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GATA-type transcription factor MrNsdD regulates dimorphic transition, conidiation, virulence and microsclerotium formation in the entomopathogenic fungus Metarhizium rileyi.

ABSTRACT: 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.

SUBMITTER: Xin C

PROVIDER: S-EPMC7415378 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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GATA-type transcription factor MrNsdD regulates dimorphic transition, conidiation, virulence and microsclerotium formation in the entomopathogenic fungus Metarhizium rileyi.

Xin Caiyan C Yang Jie J Mao Yingyu Y Chen Wenbi W Wang Zhongkang Z Song Zhangyong Z

Microbial biotechnology 20200512 5

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 ...[more]

PMID: 32395911

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Project description:APSES-type transcription factors (TFs) have analogous and diverse functions in the regulation of fungal morphogenesis processes. However, little is known about these functions in microsclerotium formation. In this study, we characterized two orthologous APSES genes (MrStuA and MrXbp) in the entomopathogenic fungus Metarhizium rileyi Deletion of either MrStuA or MrXbp impaired dimorphic transition, conidiation, fungal virulence, and microsclerotium formation. Compared with the wild-type strain, ΔMrStuA and ΔMrXbp mutants were hypersensitive to thermal and oxidative stress. Furthermore, transcriptome sequencing analysis revealed that MrStuA and MrXbp independently regulate their own distinctive subsets of signaling pathways during dimorphic transition and microsclerotium formation, but they also show an overlapping regulation of genes during these two distinct morphogenesis processes. These results provide a global insight into vital roles of MrStuA and MrXbp in M. rileyi and aid in dissection of the interacting regulatory mechanisms of dimorphism transition and microsclerotium development.IMPORTANCE Transcription factors (TFs) are core components of the signaling pathway and play an important role in transcriptional regulation of gene expression during fungal morphogenesis processes. A prevailing theory suggests an interplay between different TFs regulating microsclerotial differentiation; however, the persisting issue remains that these interplay mechanisms are not clear. Here, we analyzed two members of the APSES-type TFs in Metarhizium rileyi using a gene deletion strategy and transcriptome analysis. Mutants were significantly impaired in microsclerotium formation and dimorphic transition. Transcriptome analysis provided evidence for interacting regulatory mechanisms by the two TFs in microsclerotium formation and dimorphic transition. Furthermore, we investigated their overlapping roles in mediating the expression of genes required for different fungal morphogenesis processes. Characterization of TFs in this study will aid in dissecting the interplay between regulatory mechanisms in fungal morphogenesis processes.

Project description:G proteins are critical modulators or transducers in various transmembrane signaling systems. They play key roles in numerous biological processes in fungi, including vegetative growth, development of infection-related structures, asexual conidiation, and virulence. However, functions of G proteins in entomopathogenic fungi remain unclear. Here, we characterized the roles of MrGPA1, a G-protein subunit Gαi, in conidiation, stress resistance, and virulence in Metarhizium robertsii. MrGPA1 was localized in the mitochondria. MrGpa1 deletion resulted in a significant reduction (47%) in the conidiation capacity, and reduced expression of several key conidiation-related genes, including fluG, flbD, brlA, wetA, phiA, and stuA. Further, MrGpa1 disruption resulted in decreased fungal sensitivity to UV irradiation and thermal stress, as determined based on conidial germination of ΔMrGpa1 and wild-type (WT) strains. Chemical stress analysis indicated that MrGpa1 contributes to fungal antioxidant capacity and cell wall integrity, but is not involved in tolerance to antifungal drug and osmotic stress. Importantly, insect bioassays involving (topical inoculation and injection) of Galleria mellonella larvae revealed decreased virulence of ΔMrGpa1 strain after cuticle infection. This was accompanied by decreased rates of appressorium formation and reduced expression of several cuticle penetration-related genes. Further assays showed that MrGpa1 regulated intracellular cyclic AMP (cAMP) levels, but feeding with cAMP could not recover the appressorium formation rate of ΔMrGpa1. These observations suggest that MrGpa1 contributes to the regulation of conidiation, UV irradiation, thermal stress response, antioxidant capacity, and cell wall integrity in M. robertsii. This gene is also involved in insect cuticle penetration during infection. These findings raise the possibility of designing powerful strategies for genetic improvement of M. robertsii conidiation capacity and virulence for killing pests.

