Regulation of conidiation, polarity growth, and pathogenicity by MrSte12 transcription factor in entomopathogenic fungus, Metarhizium rileyi.

Metarhizium rileyi, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field pests, especially noctuid pests. To explore the potential factors involved in the fungal pathogenicity, MrSte12, an important and conserved functional transcription factor in mitogen-activated protein kinase pathway was carried out by functional analysis. Homologous recombination was used to disrupt the MrSte12 gene in M. rileyi. The deletant fungal strain exhibited malformed hyphae and impaired conidiogenesis, and conidia could not be collected from △MrSte12 in vitro towards SMAY medium. Although conidia could be collected again supplemented with KCl within SMAY medium, the conidial germination, growth and stress tolerance were much weaker compared with that in WT. Additionally, △MrSte12 showed a dramatic reduction in virulence in intra-hemolymph injections and no pathogenicity in topical inoculations against noctuid pests, which is due to the failure of appressorium formation. Moreover, the content of chitin and ß-1, 3-glucan in cell wall significantly reduced in mutant conidia. These results indicate that the MrSte12 gene markedly contributes to invasive growth and conidiation, as well as the major pathogenicity in M. rileyi.

MripacC regulates blastosphere budding and influences virulence of the pathogenic fungus Metarhizium rileyi.

Fungal dimorphism is the ability of certain fungi to switch between two different cellular forms, yeast and mycelial forms, in response to external environmental factors. The pacC/Pal signal transduction pathway responds to neutral and alkaline environments and is also involved in the fungal dimorphic transition. In this study, we investigated the function of the pacC homolog, MripacC, which regulates the dimorphic transition and modulates virulence of the insect pathogenic fungus Metarhizium rileyi. MripacC expression was upregulated under alkaline condition, with increased number of yeast-like cells compared to the number of hyphae cells. A MripacC deletion mutant (ΔMripacC) was obtained by homologous replacement and exhibited decreased blastospore budding, with direct development of conidia into hyphae without entering the yeast-like stage when cultured on alkaline medium. Observation of host hemolymph morphology and analysis of samples to detect the main immune factors revealed a decreased ability of ΔMripacC to evade the host immune system. The results of insect bioassays showed that ΔMripacC had decreased virulence with extended median lethality time. Together, the results suggested that MripacC not only regulated adaptation to acidic and alkaline environments, but also influenced virulence by budding blastospores. This elucidation of the function of MripacC adds to our understanding of blastospore budding and virulence of this fungal pathogen.

Pathogenicity of Metarhizium rileyi against Spodoptera litura larvae: Appressorium differentiation, proliferation in hemolymph, immune interaction, and reemergence of mycelium.

The pathogenicity of Metarhizium rileyi is a multi-faceted process that depends on many factors. This study attempts to decipher those factors of M. rileyi by investigating its pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae) larvae. Through morphogenesis analysis, we for the first time demonstrated the infection structure, appressorium, of M. rileyi that can generate a more than 4 MPa turgor pressure. The Mrpmk1 gene was found to be essential for appressorium differentiation and mycelium reemerging, ΔMrpmk1 mutant exhibited no pathogenicity towards S. litura by natural infection process. Delayed appressorium formation time, decreased appressorium formation rate and turgor pressure of ΔMrpbs2 mutant manifested itself in postponed death time and lower mortality against S. litura. Following invasion into the larval hemocoel, M. rileyi cells transformed into blastospores, which may be conducive to dispersal and propagation, moreover, the blastospore form M. rileyi may subverted phagocytic defenses. Then M. rileyi cells morphed into extended hyphal body to cope with elongated hemocytes that participated in encapsulation. In the end, M. rileyi mycelia reemerged from the larval cadaver evenly to form muscardine cadaver. Eventually, conidia were produced to complete the infection cycle. During the infection, M. rileyi triggered both cellular and humoral immunity of S. litura. Besides morphological changes, stage-specifically produced oxalic acid and F-actin arrangement may play roles in nutrient acquisition and mycelium reemerging, respectively.

