A transcription factor, MrMsn2, in the dimorphic fungus Metarhizium rileyi is essential for dimorphism transition, aggravated pigmentation, conidiation and microsclerotia formation.
ABSTRACT: 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.
Project description:BACKGROUND: Nomuraea rileyi is used as an environmental-friendly biopesticide. However, mass production and commercialization of this organism are limited due to its fastidious growth and sporulation requirements. When cultured in amended medium, we found that N. rileyi could produce microsclerotia bodies, replacing conidiophores as the infectious agent. However, little is known about the genes involved in microsclerotia development. In the present study, the transcriptomes were analyzed using next-generation sequencing technology to find the genes involved in microsclerotia development. RESULTS: A total of 4.69 Gb of clean nucleotides comprising 32,061 sequences was obtained, and 20,919 sequences were annotated (about 65%). Among the annotated sequences, only 5928 were annotated with 34 gene ontology (GO) functional categories, and 12,778 sequences were mapped to 165 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we assessed the transcriptomic differences between cultures grown in minimal and amended medium. In total, 4808 sequences were found to be differentially expressed; 719 differentially expressed unigenes were assigned to 25 GO classes and 1888 differentially expressed unigenes were assigned to 161 KEGG pathways, including 25 enrichment pathways. Subsequently, we examined the up-regulation or uniquely expressed genes following amended medium treatment, which were also expressed on the enrichment pathway, and found that most of them participated in mediating oxidative stress homeostasis. To elucidate the role of oxidative stress in microsclerotia development, we analyzed the diversification of unigenes using quantitative reverse transcription-PCR (RT-qPCR). CONCLUSION: Our findings suggest that oxidative stress occurs during microsclerotia development, along with a broad metabolic activity change. Our data provide the most comprehensive sequence resource available for the study of N. rileyi. We believe that the transcriptome datasets will serve as an important public information platform to accelerate studies on N. rileyi microsclerotia.
Project description: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 (?Mrhog1) and Mrslt2 (?Mrslt2) delayed germination and vegetative growth, displayed sensitivities to various stress, and produced morphologically abnormal clones. The ?Mrhog1 and ?Mrslt2 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 ?Mrhog1 and ?Mrslt2 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.
Project description: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 ?NrSidA 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, ?NrSidA 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 ?NrSidA strains in vivo due to a reduced capacity for iron acquisition leads to the loss of virulence in Spodoptera litura while the ?NrFtrA 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.
Project description:The GATA-type sexual development transcription factor NsdD has been implicated in virulence, secondary metabolism and asexual development in filamentous fungi. However, little is known about its function in the yeast-to-hypha transition and in microsclerotium formation. In the current study, the orthologous NsdD gene MrNsdD in the entomopathogenic fungus Metarhizium rileyi was characterized. Transcriptional analysis indicated that MrNsdD was involved in yeast-to-hypha transition, conidiation and microsclerotium formation. After targeted deletion of MrNsdD, dimorphic transition, conidiation, fungal virulence and microsclerotium formation were all impaired. Compared with the wild-type strain, the ?MrNsdD mutants were hypersensitive to thermal stress. Furthermore, transcriptome sequencing analysis revealed that MrNsdD regulated a distinct signalling pathway in M. rileyi during the yeast-to-hypha transition or microsclerotium formation, but exhibited overlapping regulation of genes during the two distinct developmental stages. Taken together, characterization of the MrNsdD targets in this study will aid in the dissection of the molecular mechanisms of dimorphic transition and microsclerotium development.
Project description:Metarhizium rileyi (Farlow) Samson is an important entomopathogenic fungus of more than 30 species of Lepidoptera larvae. The aim of this research was to characterize isolate of M. rileyi from Quivicán, Cuba on the basis of morphological and molecular approaches. The fungus was isolated from samples of S. frugiperda larvae collected from maize fields of Quivicán municipality, Mayabeque province, Cuba, and it was cultured on PDA?+?Ampicillin solid media for morphological characterization. The DNA was isolated using CTAB method and internal transcribed spacer (ITS1, ITS4) were used as the primers for the amplification. The amplified products of 1335 bp were purified and sequenced at CINVESTAV-IPN in both the directions using the above primers. A consensus sequence was obtained by alignment of the forward and reverse sequences for this region and deposited in GenBank (MG637450). The fungus produced slightly cottony colony of pale green color and dispersed conidia and septal mycelium were observed under the optical microscope. A BLAST search of the sequence in GenBank revealed a 99% of identity with several strains of N. rileyi (e.g., AF368501.1, AB268359.1 and EU553337.1) and M. rileyi (e.g., KY436756.1). This is the first report of M. rileyi isolate from maize fields of Quivicán in Cuba and this is important for biodiversity studies and is another possibility for Integrated Pest Management.
