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Autophagy induced by Vip3Aa has a pro-survival role in Spodoptera frugiperda Sf9 cells.

ABSTRACT: Vip3Aa is an insecticidal protein that can effectively control certain lepidopteran pests and has been used widely in biological control. However, the mechanism of action of Vip3Aa is unclear. In the present study, we showed that Vip3Aa could cause autophagy in Sf9 cells, which was confirmed by the increased numbers of GFP-Atg8 puncta, the appearance of autophagic vacuoles, and an elevated Atg8-II protein level. Moreover, we found that the AMPK-mTOR-ULK1 pathway is involved in Vip3Aa-induced autophagy, which might be associated with the destruction of ATP homeostasis in Vip3Aa-treated cells. Both the elevated p62 level and the increased numbers of GFP-RFP-Atg8 yellow fluorescent spots demonstrated that autophagy in Sf9 cells was inhibited at 24 h after Vip3Aa treatment. With the prolongation of Vip3Aa treatment time, this inhibition became more serious and led to autophagosome accumulation. Genetic knockdown of ATG5 or the use of the autophagy inhibitor 3-MA further increased the sensitivity of Sf9 cells to Vip3Aa. Overexpression of ATG5 reduced the cell mortality of Vip3Aa-treated cells. In summary, the results revealed that autophagy induced by Vip3Aa has a pro-survival role, which might be related to the development of insect resistance.

SUBMITTER: Hou X

PROVIDER: S-EPMC7849784 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Mitochondria and Lysosomes Participate in Vip3Aa-Induced <i>Spodoptera frugiperda</i> Sf9 Cell Apoptosis.

Project description:Vip3Aa, a soluble protein produced by certain Bacillus thuringiensis strains, is capable of inducing apoptosis in Sf9 cells. However, the apoptosis mechanism triggered by Vip3Aa is unclear. In this study, we found that Vip3Aa induces mitochondrial dysfunction, as evidenced by signs of collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, release of cytochrome c, and caspase-9 and -3 activation. Meanwhile, our results indicated that Vip3Aa reduces the ability of lysosomes in Sf9 cells to retain acridine orange. Moreover, pretreatment with Z-Phe-Tyr-CHO (a cathepsin L inhibitor) or pepstatin (a cathepsin D inhibitor) increased Sf9 cell viability, reduced cytochrome c release, and decreased caspase-9 and -3 activity. In conclusion, our findings suggested that Vip3Aa promotes Sf9 cell apoptosis by mitochondrial dysfunction, and lysosomes also play a vital role in the action of Vip3Aa.

| S-EPMC7076775 | biostudies-literature

Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells.

Project description:BackgroundSmall nucleolar RNAs (snoRNAs) function in guiding 2'-O-methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells.ResultsWe showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. frugiperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further analysis showed that a Ca2+-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was significantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15.ConclusionsWe conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expression of Ca2+-induced cell death pathway gene Cn in Sf9 cells. This is the first time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death.

| S-EPMC6458620 | biostudies-literature

Spodoptera frugiperda Sf9 cells as a model system to investigate the role of detoxification gene expression in response to xenobiotics.

Project description:Spodoptera frugiperda (fall armyworm) is a highly destructive invasive pest that feeds on numerous crops including maize and rice. It has developed sophisticated mechanisms to detoxify xenobiotics such as secondary plant metabolites as well as manmade insecticides. The aim of the study was to explore the detoxification response to plant secondary metabolites and insecticides employing a S. frugiperda Sf9 cell model exposed to indole 3-carbinol (I3C) and methoprene. The cell Inhibitory Concentration 50 (IC50) for these molecules was determined and IC10, IC20 and IC30 doses were used to monitor the induction profiles of detoxification genes. Cytochrome P450 monooxygenases (P450s) of the CYP9A subfamily were the most inducible genes of the seven examined. Our results also showed the induction of the transcription factor Cap'n'collar isoform C (CncC). Transient transformation of Sf9 cells overexpressing CncC and its partner muscle aponeurosis fibromatosis (Maf) induces overexpression of CYP4M14, CYP4M15, CYP321A9 and GSTE1 while CYP9As were not induced. Next, we determined the capacity of recombinantly expressed CYP9A30, CYP9A31 and CYP9A32 to interact with methoprene and I3C. Fluorescence-based biochemical assays revealed an interaction of methoprene with functionally expressed CYP9A30, CYP9A31 and CYP9A32 whereas almost no interaction was detected for I3C, suggesting the ability of CYP9As to metabolize methoprene. Our results showed that Sf9 cells could be a useful model to decipher detoxification pathways of S. frugiperda.

| S-EPMC9387494 | biostudies-literature

Spodoptera frugiperda isolate:Sf9

Project description:Spodoptera frugiperda isolate:Sf9 Genome sequencing and assembly

| PRJNA380964 | ENA

RNA-seq of Spodoptera frugiperda Sf9 cell line

Project description:This dataset is to provide a transcriptional status reference for the Sf9 cell line, used for ChIP assays

2022-06-29 | E-MTAB-10686 | biostudies-arrayexpress

Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin.

Project description:As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.

| S-EPMC5643380 | biostudies-literature

MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection

Project description:MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host–virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 59 end of miRNA. The 59 ends of the miRNAs were more conserved than the 39 ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect’s miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host–virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.

2015-06-11 | GSE60064 | GEO

MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection

2015-06-11 | E-GEOD-60064 | biostudies-arrayexpress

Synthesis, characterization of two matrine derivatives and their cytotoxic effect on Sf9 cell of Spodoptera frugiperda.

Project description:The invasion of Spodoptera frugiperda has imposed a serious impact on global food security. Matrine is a botanical pesticide with a broad spectrum of insecticidal activity which was recommended for controlling Spodoptera frugiperda. In order to discover effective insecticide for Spodoptera frugiperda, two matrine derivatives modified with carbon disulfide and nitrogen-containing groups were systhesized. And their inhibition activities on Sf9 cell were evaluated. The structural configuration of compounds were characterized by IR, HPLC, MS, NMR and XRD, with yields of 52% and 65%, respectively. The IC50 of the two newly synthesized compounds on Sf9 cell reduced to 0.648 mmol/L and 1.13 mmol/L, respectively, compared with that of matrine (5.330 mmol/L). In addition, microscopic observation of Sf9 cell treated with the compounds showed that the number of adherent cells decreased, the cells shrunk, vacuolated and apoptotic bodies appeared. The two newly synthesized compounds exhibited better inhibitory effect on Sf9 cell than that of the parent matrine, suggesting that the positive effect of the introduction of 1-pyrrolidinecarbodithioate and diethylcarbamodithioate groups to matrine. The morphological observation of Sf9 cell induced by derivatives indicated that apoptosis induction may be a mechanism that inhibits insect cell proliferation and exerts insecticidal effect.

| S-EPMC7581774 | biostudies-literature

Spodoptera frugiperda kinetochore co-immunoprecipitations

Project description:Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-independent kinetochores of Lepidoptera. We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

2020-02-18 | PXD016092 | Pride

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