Liriopesides B from Liriope spicata var. prolifera inhibits metastasis and induces apoptosis in A2780 human ovarian cancer cells.

Ovarian cancer is the most frequent cause of death among gynecological cancers. In the present study, the anti­cancer effect of liriopesides B, a steroidal saponin from Liriope spicata var. prolifera, against A2780 cells was investigated. Transwell chambers were adopted to assess its effect on cell invasion and chemotaxis abilities. Flow cytometry was used to analyze the cell cycle and apoptosis. Reverse transcription­quantitative PCR was employed to examine gene expression levels. Western blot analysis was performed to detect protein expression levels. Liriopesides B inhibited the invasion and chemotactic movement ability of A2780 cells in a dose­dependent manner. Furthermore, liriopesides B caused cell cycle arrest in A2780 cells at the G1 phase following incubation for 24, 48 and 72 h. Hoechst 33258 staining indicated that, following incubation for 48 h, liriopesides B induced cell apoptosis in a dose­dependent manner. Flow cytometry verified that liriopesides B induced apoptosis in A2780 cells and induced late apoptosis in a dose­dependent manner. Furthermore, liriopesides B significantly increased the mRNA expression levels of E­CADHERIN, p21 and p27 and decreased the gene expression levels of BCL­2, which was consistent with its protein expression levels. In conclusion, liriopesides B possess anti­cancer properties, including inhibition of metastasis­associated behaviors, cell cycle arrest and induction of apoptosis. Therefore, liriopesides B may be considered as a candidate drug against ovarian cancer.

In silico studies reveal RSc1154 and RhlE as temperature-related pathogenic proteins of Ralstonia solanacearum.

This study aimed to explore the temperature-related pathogenic mechanism of Ralstonia solanacearum infection in tomato (Lycopersicon esculentum Mill.). Based on bioinformatics analysis of microarray dataset (GSE33657), the co-differentially expressed genes (co-DEGs) ribonucleic acids were identified in R. solanacearum GMI1000-infected L. esculentum Mill., which was cultured at 20°C and 28°C, in rich medium containing casamino acids, peptone, and glucose (CPG) and planta. In total, 63 upregulated co-DEGs and 57 downregulated co-DEGs were identified between 20°C and 28°C in the CPG and planta groups. Protein-protein interaction network revealed 70 protein interaction pairs and 59 nodes. Notably, iolG, iolE, ioll and RSc1248 played critical roles in the network. The subcellular localization and functional annotation showed that the increased expressed proteins were mainly localized in the inner cell membrane, while those with decreased expression were localized in the cytoplasm. Furthermore, these proteins were mainly enriched in regulation of DNA-templated transcription. RSc1154 and RhlE were predicted to be temperature-related pathogenic genes for R. solanacearum in tomato. Furthermore, phosphorelay signal transduction system function might play an important role in R. solanacearum infection. The candidate genes were verified by quantitative real-time PCR, and the results were consistent with gene expression profile.

Regulation of conidiation, dimorphic transition, and microsclerotia formation by MrSwi6 transcription factor in dimorphic fungus Metarhizium rileyi.

Microsclerotia (MS) produced in the liquid culture of the dimorphic insect pathogen Metarhizium rileyi can be used as a mycoinsecticide. Bioinformatics analysis demonstrated that the cell cycle signaling pathway was involved in regulating MS formation. To investigate the mechanisms by which the signaling pathway is regulated, a cell cycle box binding transcription factor MrSwi6 of M. rileyi was characterized. MrSwi6 was highly expressed during periods of yeast-hypha transition and conidia and MS formation. When compared with wild-type and complemented strains, disruption of MrSwi6 significantly reduced conidia (15-36%) and MS formation (96.2%), and exhibited decreased virulence levels. Digital expression profiling revealed that genes involved in antioxidation, pigment biosynthesis, and ion transport and storage were regulated by MrSwi6 during conidia and MS development. These results confirmed the significance of MrSwi6 in dimorphic transition, conidia and MS formation, and virulence in M. rileyi.

