nox expression following rotenone inhibition and Nox silencing. TUB and TEF genes were used as the internal controls. The results are the mean relative expression ± SD. *P \ 0.05, **P \ 0.01, when compared with the control

but the siRNA concentration of 200 nM had little effect on it (Fig. 4). Role of Nox in MS formation The MS phenotypes were monitored by culturing RNAi transformants in AM. There were obvious differences in morphological features after gene silencing. Following the treatment with 500 nM siRNA, the fermentation broth exhibited that the dark purple pigment disappeared, viscosity declined and the size of the MS was about tenfold larger than that noted in the controls or sample treated with 200 nM siRNA (Fig. 5). Furthermore, consistent with the pharmacological study, on day 3 after transformation by using 500 nM siRNA, the Nox-RNAi strain could hardly form MS, in which the MS yields sharply decreased by 98.5 % (Fig. 6a). In contrast, the wild-type and negative control showed normal MS development. Hence, the 500 nM siRNA transformant was used in the virulence test.

Fig. 6 Silencing of the Nox gene in vitro. a The MS yields in AM obtained 3 days after Nox gene silencing. b The intracellular H2O2 concentration of MS after 3 days of inoculation in the RNAi system. The results are the mean ± SD. *P \ 0.05, **P \ 0.01, when compared with the control

Production of H2O2 in RNAi To examine whether inactivation of Nox leads to alter ROS accumulation, the H2O2 production was measured after the transformants culturing in AM for 3 days. As expected, the intracellular H2O2 concentration decreased by 38 % following the 500 nM siRNA treatment (Fig. 6b). The

Fig. 5 MS phenotypes of the control and siRNA-transformation treatments after 3 days of inoculation in AM. Scale bars 1 mm (first three images) and 5 mm (last image)

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Fig. 7 Survival of the cabbage caterpillar was an application of 5 ll suspensions of 1 9 107 MS ml-1 from wild-type, eGFP and NoxRNAi strains, respectively. The control insects were inoculated with 5 ll cottonseed oil

significant reduction in intracellular H2O2 indicated that H2O2 production was impaired in 500 nM siRNA transformant, which was co-related to RNAi-induced decrease in Nox transcription and MS yields. These results indicated that Nox was essential for MS formation. Influence of Nox in the virulence of N. rileyi The three strains were inoculated into the cabbage caterpillar larvae for virulence assays. Based on the survival curve of cabbage caterpillar (Fig. 7), it was subjected to probit analysis. The average LT50 values for wild-type, eGFP and Nox-RNAi strains were estimated at 8.53, 8.73 and 10.36 days, respectively. The increase of LT50 for RNAi strain indicated that Nox was required for N. rileyi virulence.

Discussion Although it has been hypothesized that oxidative stress may be involved in MS development in N. rileyi (Song et al. 2013), the mechanism of MS differentiation in N. rileyi is not yet very clear. In the present study, oxidative stress producing agents—H2O2 and menadione had similar effects on growth and differentiation of N. rileyi MS. It indicated that a certain concentration of H2O2 and menadione causing a higher oxidative stress contributed to MS differentiation (Table 1). Menadione as an oxidant can be reduced to O2- by the catalysis of flavoenzymes (Singh et al. 2012). Besides, it has been demonstrated that H2O2 and O2- as directors of oxidative stress are involved in sclerotial differentiation of filamentous phytopathogenic fungi (Papapostolou and Georgiou 2010a, b). Zhou et al.

(2011) also demonstrated that exogenous low dose of (10-13) H2O2 could stimulate cell proliferation as well as the findings in the present study. However, ascorbic acid is known to be an antioxidant capable of scavenging ROS. Exogenous ascorbic acid, as an antioxidant decrease oxidative stress, caused up to 100 % inhibition of MS formation in a dose-dependent manner (Table 1). Therefore, it concluded that artificial antioxidants that eliminate oxidative stress could inhibit MS metamorphosis. Similar results were also reported that ascorbic acid could decrease lipid peroxidation and the number of sclerotia formed in Sclerotium rolfsii and Rhizoctonia solani (Ellil 1999; Georgiou and Petropoulou 2001). The present study showed that the oxidative stress–H2O2 was involved in N. rileyi MS differentiation. During MS development, the H2O2 level peaked in the MI stage, representing the transition from pre-differentiation to differentiation stage (Fig. 1). As the MI stage was characterized by highly proliferating hyphae, the increasing level of H2O2 suggested that a higher oxidative stress in the MI stage than that noted in the UD stage, and H2O2 might play a role in both cell proliferation and differentiation. The higher level of H2O2 produced in the MI stage was also supported by the expression pattern of catalase (CAT) (Song et al. 2013), furthermore, the increasing CAT transcription could protect MS from excess levels of H2O2 during its formation. The results further affirmed the conclusion that oxidative stress occurred during MS development. NADH oxidases always play important roles in mediating O2- and H2O2 generation; in particular, it is a primary source for H2O2 generation in aerobes. Although H2O2-forming and H2O-forming oxidases have been purified and characterized from several bacterial species, there are few reports on NADH oxidase of N. rileyi (Schmidt et al. 1986; Heux et al. 2006; Gao et al. 2012). In accordance with the theory of oxidative stress, we cloned and analyzed the Nox gene from MS in N. rileyi for the first time. Although flavin-containing NADH oxidases have been purified and characterized from several bacteria. They commonly contain the flavin adenine dinucleotide (FAD)and NADH-binding regions (Higuchi et al. 2000; Reed et al. 2001; Gao et al. 2012), with the exception of the NADH oxidase from Acholeplasma laidlawii (Reinards et al. 1981). The Nox coding region from N. rileyi contains a flavin mononucleotide (FMN) binding domain that catalyzes the reduction of a quinone, which is similar to those of A. laidlawii and TIM phosphate binding superfamily (Reinards et al. 1981; Nagano et al. 2002; Raushel et al. 2003). Furthermore, we showed that Nox played a critical role in the control of MS development in N. rileyi. It was found that the Nox transcription level was up-regulated in the MI

