Differential Functions of Two Metalloproteases, Mrmep1 and Mrmep2, in Growth, Sporulation, Cell Wall Integrity, and Virulence in the Filamentous Fungus Metarhizium robertsii

The Metarhizium genus of filamentous entomopathogenic fungi plays a pivotal role in regulating insect populations. Metalloproteases (MEPs) are a widely distributed and diverse family of hydrolytic enzymes that are important toxicity factors in the interactions between fungi and their hosts. Herein, we characterized two MEPs, Mrmep1 and Mrmep2, in Metarhizium robertsii using gene deletion. Growth rates of the resulting ΔMrmep1 and ΔMrmep2 mutants decreased by 16.2 and 16.5%, respectively, relative to the wild-type (WT) strain. Both mutants were less sensitive to cell wall-perturbing agents, sodium dodecyl sulfate and Congo red than the WT strain, whereas did not show any obvious changes in fungal sensitivity to ultraviolet B irradiation or heat stress. The conidial yield of ΔMrmep1, ΔMrmep2, and ΔMrmep1ΔMrmep2 mutants decreased by 56.0, 23, and 53%, respectively. Insect bioassay revealed that median lethal time values against Galleria mellonella increased by 25.5% (ΔMrmep1), 19% (ΔMrmep2), and 28.8% (ΔMrmep1ΔMrmep2) compared with the WT strain at a concentration of 1 × 107 conidia mL-1, suggesting attenuated fungal virulence in the ΔMrmep1, ΔMrmep2, and ΔMrmep1ΔMrmep2 strains. During fungal infection, transcription levels of Mrmep1 was 1.6-fold higher than Mrmep2 at 36 h post inoculation. Additionally, transcription levels of gallerimycin gene were 1.2-fold, 2.18-fold, and 2.5-fold higher in insects infected with the ΔMrmep1, ΔMrmep2, or ΔMrmep1ΔMrmep2 mutant than those infected with the WT strain, respectively. Our findings suggest that Mrmep1 and Mrmep2 are differentially contributed to the growth, sporulation, cell wall integrity, and virulence of M. robertsii

Modularity-Guided Graph Topology Optimization And Self-Boosting Clustering

Existing modularity-based community detection methods attempt to find community memberships which can lead to the maximum of modularity in a fixed graph topology. In this work, we propose to optimize the graph topology through the modularity maximization process. We introduce a modularity-guided graph optimization approach for learning sparse high modularity graph from algorithmically generated clustering results by iterative pruning edges between two distant clusters. To the best of our knowledge, this represents a first attempt for using modularity to guide graph topology learning. Extensive experiments conducted on various real-world data sets show that our method outperforms the state-of-the-art graph construction methods by a large margin. Our experiments show that with increasing modularity, the accuracy of graph-based clustering algorithm is simultaneously increased, demonstrating the validity of modularity theory through numerical experimental results of real-world data sets. From clustering perspective, our method can also be seen as a self-boosting clustering method

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

Background: Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs. Methodology and Results: We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sjabantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in ou

An “In-Depth” Description of the Small Non-coding RNA Population of Schistosoma japonicum Schistosomulum

Parasitic flatworms of the genus Schistosoma are the causative agents of schistosomiasis, which afflicts more than 200 million people yearly in tropical regions of South America, Asia and Africa. A promising approach to the control of this and many other diseases involves the application of our understanding of small non-coding RNA function to the design of safe and effective means of treatment. In a previous study, we identified five conserved miRNAs from the adult stage of Schistosoma japonicum. Here, we applied Illumina Solexa high-throughput sequencing methods (deep sequencing) to investigate the small RNAs expressed in S. japonicum schistosomulum (3 weeks post-infection). This has allowed us to examine over four million sequence reads including both frequently and infrequently represented members of the RNA population. Thus we have identified 20 conserved miRNA families that have orthologs in well-studied model organisms and 16 miRNA that appear to be specific to Schistosoma. We have also observed minor amounts of heterogeneity in both 3′ and 5′ terminal positions of some miRNA as well as RNA fragments resulting from the processing of miRNA precursor. An investigation of the genomic arrangement of the 36 identified miRNA revealed that seven were tightly linked in two clusters. We also identified members of the small RNA population whose structure indicates that they are part of an endogenously derived RNA silencing pathway, as evidenced by their extensive complementarities with retrotransposon and retrovirus-related Pol polyprotein from transposon

The Polyubiquitin Gene MrUBI4 Is Required for Conidiation, Conidial Germination, and Stress Tolerance in the Filamentous Fungus Metarhizium robertsii

The polyubiquitin gene is a highly conserved open reading frame that encodes different numbers of tandem ubiquitin repeats from different species, which play important roles in different biological processes. Metarhizium robertsii is a fungal entomopathogen that is widely applied in the biological control of pest insects. However, it is unclear whether the polyubiquitin gene is required for fungal development, stress tolerance, and virulence in the entomopathogenic fungus. In the present study, the polyubiquitin gene (MrUBI4, MAA_02160) was functionally characterized via gene deletion in M. robertsii. Compared to the control strains, the MrUBI4 deletion mutant showed delayed conidial germination and significantly decreased conidial yields (39% of the wild-type 14 days post-incubation). Correspondingly, the transcript levels of several genes from the central regulatory pathways associated with conidiation, including brlA, abaA, and wetA, were significantly downregulated, which indicated that MrUBI4 played an important role in asexual sporulation. Deletion of MrUBI4 especially resulted in increased sensitivity to ultraviolet (UV) and heat-shock stress based on conidial germination analysis between mutant and control strains. The significant increase in sensitivity to heat-shock was accompanied with reduced transcript levels of genes related to heat-shock protein (hsp), trehalose, and mannitol accumulation (tps, tpp, nth, and mpd) in the MrUBI4 deletion mutant. Deletion of MrUBI4 has no effect on fungal virulence. Altogether, MrUBI4 is involved in the regulation of conidiation, conidial germination, UV stress, and heat-shock response in M. robertsii