Discrimination of Burkholderia gladioli pv.alliicola and B. cepacia complex using the gyrB gene of B. gladioli pv. alliicola.

The aim of the present study was to investigate the efficiency of the gyrB gene derived from Burkholderia gladioli pv.Alliicola (Bga) on the identification of Bga from the B. cepacia complex (Bcc) based on the COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy. A set of primers used for the specific amplification of the gyrB gene in Bga were designed according to the CODEHOP principle. A total of 1,644 bp of the gyrB gene sequence of Bga were acquired by CODEHOP amplification. The sequence was blasted in GenBank and it revealed an average of 86% similarity with the gyrB gene of nine genomovars of Bcc. A phylogenetic tree was constructed using the gyrB gene sequences. The microarray method was adopted to discriminate Bga from Bcc based on the specific probes designed upon the gyrB gene, and five genomovars of Bcc demonstrated a good discrimination from Bga on the microarray chip. CODEHOP strategy succeeded in amplification of the gyrB gene of Bga, which made it possible for the identification of Bga from five genomovars of Bcc.

A transcription factor, MrMsn2, in the dimorphic fungus Metarhizium rileyi is essential for dimorphism transition, aggravated pigmentation, conidiation and microsclerotia formation.

Microsclerotia (MS) are pseudoparenchymatous aggregations of hyphae of fungi that can be induced in liquid culture for biocontrol applications. Previously, we determined that the high-osmolarity glycerol (HOG) signalling pathway was involved in regulating MS development in the dimorphic insect pathogen Metarhizium rileyi. To further investigate the mechanisms by which the signalling pathway is regulated, we characterized the transcriptional factor MrMsn2, a homologue of the yeast C2 H2 transcriptional factor Msn2, which is predicted to function downstream of the HOG pathway in M. rileyi. Compared with wild-type and complemented strains, disruption of MrMsn2 increased the yeast-to-hypha transition rate, enhanced conidiation capacity and aggravated pigmentation in M. rileyi. The ▵MrMsn2 mutants were sensitive to stress, produced morphologically abnormal clones and had significantly reduced MS formation and decreased virulence levels. Digital expression profiling revealed that genes involved in antioxidation, pigment biosynthesis and ion transport and storage were regulated by MrMsn2 during conidia and MS development. Taken together, our findings confirm that MrMsn2 controlled the yeast-to-hypha transition, conidia and MS formation, and virulence.

Adaption to stress via Pbs2 during Metarhizium rileyi conidia and microsclerotia development.

The high osmolarity glycerol (HOG) pathway plays important role in Metarhizium rileyi microsclerotia (MS) development. To investigate how M. rileyi transduce growth stress and regulate MS development via mitogen-activated protein kinase kinase (MAPKK) Pbs2, phenotypic characterization of the yeast Pbs2 homolog were performed. Expression of pbs2 peaked when MS formation occurred day 3 in liquid amended medium. Compared with wild-type and complemented strains, deletion mutant of pbs2 (Δpbs2) delayed dimorphic switch and vegetative growth, displayed sensitivities to various stress, and significantly reduced conidial (98%) and MS (40%) yields. Furthermore, transcription analysis showed that other genes of HOG signaling pathway were down-regulated in Δpbs2 mutants. Insect bioassays revealed that Δpbs2 mutants had decreased virulence levels in topical (24%) and injection (53%) bioassays. This study confirmed that Pbs2 play important roles in colony morphology, conidiation, stresses response and MS development in M. rileyi.

The bZIP transcriptional factor activator protein-1 regulates Metarhizium rileyi morphology and mediates microsclerotia formation.

