ß-1,3-Glucan recognition protein 3 activates the prophenoloxidase system in response to bacterial infection in Ostrinia furnacalis Guenée.

Pattern recognition receptors (PRRs) are biosensor proteins that bind to non-self pathogen associated molecular patterns (PAMPs). ß-1,3-glucan recognition proteins (ßGRPs) play an essential role in immune recognition and signaling pathway of insect innate immunity. Here, we report the cloning and characterization of cDNA of OfßGRP3 from Ostrinia furnacalis larvae. The OfßGRP3 contains 1455 bp open reading frame, encoding a predicted 484 amino acid residue protein. In hemocytes, the expression levels of OfßGRP3 in Escherichia coli-challenged group were higher than those of Bacillus subtilis-challenged group at 2, 4, 8, 10 and 12 h post injection (HPI). In fat body, OfßGRP3 expression in both B. subtilis and E. coli-challenged group was significantly higher than that in untreated group from 4 to 10 HPI, and then the expression continuously dropped from 12 to 36 HPI. The OfßGRP3 expression in laminarin-injected group was higher than that in lipopolysaccharides (LPS)-injected group in various test tissues from 4 to 24 HPI. The LT50 of E. coli-infected OfßGRP3-RNAi larvae (1.0 days) was significantly lower compared with that of E. coli infected wild-type larvae (3.0 days) (p < 0.01). Only 10.2% Sephadex G50 beads (degree 3) were completely melanized in the larvae inoculated with OfßGRP3 dsRNA, as compared to 48.8% in control larvae (p < 0.01). A notable reduction in the PO activity and IEARase activity in hemolymph was also detected in the OfßGRP3 knockdown larvae. Our study demonstrates that OfßGRP3 is one of PRR members involved the PPO-activating system in O. furnacalis larvae.

Cloning, expression and characterization of Ostrinia furnacalis serpin1, a regulator of the prophenoloxidase activation system.

Serine protease inhibitors of the serpin superfamily are regulators of proteases involved in a variety of physiological processes including immune responses. In this study, we have isolated a full-length serpin cDNA from Ostrinia furnacalis. The 1188 bp open reading frame encodes a 395-residue protein with a theoretical molecular mass of 43.3 kDa and an isoelectric point of 4.92. Ofserpin1 contains a putative signal peptide followed by a conserved domain including a reactive center loop (RCL) with a hinge region (E(344) to S(353)) and a predicted P1-P1' cleavage site (Leu(360)-Ser(361)). Ofserpin1 mRNA and protein were detected in all the tested tissues, particularly in hemocytes and integument. The recombinant protein inhibited chymotrypsin and trypsin in a dose-dependent manner, and were significantly cleaved by the enzyme trypsin and chymotrypsin. Ofserpin1 impeded the prophenoloxidase activation cascade by 45.6% at 16.5 µg, and affected activity of prophenoloxidase activating protease. Levels of Ofserpin1 transcripts in the integument were higher than those in hemocytes, fat body and midgut. After an immune challenge with Staphylococcus aureus and Escherichia coli, the relative mRNA levels of Ofserpin1 decreased in 2-10h post-infection (hpi) in integument and hemocytes compared to the untreated control. Our results suggested that Ofserpin1 has serine protease inhibitory activity and is likely involved in the regulation of prophenoloxidase activation system in O. furnacalis.