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Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenee to defend against bacterial infection.

ABSTRACT: The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated Mr of 44.8?kDa and pI of 6.66. Multiple sequence alignment and phylogenetic analysis indicated that OfPAP was orthologous to the PAPs in the other lepidopterans. A large increase of the transcript levels was observed in hemocytes at 4?h post injection (hpi) of killed Bacillus subtilis, whereas its level in integument increased continuously from 4 to 12 hpi in the challenged larvae and began to decline at 24 hpi. After OfPAP expression had been silenced, the median lethal time (LT50) of Escherichia coli-infected larvae (1.0 day) became significantly lower than that of E. coli-infected wild-type (3.0 days, p?

SUBMITTER: Feng C

PROVIDER: S-EPMC6093219 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection.

Feng Congjing C Zhao Ya Y Chen Kangkang K Zhai Huifeng H Wang Zhenying Z Jiang Haobo H Wang Yingjuan Y Wang Libao L Zhang Yiqiang Y Tang Tai T

Developmental and comparative immunology 20180702

The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated ...[more]

PMID: 30017863

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Project description:Ostrinia furnacalis, an important pest of corn, has substantial detrimental effects on corn production. The mitogen-activated protein kinase (MAPK) signaling pathway plays a pivotal role in an insect's resistance to environmental stress. The expression levels of JNK and p38 have been well recorded in several insects under different environmental stressors, at different developmental stages, and in various tissue types; however, there is limited information on JNK and p38 in agricultural insects. To clarify the mechanism whereby O. furnacalis responds to environmental stress, we cloned JNK and p38 from O. furnacalis and subsequently named them OfJNK and Ofp38, respectively. Further, we examined the expression levels of OfJNK and Ofp38 under different environmental stressors. In this study, we obtained full-length sequences of OfJNK and Ofp38, and RT-qPCR results showed that these genes were expressed at all developmental stages, in various tissues (head, chest, abdomen, leg, wing, antennae, compound eye, midgut, and ovary) and under different environmental stressors (4°C and ultraviolet A treatment for 0, 30, 60, 90, and 120 min). The expression levels of OfJNK and Ofp38 were relatively higher in eggs and 3-day-old adult females than in other developmental stages. Moreover, the expression level of OfJNK was higher in the wings than in other tissues, whereas that of Ofp38 was significantly higher in the ovaries than in other tissues. OfJNK and Ofp38 showed high expression 90 min after being subjected to treatment at 4°C and ultraviolet A irradiation; the expression of Ofp38 peaked at 30 min, whereas that of OfJNK peaked at 60 min. These results indicate that O. furnacalis differs in terms of its response under different environmental stressors. In summary, our results will provide a foundation for additional research needed to determine the role of the MAPK signaling pathway and the underlying mechanisms by which it shows resistance to environmental stresses in insects.

Project description:BackgroundOrthopteran migratory locust, Locusta migratoria, and lepidopteran Asian corn borer, Ostrinia furnacalis, are two types of insects undergoing incomplete and complete metamorphosis, respectively. Identification of candidate genes regulating wing development in these two insects would provide insights into the further study about the molecular mechanisms controlling metamorphosis development. We have sequenced the transcriptome of O. furnacalis larvae previously. Here we sequenced and characterized the transcriptome of L. migratoria wing discs with special emphasis on wing development-related signaling pathways.Methodology/principal findingsIllumina Hiseq2000 was used to sequence 8.38 Gb of the transcriptome from dissected nymphal wing discs. De novo assembly generated 91,907 unigenes with mean length of 610 nt. All unigenes were searched against five databases including Nt, Nr, Swiss-Prot, COG, and KEGG for annotations using blastn or blastx algorithm with an cut-off E-value of 10-5. A total of 23,359 (25.4%) unigenes have homologs within at least one database. Based on sequence similarity to homologs known to regulate Drosophila melanogaster wing development, we identified 50 and 46 potential wing development-related unigenes from L. migratoria and O. furnacalis transcriptome, respectively. The identified unigenes encode putative orthologs for nearly all components of the Hedgehog (Hh), Decapentaplegic (Dpp), Notch (N), and Wingless (Wg) signaling pathways, which are essential for growth and pattern formation during wing development. We investigated the expression profiles of the component genes involved in these signaling pathways in forewings and hind wings of L. migratoria and O. furnacalis. The results revealed the tested genes had different expression patterns in two insects.Conclusions/significanceThis study provides the comprehensive sequence resource of the wing development-related signaling pathways of L. migratoria. The obtained data gives an insight into better understanding the molecular mechanisms involved in the wing development in L. migratoria and O. furnacalis, two insect species with different metamorphosis types.

Project description:Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high Mr cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high Mr complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated in vitro. To complement the in vitro analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.

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Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenee to defend against bacterial infection.

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Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection.

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