Internal oxidative stress can trigger microsclerotia (MS) formation of Metarhizium rileyi in liquid culture. Activator protein 1 (AP1) is a transcription factor and an important determinant of the response to oxidative stress. To investigate how M. rileyi responds to internal oxidative stress and how MS development is regulated, the Mrap1 gene was characterized. Mrap1 was highly expressed during periods of invasive hyphal growth and in response to changing culture conditions during MS development. Compared with the wild-type and complemented strains, ΔMrap1 mutants exhibited various defects in aerial hyphal growth, yeast-to-hypha transition, and conidia and MS formation. ΔMrap1 mutants also displayed sensitivity to oxidative stress, were morphologically abnormal, and responded differently to oxidative stress during MS development. ΔMrap1 mutants had significantly reduced conidial (74-82%) and MS (99%) yields. Insect bioassays revealed that ΔMrap1 mutants displayed reduced virulence in topical (43-76%) and injection (45-70%) bioassays. Moreover, the ability of ΔMrap1 mutants to grow out of the cuticle was reduced due to impaired conidiation on the host cadaver. Digital gene expression profiling revealed that genes involved in antioxidation, pigment biosynthesis, and ion transport were regulated by Mrap1 during MS development. Taken together, our results confirm the importance of Mrap1 in vegetative growth, conidia and MS formation, and virulence.

An efficient gene disruption method using a positive-negative split-selection marker and Agrobacterium tumefaciens-mediated transformation for Nomuraea rileyi.

Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The ß-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0-4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.

Global Transcriptome Sequencing Reveals Molecular Profiles of Summer Diapause Induction Stage of Onion Maggot, Delia antiqua (Diptera: Anthomyiidae).

The onion maggot, Delia antiqua, is a worldwide subterranean pest and can enter diapause during the summer and winter seasons. The molecular regulation of the ontogenesis transition remains largely unknown. Here we used high-throughput RNA sequencing to identify candidate genes and processes linked to summer diapause (SD) induction by comparing the transcriptome differences between the most sensitive larval developmental stage of SD and nondiapause (ND). Nine pairwise comparisons were performed, and significantly differentially regulated transcripts were identified. Several functional terms related to lipid, carbohydrate, and energy metabolism, environmental adaption, immune response, and aging were enriched during the most sensitive SD induction period. A subset of genes, including circadian clock genes, were expressed differentially under diapause induction conditions, and there was much more variation in the most sensitive period of ND- than SD-destined larvae. These expression variations probably resulted in a deep restructuring of metabolic pathways. Potential regulatory elements of SD induction including genes related to lipid, carbohydrate, energy metabolism, and environmental adaption. Collectively, our results suggest the circadian clock is one of the key drivers for integrating environmental signals into the SD induction. Our transcriptome analysis provides insight into the fundamental role of the circadian clock in SD induction in this important model insect species, and contributes to the in-depth elucidation of the molecular regulation mechanism of insect diapause induction.

Cloning and functional analysis of farnesyl pyrophosphate synthase (FPPS) gene from Mylabris cichorii.

Cantharidin, a defensive terpene compound synthesized by the meloid beetle (Coleoptera, Meloidae), is an important anticancer agent. However, there has been little study done on how this compound synthesized by the beetle. In this paper, a farnesyl pyrophosphate synthase (FPPS) gene, designated McFPPS, was isolated from Mylabris cichorii by reverse transcription PCR based on conserved domains in other organisms. Multiple alignment analysis showed that the deduced amino acids shared >70% homology with FPPSs from other species and contained typically seven conservative regions. Gene expression profile analysis revealed that McFPPS was expressed throughout the tested growth stages of M. cichorii adults, whereas the transcripts accumulated to the highest level at 20 days in male adults while the highest expression level appeared at 15 days in females. Tissue expression pattern analysis showed that McFPPS was expressed constitutively in all tested tissues and a relatively higher expression level in the alimentary canal of males, but no significant tissue difference in the females. For the first time, a RNA interference strategy was employed to induce a greater suppression of McFPPS mRNA, and thus a sharp decrease in the expression levels of downstream genes and the concentration of product. All these results indicated that McFPPS may be directly involved or play an essential role in the biosynthesis of cantharidin.

