Dysregulation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3)inflammasome is involved in many chronic inflammatory diseases, including gouty arthritis. Activation of the NLRP3 inflammasome requires priming and activation signals: the priming signal controls the expression of NLRP3 and interleukin (IL)-1β precursor (proIL-1β), while the activation signal leads to the assembly of the NLRP3 inflammasome and to caspase-1 activation. Here, we reported the effects of the alcoholic extract of Taiwanese green propolis (TGP) on the NLRP3 inflammasome in vitro and in vivo. TGP inhibited proIL-1β expression by reducing nuclear factor kappa B activation and reactive oxygen species (ROS) production in lipopolysaccharide-activated macrophages. Additionally, TGP also suppressed the activation signal by reducing mitochondrial damage, ROS production, lysosomal rupture, c-Jun N-terminal kinases 1/2 phosphorylation and apoptosis-associated speck-like protein oligomerization. Furthermore, we found that TGP inhibited the NLRP3 inflammasome partially via autophagy induction. In the in vivo mouse model of uric acid crystal-induced peritonitis, TGP attenuated the peritoneal recruitment of neutrophils, and the levels of IL-1β, active caspase-1, IL-6 and monocyte chemoattractant protein-1 in lavage fluids. As a proof of principle, in this study, we purified a known compound, propolin G, from TGP and identified this compound as a potential inhibitor of the NLRP3 inflammasome. Our results indicated that TGP might be useful for ameliorating gouty inflammation via inhibition of the NLRP3 inflammasome.

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DNA methyltransferases (DNMTs) are a group of proteins that catalyze DNA methylation by transferring a methyl group to DNA. The genetic variation in DNMTs results in differential DNA methylation patterns associated with various biological processes. In fungal species, DNMTs and their DNA methylation profiles were found to be very diverse and have gained many research interests. We reviewed fungal DNMTs in terms of their biological functions, protein domain structures, and their associated epigenetic regulations compared to those known in plant and animal systems. In addition, we summarized recent reports on potential RNA-directed DNA methylation (RdDM) related to DNMT5 in fungi. We surveyed up to 40 fungal species with published genome-wide DNA methylation profiles (methylomes) and presented the associations between the specific patterns of fungal DNA methylation and their DNMTs based on a phylogenetic tree of protein domain structures. For example, the main DNMTs in Basidiomycota, DNMT1 with RFD domain + DNMT5, contributing to CG methylation preference, were distinct from RID + Dim-2 in Ascomycota, resulting in a non-CG methylation preference. Lastly, we revealed that the dynamic methylation involved in fungal life stage changes was particularly low in mycelium and DNA methylation was preferentially located in transposable elements (TEs). This review comprehensively discussed fungal DNMTs and methylomes and their connection with fungal development and taxonomy to present the diverse usages of DNA methylation in fungal genomes.

The Toll signaling pathway is responsible for defense against both Gram-positive bacteria and fungi. Gram-negative binding protein 3 (GNBP3) has a strong affinity for the fungal cell wall component, β-1,3-glucan, which can activate the prophenoloxidase (proPO) cascade and induce the Toll signaling pathway. Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in the Toll signaling pathway. In this study, we monitored the response of 5 key genes (TmGNBP3, TmMyD88, and Tenecin 1, 2, and 3) in the Toll pathway of the mealworm Tenebrio molitor immune system against the fungus Beauveria bassiana JEF-007 using RT-PCR. TmGNBP3, Tenecin 1, and Tenecin 2 were significantly upregulated after fungal infection. To better understand the roles of the Toll signaling pathway in the mealworm immune system, TmGNBP3 and TmMyD88 were knocked down by RNAi silencing. Target gene expression levels decreased at 2 d postknockdown and were dramatically reduced at 6 d post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 d post-dsRNA injection. Silencing of TmMyD88 and TmGNBP3 resulted in reduced resistance of the host to fungal infection. Particularly, reducing TmGNBP3 levels obviously downregulated Tenecin 1 and Tenecin 2 expression levels, whereas silencing TmMyD88 expression resulted in decreased Tenecin 2 expression. These results indicate that TmGNBP3 is essential to induce downstream antifungal peptide Tenecin 1 expression against B. bassiana JEF-007.

BackgroundOutbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylina larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed.ResultsThe genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hrs) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV.ConclusionThe gene parity plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that LyxyMNPV is a Group II NPV that is most closely related to LdMNPV but with a highly distinct genomic organisation.

Chemical pesticides have been used for pest control for many decades, but they cause serious problems, including insecticide resistance, secondary pest resurgence, and negative environmental impacts. Therefore, sustainable alternatives to chemical pesticides are necessary for pest control. Entomopathogenic fungi (EPF) are natural epizootic pathogens of insects, and some of them have been used as microbial biocontrol agents. Herein, we attempted to construct an entomopathogenic fungal library (EFLib) via the “Tenebrio molitor pathogenicity-based fungal collection method (TmPC)” to select EPF for control of the serious agricultural pest Spodoptera litura. A total of 172 soil samples were collected in northern and central Taiwan for the EFLib construction. The isolation efficiency of TmPC was 64.02%. The EFLib consisted of 101 isolates and was designated as the National Chung Hsing University (NCHU) EFLib. Among these isolates, 26 showed high virulence (mortality = 100%) to T. molitor larvae. Based on the results of molecular identification, the highly virulent isolates belonged to seven genera, including Beauveria, Clonostachys, Fusarium, Cordyceps, Penicillium, Purpureocillium, and Metarhizium. To evaluate the potential of these isolates for Spodoptera litura control, 12 isolates were selected for pathogenicity screening against S. litura larvae. A total of six EPF isolates belonging to the genera Beauveria and Metarhizium showed rapid eradication of the S. litura larvae. To rank the potential of these fungal strains for pest management, the six isolates were subjected to thermotolerance and conidial production assays, and a novel effective conidia number (ECN) formula was applied. The results indicated that the ECN index of Beauveria australis (NCHU-113) was much higher than that of Metarhizium isolates. However, among the Metarhizium isolates, NCHU-95 showed the highest ECN index. Altogether, NCHU-69 and NCHU-113 should be further tested in field trials. To our knowledge, this is the first attempt to integrate pathogenicity or virulence and ECN data into EPF screening and ranking, providing a baseline for mass selection of potential EPF strains for further applications.

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