Project description:Verticillium dahliae is a soilborne fungus that causes wilt disease in plants. The microsclerotia of V. dahliae produce infectious hyphae that give rise to primary infections. In this study, RNA-seq libraries were prepared from microsclerotia (MS)-producing cultures of V. dahliae (ave = 52.23 million reads), and those not producing microsclerotia (NoMS, ave = 50.58 million reads) and analyzed for differential gene expression.
Project description:Verticillium dahliae is a soil-borne, hemibiotrophic phytopathogenic fungus that causes wilting in crop plants. Here, we constructed a random insertional mutant library using Agrobacterium tumefaciens-mediated transformation to study the pathogenicity and regulatory mechanisms of V. dahliae. The fungal-specific transcription factor-encoding gene Vdpf was shown to be associated with vegetative growth and virulence, with the highest transcript expression occurring during conidia formation in the V991 strain. The deletion mutants (?Vdpf) and insertion mutants (IM?Vdpf) produced fewer conidia than did the wild-type (WT) fungi, which contributed to the reduced virulence. Unlike the WT, the complemented strains and IM?Vdpf, ?Vdpf formed swollen, thick-walled and hyaline mycelium rather than melanized microsclerotia. The ?Vdpf mutants were melanin deficient, with undetectable expression of melanin biosynthesis-related genes (Brn1, Brn2 and Scd1). The melanin deficiency was related to cyclic adenosine monophosphate (cAMP) and the G-protein-coupled signalling pathways in this study. Similar to the WT and complemented strains, the ?Vdpf and IM?Vdpf mutants could also successfully penetrate into cotton and tobacco roots, but displayed reduced virulence because of lower biomass in the plant roots and significantly reduced expression of pathogenicity-related genes in V. dahliae. In conclusion, these results provide insights into the role of Vdpf in melanized microsclerotia formation, conidia production and pathogenicity.
Project description:BACKGROUND: The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to 15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae. RESULTS: To shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 50.58 million reads). Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations. CONCLUSION: Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae. The identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development.
Project description:The tick Rhipicephalus microplus poses a serious threat to the cattle industry, resulting in economic losses aggravated by tick resistance to chemical acaricides. Strains of Metarhizium spp., a well-known group of entomopathogenic fungi, can contribute to managing this ectoparasite. We explored two novel granular, microsclerotia- or blastospores-based formulations of Metarhizium robertsii for R. microplus control under semi-field conditions. Fungal persistence in soil was also observed for 336 days. The experiment used pots of Urochloa decumbens cv. Basilisk grass, treated with 0.25 or 0.5 mg of granular formulation/cm<sup>2</sup> (25 or 50 kg/ha) applied to the soil surface prior to transferring engorged tick females onto the treated soil. The fungal granules yielded more conidia with subsequent sporulation under controlled indoor conditions than in the outdoor environment, where the levels of fungus rapidly declined over time. Metarhizium-root colonization ranged from 25 to 66.7% depending on the propagule and rate. Fungal formulations significantly reduced the number of tick larvae during the humid season, reaching at least 64.8% relative efficacy. Microsclerotia or blastospores-granular formulations of M. robertsii can reduce the impact of R. microplus, and thus prove to be a promising tool in the control of ticks.
Project description:Metarhizium rileyi (formerly known as Nomuraea rileyi) is a potential agent for microbial control of many insect pests from the order Lepidoptera, the damages of which can cause considerable loss of productivity in agriculture. We report the genome sequence and annotation of M. rileyi strain Cep018-CH2/ARSEF 7053.