The high osmotic response and cell wall integrity pathways cooperate to regulate morphology, microsclerotia development, and virulence in Metarhizium rileyi.

Microsclerotia (MS) formation was successfully induced in Metarhizium rileyi under changing liquid culture conditions. Mitogen-activated protein kinases (MAPKs) play important roles in fungal development and in coordinating many stress responses. To investigate how M. rileyi transduces growth stress and regulates MS differentiation, we characterized the roles of two MAPKs, Hog1- and Slt2-type orthologues, in M. rileyi. Compared with the wild-type strain, the deletion mutants of Mrhog1 (ΔMrhog1) and Mrslt2 (ΔMrslt2) delayed germination and vegetative growth, displayed sensitivities to various stress, and produced morphologically abnormal clones. The ΔMrhog1 and ΔMrslt2 mutants significantly reduced conidial (42-99%) and MS (96-99%) yields. A transcriptional analysis showed that the two MAPKs regulate MS development in a cooperative manner. Insect bioassays revealed that ΔMrhog1 and ΔMrslt2 had decreased virulence levels in topical (36-56%) and injection (78-93%) bioassays. Our results confirmed the roles of MrHog1 and MrSlt2 in sensing growth-related stress and in regulating MS differentiation.

Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications.

Siderophore Biosynthesis but Not Reductive Iron Assimilation Is Essential for the Dimorphic Fungus Nomuraea rileyi Conidiation, Dimorphism Transition, Resistance to Oxidative Stress, Pigmented Microsclerotium Formation, and Virulence.

Iron is an indispensable factor for the dimorphic insect pathogenic Nomuraea rileyi to form persistent microsclerotia which can replace conidia or blastospores for commercial mass production. There are two high affinity iron acquisition pathways in N. rileyi, siderophore-assisted iron mobilization and reductive iron assimilation systems. Transcription of the two iron uptake pathways related genes is induced under iron-limiting conditions. Stage-specific iron uptake-related genes expression during microsclerotia development shows siderophore-mediated iron acquisition genes are rigorously upregulated specifically during the formation and mature period while reductive iron assimilation related genes just display a higher expression at the late maturation period. Abrogation of reductive iron assimilation, by the deletion of the high affinity iron permease (NrFtrA), has no visible effect on microsclerotia biogenesis in N. rileyi. In sharp contrast, N. rileyi L-ornithine-N(5)-monooxygenase (NrSidA), required for synthesis of all siderophores, is absolutely necessary for the development of pigmented microsclerotia. In agreement with the lower intracellular iron contents of microsclerotia in ΔNrSidA strains, not only the pigments, but both the number and the biomass are also noticeably reduced. Certain concentration of ROS is required for promoting microsclerotia biogenesis. Combined with expression pattern analysis of related genes and quantitative of intracellular iron or extracellular siderophore in WT and mutants, these data demonstrate the lack of adequate intracellular iron caused by the loss of the siderophore results in the deficiency of ROS detoxication. Furthermore, ΔNrSidA strains show significantly increased sensitivity to hydrogen peroxide. Besides, NrSidA, but not NrFtrA, play a crucial role in vegetative growth under iron-limiting conditions, conidiation, and dimorphic switching. Remarkably, the slower growth of the ΔNrSidA strains in vivo due to a reduced capacity for iron acquisition leads to the loss of virulence in Spodoptera litura while the ΔNrFtrA mutants behaved as WT during infection. Together, these results prove siderophore-assisted iron mobilization is the major pathway of cellular iron uptake and essential for conidiation, dimorphism transition, oxidative stress resistance, pigmented microsclerotium formation and full virulence.