The APSES Gene MrStuA Regulates Sporulation in Metarhizium robertsii

The APSES family is a unique family of transcription factors with a basic helix-loop-helix structure (APSES: Asm1p, Phd1p, Sok2p, Efg1p, and StuAp), which are key regulators of cell development and sporulation-related processes. However, the functions of the APSES family of genes in the entomopathogenic fungus Metarhizium robertsii have not been reported. Here, we report the identification and characterization of the MrStuA gene, a member of the APSES family, in M. robertsii. The selected gene was identified as StuA in M. robertsii (MrStuA) because the gene product contains two conserved sequences, an APSES-type DNA-binding domain and a KilA DNA-binding domain, and has the highest homology with the StuA in the C-II clade of the APSES family. We found that the number of conidia produced by the ΔMrStuA strain was 94.45% lower than that in the wild type. Additionally, in the mutant, the conidia displayed an elongated shape, the sporulation was sparse and the phialide were slender. In addition, transcription levels of two central regulators of asexual development, AbaA and WetA, were significantly reduced in the mutant; furthermore, the transcription levels of other sporulation related genes, such as Mpk, Phi, Med, Aco, Flu, and FlbD, also decreased significantly. We also show that the median lethal time (LT50) of the mutant increased by 19%. This increase corresponded with a slower growth rate and an earlier conidia germination time compared to that of the wild strain. However, the resistance of the mutant to chemicals or physical stressors, such as ultraviolet radiation or heat, was not significantly altered. Our results indicate that in M. robertsii, MrStuA may play a crucial role in regulating sporulation as well as virulence, germination, and vegetative growth. This study improves our understanding of the impact of the transcription factor StuA on sporulation processes in filamentous fungi and provides a basis for further studies aimed at improving sporulation efficiency of these fungi for use as a biocontrol agent

Differential DNA methylation may contribute to temporal and spatial regulation of gene expression and the development of mycelia and conidia in entomopathogenic fungus Metarhizium robertsii

© 2017 British Mycological Society Conidia and mycelia are two important developmental stages in the asexual life cycle of entomopathogenic fungus Metarhizium. Despite the crucial role that DNA methylation plays in many biological processes, its role in regulation of gene expression and development in fungi is not yet fully understood. We performed genome-wide analysis of DNA methylation patterns of an M. robertsii strain with single base pair resolution. Specifically, we examined for changes in methylation patterns between the conidia and mycelia stages. The results showed that approximately 0.38 % of cytosines are methylated in conidia, which is lower than the DNA methylation level (0.42 %) in mycelia. We found that DNA methylation undergoes genome-wide reprogramming during fungal development in M. robertsii. 132 differentially methylated regions (DMRs), which were mostly distributed in gene regions, were identified. KEGG analysis revealed that the DMR-associated genes belong to metabolic pathways. Intriguingly, in contrast to most other eukaryotes, promoter activities in M. robertsii seemed differentially modulated by DNA methylation levels. We found that transcription tended to be enhanced in genes with moderate promoter methylation, while gene expression was decreased in genes with high or low promoter methylation

Ordered Nafion (R) ionomers decorated polypyrrole nanowires for advanced electrochemical applications

Fabrication of novel electrode materials with ordered proton-migration channels is an effective strategy to enhance the proton conductivity of the electrode for polymer electrolyte membrane fuel cells. Here we report the electrochemical fabrication of ordered Nafion (R) ionomers decorated polypyrrole nanowires to construct the ordered proton-migration channels. Based on the electrostatic interaction between Nafion (R) ionomers and the polymer intermediate, ordered Nafion (R) ionomers decorated polypyrrole nanowires could be fabricated via chronoamperometry with varying contents of Nafion (R) ionomers. The morphologies, charge-storage performances, electron conductivity and proton conductivity of the composites are investigated by scanning electron microscopy, cyclic-voltammetry, galvanostatic charge-discharge measurement and electrochemical impedance spectroscopy. With the modification effect of Nafion (R) ionomers on polypyrrole nanowires, the composite shows greater ordered structure relative to another without Nafion (R) ionomers and the electrochemical performances change with the content of Nafion (R) ionomers. The composite could achieve a high specific capacitance of 356 F/g at 1 A/g with a 0.62-fold enhancement compared to polypyrrole nanowires without Nafion (R) ionomers. It also displays a superior electrical conductivity of 49 S/cm and a quite high proton conductivity of 0.014 S/cm at working conditions of fuel cells, which are associated with the requirements of fuel cells and have the potential to be the electrode material for a large range of electrochemical energy conversion devices. (C) 2017 Science Press and Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier B.V. and Science Press. All rights reserved

Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis