Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest.

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches.

BACKGROUND: The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approaches. RESULTS: A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices, Ophiocordyceps, Sebacinia and Archaeorhizomyces were predominant genera with the abundance of 95.86, 1.14, 0.85%, respectively. A total of 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota and unclassified fungi were predominant genera with an average abundance of 53.32, 8.69, 8.12, 8.12, 7.21, 4.6, 3.08 and 3.05%, respectively. The fungal communities in external mycelial cortices were significantly distinct from soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16 °C, resulted in 77 fungal strains identified by rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora and Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera. CONCLUSIONS: The significantly differences and overlap in fungal community structure between two approaches highlight that the integration of high-throughput sequencing and culture-dependent approaches would generate more information. Our result reveal a comprehensive understanding of fungal community structure of natural O. sinensis, provide new insight into O. sinensis associated fungi, and support that microbiota of natural O. sinensis is an untapped source for novel bioactive metabolites discovery.

The Salvia miltiorrhiza NAC transcription factor SmNAC1 enhances zinc content in transgenic Arabidopsis.

NAC transcription factors play important roles in plant biological processes, including plant development, environmental stress responses and element enrichment. A novel NAC transcription factor gene, designated SmNAC1, was isolated from Salvia miltiorrhiza. SmNAC1 was localized in the nucleus in onion protoplasts and exhibited transcriptional activation activities in yeast. In addition, the SmNAC1 protein could specifically bind to the cis-elements of the NAC proteins. SmNAC1 was expressed at a higher level in the leaves of S. miltiorrhiza, indicating that SmNAC1 might be involved in the transportation of zinc. To examine the function of SmNAC1, transgenic Arabidopsis plants overexpressing SmNAC1 were generated. Zinc content assays in the transgenic plants demonstrated that overexpressed SmNAC1 plants had enhanced tolerance to high zinc concentrations, and zinc was enriched in the shoot tissues. Our results demonstrate that SmNAC1 plays important roles in the response to zinc stress. Zinc was mainly enriched in the leaves of S. miltiorrhiza and the shoot tissues of transgenic Arabidopsis plants. SmNAC1 might participate in zinc transportation from the roots to the shoots, that constitutes a useful gene for improving zinc content in plants.

[Establishment of SeMNPV persistent infection in Spotoptera exigua cells].

Persistent baculovirus infection is observed frequently in insect populations. Persistent infection can be transformed to a replicative and infective state caused by stress factors and plays an important role in regulating the size of insect population and in epizoology of baculoviruses. The aim of this study is to establish a persistently baculovirus-infected cell system to explore the molecular mechanisms of baculoviral persistence. Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was serially undiluted passaged in Se301 cells to reduce virulence. Upon infection of Se301 cells with the SeMNPV up to passage 8, a few cells survived even if most of cells died due to virus infection. The surviving cells were passaged and designated as P8-Se301 cell strain. P8-Se301 cells had a population doubling time of 58-65 hours and grew slower than Se301 cells. Light microscopy and electron microscopy observation showed symptom of baculovirus infection, such as virogenic stroma, viral particles and occlusion bodies, in some of P8-Se301 cells. End-point dilution assay and infectious center assay showed that 4.14% +/- 0.99% cells continually released infectious progeny virus which replicated slower than SeMNPV in Se301 cells. The result indicated that P8-Se301 cells show a typical character trait of baculovirus persistent infection.