Synthesis and antifungal activity of hexahydropyrrolidoindole alkaloids.

Twenty-one hexahydropyrrolidoindole alkaloids were designed and synthesised via acylation reaction at the 3-N position from the commercially available indole-3-acetonitrile as the starting material in excellent yields. The effects of all target compounds against Verticillium dahlia, Fusarium oxysperium sp., Cytospora juglandis, Aspergillu sflavu, Aspergillus niger and Fusarium oxysporum were determined. The results of bioassays indicated that the majority of tested compounds displayed comparable or better in vitro bioactivity than the positive control. Notably, compounds 8 and 17 revealed potent activity against C. juglandis and A. sflavu, both with the same minimum inhibitory concentration value of 1.9 µg mL-1, which has fungicidal activity far exceeded that of amphotericin B and chlorothalonil.

Metabolomic analysis of lipid changes in Bombyx mori infected with Nosema bombycis.

The silkworm (Bombyx mori) is a model species of lepidopteran insect. Microsporidium spp. are obligate intracellular eukaryotic parasites. Infection by the microsporidian Nosema bombycis (Nb) results in an outbreak of Pébrine disease in silkworms and causes substantial losses to the sericulture industry. It has been suggested that Nb depends on nutrients from host cells for spore growth. However, little is known about changes in lipid levels after Nb infection. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was performed to analyze the effect of Nb infection on lipid metabolism in the midgut of silkworms. A total of 1601 individual lipid molecules were detected in the midgut of silkworms, of which 15 were significantly decreased after Nb challenge. Classification, chain length, and chain saturation analysis revealed that these 15 differential lipids can be classified into different lipid subclasses, of which 13 belong to glycerol phospholipid lipids and two belong to glyceride esters. The results indicated that Nb uses the host lipids to complete its own replication, and the acquisition of host lipid subclasses is selective; not all lipid subclasses are required for microsporidium growth or proliferation. Based on lipid metabolism data, phosphatidylcholine (PC) was found to be an important nutrient for Nb replication. Diet supplementation with lecithin substantially promoted the replication of Nb. Knockdown and overexpression of the key enzyme phosphatidate phosphatase (PAP) and phosphatidylcholine (Bbc) for PC synthesis also confirmed that PC is necessary for Nb replication. Our results showed that most lipids in the host midgut decreased when silkworms were infected with Nb. Reduction of or supplementation with PC may be a strategy to suppress or promote microsporidial replication.

Involvement of the neddylation modification system in Bombyx mori nucleopolyhedrovirus replication.

Neddylation is a posttranslational modification that is similar to ubiquitination, and involved in some critical biological processes, such as DNA repair, transcription regulation, and ubiquitin-proteasome pathway. Recently, it was found that neddylation inhibitor MLN4924 has potent antiviral activity against human viruses including herpes simplex virus (HSV)-1, HSV-2, and influenza viruses. Here, we report that MLN4924 could dramatically and dose-dependently inhibits the propagation, formation of budding virus (BV) and occlusion body (OB) of a lepidopteran virus-Bombyx mori nucleopolyhedrovirus (BmNPV), impaired OB assembly. In addition, the neddylation modification protein NEDD8 is colocalized with aggresome and autophagosome. Our findings suggest that inhibiting neddylation could be an antibaculovirus strategy and MLN4924 may be used as candidate drug for that purpose.

iTRAQ-based quantitative proteomic analysis of silkworm infected with Beauveria bassiana.