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:BackgroundMost fungi, including entomopathogenic fungi, have two different conidiation patterns, normal and microcycle conidiation, under different culture conditions, eg, in media containing different nutrients. However, the mechanisms underlying the conidiation pattern shift are poorly understood.ResultsIn this study, Metarhizium acridum undergoing microcycle conidiation on sucrose yeast extract agar (SYA) medium shifted to normal conidiation when the medium was supplemented with sucrose, nitrate, or phosphate. By linking changes in nutrients with the conidiation pattern shift and transcriptional changes, we obtained conidiation pattern shift libraries by Solexa/Illumina deep-sequencing technology. A comparative analysis demonstrated that the expression of 137 genes was up-regulated during the shift to normal conidiation, while the expression of 436 genes was up-regulated at the microcycle conidiation stage. A comparison of subtractive libraries revealed that 83, 216, and 168 genes were related to sucrose-induced, nitrate-induced, and phosphate-induced conidiation pattern shifts, respectively. The expression of 217 genes whose expression was specific to microcycle conidiation was further analyzed by the gene expression profiling via multigene concatemers method using mRNA isolated from M. acridum grown on SYA and the four normal conidiation media. The expression of 142 genes was confirmed to be up-regulated on standard SYA medium. Of these 142 genes, 101 encode hypothetical proteins or proteins of unknown function, and only 41 genes encode proteins with putative functions. Of these 41 genes, 18 are related to cell growth, 10 are related to cell proliferation, three are related to the cell cycle, three are related to cell differentiation, two are related to cell wall synthesis, two are related to cell division, and seven have other functions. These results indicate that the conidiation pattern shift in M. acridum mainly results from changes in cell growth and proliferation.ConclusionsThe results indicate that M. acridum shifts conidiation pattern from microcycle conidiation to normal conidiation when there is increased sucrose, nitrate, or phosphate in the medium during microcycle conidiation. The regulation of conidiation patterning is a complex process involving the cell cycle and metabolism of M. acridum. This study provides essential information about the molecular mechanism of the induction of the conidiation pattern shift by single nutrients.

Project description:BACKGROUND:Metarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Unfortunately, its worldwide commercial use has been restricted by a short shelf life and inconsistencies in virulence. In our previous study, a gene (GenBank accession number EFZ01626) was found to be significantly upregulated in heat-treated conidia. In the present study, this gene was characterized via gene disruption and complementation strategies. RESULTS:The gene (amplified by rapid amplification of cDNA ends PCR) was 1219 bp long and contained an open reading frame (ORF) of 777 bp. It encoded a protein of 234 amino acid residues with a 26-residue signal peptide. Bioinformatics analyses did not identify conserved functional domains; therefore, it was assumed to be a secreted virulence-associated protein according to its signal peptide and bioassay results. We found that the conidial germination rate of the ΔMrSVP mutant fungi dramatically decreased after heat shock treatment in a thermotolerance test. In addition, transcription levels of all tested heat shock-related genes were significantly lower in the mutant than in the wild type. We also demonstrated that the mean lethal time to death (LT50) of ΔMrSVP significantly increased relative to the wild type in insect bioassays (both topical inoculation and injection) involving Galleria mellonella. Moreover, similar rates of appressorium formation between ΔMrSVP and the wild type-and the significantly different expression of virulence-related genes such as acid trehalase and sucrose nonfermenting protein kinase in the haemocoel after injection-revealed that MrSVP is required for virulence in the insect haemocoel. CONCLUSIONS:Overall, our data suggest that the Mrsvp gene contributes to thermotolerance and virulence of M. robertsii. Furthermore, this gene is deeply involved in the mycosis of insect cadavers and in immune escape rather than insect cuticle penetration during infection.

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Project description:The opportunistic human pathogen Talaromyces marneffei exhibits a temperature-dependent dimorphic transition, which is closely related with its pathogenicity. This species grows as multinucleate mycelia that produce infectious conidia at 25°C, while undergoes a dimorphic transition to generate uninucleate yeast form cells at 37°C. The mechanisms of phenotype switching are not fully understood. The transcription factor madsA gene is a member of the MADS-box gene family. Previously, it was found that overexpression of madsA gene resulted in mycelial growth instead of yeast form at 37°C. In the current study, the madsA deletion mutant (ΔmadsA) and complemented strain (CMA) were constructed by genetic manipulation. We compared the phenotypes, growth, conidiation, conidial germination and susceptibility to stresses (including osmotic and oxidative) of the ΔmadsA with the wild-type (WT) and CMA strains. The results showed that the ΔmadsA displayed a faster process of the yeast-to-mycelium transition than the WT and CMA. In addition, the deletion of madsA led to a delay in conidia production and conidial germination. The tolerance of ΔmadsA conidia to hydrogen peroxide was better than that of the WT and CMA strains. Then, RNA-seq was performed to identify differences in gene expression between the ΔmadsA mutant and WT strain during the yeast phase, mycelium phase, yeast-to-mycelium transition and mycelium-to-yeast transition, respectively. Gene ontology functional enrichment analyses indicated that some important processes such as transmembrane transport, oxidation-reduction process, protein catabolic process and response to oxidative stress were affected by the madsA deletion. Together, our results suggest that madsA functions as a global regulator involved in the conidiation and germination, especially in the dimorphic transition of T. marneffei. Its roles in the survival, pathogenicity and transmission of T. marneffei require further investigation.

Dataset Information

GATA-type transcription factor MrNsdD regulates dimorphic transition, conidiation, virulence and microsclerotium formation in the entomopathogenic fungus Metarhizium rileyi.

Publications

GATA-type transcription factor MrNsdD regulates dimorphic transition, conidiation, virulence and microsclerotium formation in the entomopathogenic fungus Metarhizium rileyi.

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