De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii.

The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20-25 day-old adult males and 20-25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles.

Agrobacterium tumefaciens-mediated transformation of the entomopathogenic fungus Nomuraea rileyi.

An Agrobacterium-mediated genetic transformation system for the entomopathogenic fungus Nomuraea rileyi was established. Three binary T-DNA vectors, pPZP-Hph, pPZP-Hph-RNAi and pPZP-Hph-DsRed2, were constructed. The trpc promoter from Aspergillus nidulans was used as the cis-regulatory element to drive the expression of hygromycin phosphotransferase (hph) gene and DsRed2, which conferred the hygromycin B (Hyg B) resistance and red fluorescence visualization, respectively. The blastospores and conidia were used as the recipients. The blastospores' transformation efficiency reached ∼20-40 transformants per 10(6) blastospores, whereas the conidia were not transformed. Based on an analysis of five generations of subcultures, PCR and Southern blotting assays, the Ptrpc-hph cassette had integrated into the genomes of all transformants, which contained single copy of the hph gene and showed mitotic stability. Abundant altered morphologic phenotypes in colonies, blastospores and hyphae formations were observed in the arbitrary insertional mutants of N. rileyi, which made it possible to study the relationships between the functions and the interrupted genes over the whole genome. The transformation protocol will promote the functional characterization of genes, and the construction of genetically engineered strains of this important entomopathogenic fungus, and potentially of other similar fungal pathogens.

Involvement of an alternative oxidase in the regulation of hyphal growth and microsclerotial formation in Nomuraea rileyi CQNr01.

Mitochondria of Nomuraea rileyi contain an alternative oxidase (Aox), which reduces oxygen to water by accepting electrons directly from ubiquinol. Furthermore, through a transcriptional analysis, we found that an alternative oxidase (Nraox) was up-regulated during microsclerotial formation. To study the function of NrAox, Nraox was cloned from N. rileyi CQNr01. The full-length cDNA was 1266 bp with an open reading frame of 1068 bp encoding 355 amino acids. A phylogenetic analysis revealed that the NrAox of N. rileyi was closely related to Metarhizium acridum Aox. The relative expression level of the Nraox was up-regulated during microsclerotial (MS) initiation. A salicylhydroxamic acid, a specific alternative oxidase inhibitor, application to the culture media severely decreased MS yields, changed the hyphae morphology and slowed the H2O2 removal. Nraox silencing caused mycelial deformations, reduced the MS yields by 97.3 % and increased MS size compared with those of the control. MS virulence was decreased to 26.2 % after Nraox was silenced. However, the Nraox-silenced strain was sensitive to environmental stress, and the growth rate was reduced under stress conditions. The results obtained suggested that Nraox is required for MS differentiation by regulating the intracellular H2O2 concentration and hypha growth. Additionally, Nraox had a great impact on the virulence of N. rileyi.

Role of two Nomuraea rileyi transmembrane sensors Sho1p and Sln1p in adaptation to stress due to changing culture conditions during microsclerotia development.

Microsclerotia (MS) formation was successfully induced in Nomuraea rileyi in liquid amended medium (AM) culture. To investigate how N. rileyi senses growth stress and regulates MS differentiation, based on transcriptome library, sho1 and sln1 genes were cloned. The transcription levels of sho1 and sln1 were upregulated in response to the changing culture conditions. To determine the functions of sho1 and sln1, gene-silencing mutants (sholi, sln1i and shol&sln1i) were generated using RNA silencing technology. The significant phenotypic changes in the mutants included reduced conidial yields by 22.72, 40.27, and 63.67 % and virulence by 24.53, 25.74, and 59.04 %, respectively. Furthermore, the mutants presented decreased MS yields by approximately 96 % under changing culture conditions. Our results confirmed the crucial role of Sho1p and Sln1p in sensing growth stress due to changing culture conditions and regulating MS differentiation.