Beauveria bassiana is a harmful pathogen to the economically important insect silkworm, always causes serious disease to the silkworm, which results in great losses to the sericulture industry. In order to explore the silkworm (Bombyx mori) response to B. bassiana infection, differential proteomes of the silkworm responsive to B. bassiana infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) at the different stage of the 3rd instar silkworm larvae. Among the 5040 proteins identified with confidence level of ≥95 %, total 937 proteins were differentially expressed, of which 488 proteins were up-regulated and 449 proteins were down-regulated. 23, 15, 250, 649 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the B. bassiana infected larvae at 18, 24, 36, 48 h post infection (hpi) respectively. Based on GO annotations, 6, 4, 128, 316 DEPs were involved in biological processes, 12, 5, 143, 376 DEPs were involved in molecular functions, and 6, 3, 108, 256 DEPs were involved in cell components at 18, 24, 36, 48 hpi respectively. KEGG pathway analysis displayed that 18, 12, 210, 548 DEPs separately participated in 63, 35, 201, 264 signal transduction pathways at different time of infection, and moreover a higher proportion of DEPs involved in metabolic pathways. The cluster analysis on the DEPs of different infection stages distinguished a co-regulated DEP, lysozyme precursor, which was up-regulated at both the mRNA level and the protein level, indicating that the lysozyme protein kept playing an important role in defending the silkworm against B. bassiana infection. This was the first report using an iTRAQ approach to analyze proteomes of the whole silkworm against B. bassiana infection, which contributes to better understanding the defense mechanisms of silkworm to B. bassiana infection and provides important experimental data for the identification of key factors involved in the interaction between the pathogenic fungus and its host.

High efficiency transformation and mutant screening of the laurel wilt pathogen, Raffaelea lauricola.

The fungal pathogen, Raffaelea lauricola, is the causative agent of laurel wilt, a devastating disease affecting the Lauraceae family. The fungus is vectored by ambrosia beetles that carry the fungus in specialized structures (mycangia), with the fungus acting as a symbiont and food source for beetle larvae growing in tree galleries. In order to probe the molecular basis for plant pathogenicity and insect symbiosis of the laurel wilt fungus, molecular tools including establishment of efficient transformation protocols are required. Resistance marker profiling revealed susceptibility of R. lauricola to phosphinothricin, chlorimuron ethyl, hygromycin, and benomyl. Agrobacterium-mediated transformation using either the bar or sur marker resulted in 1-200 transformants/105 spores. A second protocol using lithium acetate-polyethylene glycol (LiAc-PEG) treatment of fungal blastospores yielded 5-60 transformants/µg DNA/108 cells. Transformants were mitotically stable (at least 5 generations), and > 95% of transformants showed a single integration event. R. lauricola strains expressing green and red fluorescent proteins (EGFP and RFP), as well as glucuronidase (GUS), were constructed. Using the Agrobacterium-mediated method, a random T-DNA insertion library was constructed, and genetic screens led to the isolation of developmental mutants as well as mutants displaying enhanced resistance to sodium dodecyl sulfate (SDS) or fluconazole, and those showing decreased susceptibility to biphenol. These results establish simple and reliable genetic tools for transformation of R. lauricola needed for genetic dissection of the symbiotic and virulent lifestyles exhibited by this fungus and establish a library of insertion mutants that can be used in various genetic screens to dissect molecular pathways. KEY POINTS: • Vectors and transformation protocols were developed for Raffaelea lauricola. • Method was used for construction of a random insertion mutant library. • Mutant library was validated by phenotypic screens for resistance and susceptibility to various agents.

Label-free LC-MS/MS proteomic analysis of the hemolymph of silkworm larvae infected with Beauveria bassiana.

Beauveria bassiana, a pathogen of the economically important silkworm (Bombyx mori), causes serious losses in the sericulture industry; however, the mechanisms underlying B. bassiana infection and the silkworm response are not fully understood. To obtain new insights into the interaction between B. bassiana and its host, hemolymph samples from fifth instar silkworm larvae infected with B. bassiana were analyzed at 36-h post-inoculation using a label-free LC-MS/MS proteomic technique. In total, 671 proteins were identified in the hemolymph, including 87 differentially expressed proteins, 42 up-regulated and 45 down-regulated in infected larvae. Six were detected only in infected larvae, and five were detected only in uninfected larvae. Based on GO annotations, 48 of the differentially expressed proteins were involved in molecular functions, 42 were involved in biological processes, and 39 were involved in cell components. A KEGG pathway analysis indicated that these differentially expressed proteins participate in 85 signal transduction pathways, including the amoebiasis, MAPK signaling, Hippo signaling, Toll and Imd signaling, and lysosome pathways. The silkworm hemolymph is the main site for B. bassiana replication. We identified differentially expressed proteins involved in the regulation of the host response to B. bassiana infection, providing important experimental data for the identification of key factors contributing to the interaction between the pathogenic fungus and its host.