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stage (Fig. 3), which was consistent with the reported by Kim et al. (2011), also co-related to the intracellular H2O2 concentration (Fig. 1). As MS initiation was associated with the generation of ROS (Fig. 1), inhibition of ROS might have negative impacts on MS development. Under conditions with treatment by rotenone, an inhibitor of NADH oxidase, the Nox transcription and MS yields decreased. It indicated that the Nox was involved in the regulation of MS formation. Similar results were also observed when S. sclerotiorum was grown in diphenyleneiodonium to inhibit NADPH oxidase (Kim et al. 2011). In addition, owing to laboratory restrictions, the role of Nox in MS formation was verified by RNAi technology, instead of knockout technology. Down-regulation of Nox in N. rileyi resulted in limited MS development (Fig. 5). Besides, 500 nM siRNA appeared to be effective for Nox silencing, and partial silencing of Nox did not affected N. rileyi MS development. These findings were similar to the results of rotenone treatments, and consistent with those reported for Botrytis cinerea NADPH oxidase mutants (Segmu¨ller et al. 2008), where NADPH oxidase genes were involved in sclerotial formation. The above-mentioned results indicated that Nox was required for proper MS development in N. rileyi. During the course of these studies, we noticed that Nox expression was linked to H2O2 production. Hence, the intracellular H2O2 concentration of MS was measured in RNAi background. It was found that H2O2 level produced by Nox-RNAi strain was noticeably lower than that those produced by the controls (Fig. 6b). The findings suggested that inhibition of MS differentiation might result from the down-regulation of NADH oxidase, which disrupted the accumulation of H2O2 during MS development. The results were analogous to what was reported for S. sclerotiorum NADPH oxidase RNAi mutants (Kim et al. 2011). MS served as a reproductive structure with a diameter of 200–600 lm for fungi (Song et al. 2013), which also exhibited insecticidal activity against insects, such as MS produced by M. anisopliae (Jackson and Jaronski 2009) and N. rileyi (Yin et al. 2012) in submerged liquid culture fermentation showed high infection and mortality to Tetanops myopaeformis and S. litura, respectively. In the present study, silencing of Nox significantly decreased the virulence in N. rileyi. The LT50 value was approximately increased by 21.5 % after inoculating cabbage caterpillar with MS from Nox-RNAi strain. The result suggested that Nox had an influence on virulence in N. rileyi. So far, how ROS generated genes-NADPH oxidases influenced the virulence of Magnaporthe oryzae have been demonstrated that NADPH oxidases-generated ROS within Magnaporthe oryzae appressorium facilitated oxidative cross-linking of proteins, thereby strengthening the appressorium cell wall to penetrate and cause infection (Thines et al. 2000; Egan

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et al. 2007). However, N. rileyi does not form appressoria in penetration into insect tissue, but rather a mucilaginous mass around the germinal tubes, which would also function as an adhesive and facilitate enzyme production to directly penetrate the cuticle of insects (Kumar et al. 1997; Srisukchayakul et al. 2005). Hence, how the Nox influences the virulence of N. rileyi may be different from that of M. oryzae and it is needed to further study. In summary, our results further validated the involvement of ROS in MS development. Furthermore, the Nox may control MS differentiation by regulating the intracellular H2O2 accumulation and be essential for virulence in N. rileyi. These findings provide a valuable insight into the molecular mechanism of MS differentiation in N. rileyi and other entomogenous fungi, as well as may contribute to the large-scale production of MS to control insects, because of the great agricultural and economic importance of N. rileyi. Acknowledgments This research was financially supported by State High Technique Program (863) of China (Project No. 2011AA10A201) and Special Fund for Agro-Scientific Research in the Public Interest (Project No. 201103002).

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