Molecular Cloning, Bioinformatic Analysis, and Expression of Bombyx mori Lebocin 5 Gene Related to Beauveria bassiana Infection.

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori, by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana, the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.

Corrigendum to "Molecular Cloning, Bioinformatic Analysis, and Expression of Bombyx mori Lebocin 5 Gene Related to Beauveria bassiana Infection".

[This corrects the article DOI: 10.1155/2017/9390803.].

Expression profiling of Bombyx mori gloverin2 gene and its synergistic antifungal effect with cecropin A against Beauveria bassiana.

Gloverin2 is a cationic and glycine-rich antimicrobial peptide whose expression can be induced in fat body of silkworm (Bombyx mori) larvae exposed to bacteria. The purpose of this study is to identify the roles of Bombyx mori gloverin2 (Bmgloverin2) during entomopathogenic fungus Beauveria bassiana infection. Fluorescent quantitative real-time PCR analysis indicated that the relative expression level of Bmgloverin2 gene was up-regulated in the silkworm larvae infected by B. bassiana. The cDNA of Bmgloverin2 was cloned from the silkworm by RT-PCR and the DNA segment of the Bmgloverin2 peptide (without signal peptide sequence) was inserted into pCzn1 expression plasmid and expressed in E. coli ArcticExpress (DE3). SDS-PAGE results revealed that soluble recombinant Bmgloverin2 was successfully expressed and purified. Polyclonal antibody against the Bmgloverin2 was successfully produced with the expressed recombinant protein. Western blot analysis indicated that Bmgloverin2 could be detected in the fat body of silkworm larvae infected with B. bassiana, suggesting that the expression of Bmgloverin2 could be induced by B. bassiana infection in silkworm. Antifungal assays indicated that the Bmgloverin2 had a synergistic antifungal activity with B. mori cecropin A (BmCecA) to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that Bmgloverin2 exhibits synergistic effect with BmCecA in antifungal activity against B. bassiana. The study demonstrates that Bmgloverin2 is an antifungal protein which plays an important role in synergistic antifungal activity with other antimicrobial peptide in silkworm.

Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana.

A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm.

Melt blending in situ enhances the interaction between polystyrene and graphene through π-π stacking.

The effect of melt blending on the interaction between graphene and polystyrene (PS) matrix has been investigated in this paper. The interaction between graphene and PS was significantly enhanced by melt blending, which led to an increased amount of PS-functional graphene (PSFG) exhibiting good solubility in some solvents. The PS chains on PSFG could effectively prevent the graphene sheets from aggregating and the prepared PS/PSFG composites exhibited a homogeneous dispersion and an improved electrical property. The mechanism of melt blending on this enhanced interaction was attributed to the formation of π-π stacking during the melt blending. Moreover, the formation of chemical bonding during melt blending may have also enhanced the interaction.

Insecticidal evaluation of Beauveria bassiana engineered to express a scorpion neurotoxin and a cuticle degrading protease.

To improve the insecticidal efficacy of the entomopathogen Beauveria bassiana, the fungus was genetically modified with an insect-specific scorpion neurotoxin AAIT and an insect cuticle degrading protease PR1A from another insect pathogen (Metarhizium anisopliae). The wild-type and the transformants were bioassayed against the larvae of Masson's pine caterpillar Dendrolimus punctatus and the wax moth Galleria mellonella. In comparison to the wild-type strain, engineered isolates took fewer spores to kill 50% of pine caterpillars, 15-fold less for the aaIT single transformant Bb13T and eightfold less for the double transformant Bb13TPR1A, respectively. The median lethal times for Bb13T and Bb13TPR1A were reduced by 40% and 36.7%, respectively against D. punctatus and 24.4% and 20.9%, respectively against G. mellonella. Our data showed that the cotransformation of these two genes produced no synergistic effects on virulence improvement. It is evident from this study that AAIT could be degraded by the protease PR1A when they are expressed together, emphasizing that protein interactions need to be evaluated when working with multiple genes, particularly if they include proteases.