Function Studies On Pattern Recognition Receptor PGRP1?IML-1?CTL-S4 In Asian Corn Borer,Ostrinia Furnacalis(Guenée)

Insects lack the acquired immune system and mainly rely on their own effective innate immune system for defense against the invasion of bacteria,fungi and other pathogens.Insect innate immune response is induced by the specific recognition of common components bearing on the microbial surfaces,known as pathogen-associated molecular patterms(PAMPs),by pattern recognition receptors(PRRs).Peptidoglycan recognition proteins(PGRPs)are members of a large superfamily of PRRs that specially recognize peptidoglycan molecules bearing on the surfaces of pathogen.All PGRPs share a highly conserved carboxyl-terminal PGRP domain with approximately 160 amino acids length,which is homologous to N-acetylmuramyl-alanine amidases.C-type lectins(CTLs)known as a group of calcium-dependent carbohydrate binding proteins,are defined by the possession of at least one carbohydrate recognition domain(CRD)also called as CTLD(C-type lectin domain).Recognition of invational signal is the first step in innate immune response.The studies on the functions and functioning mechanism of PGRPs and C-type lectins in signal recognition pathway is of great significance for understanding the molecular mechanism of innate immune response in insects.Asian corn borer,Ostrinia furnacalis(Guenee),is an important agricultural pest in China,and cause serious damages on various crops.In this study,we firstly clarified that the infection of Beauveria bassiana indeed could induce the cellular immune responses in Asian corn borer.Then we sequenced and analyzed the transcriptomes from Asian corn borer larvae induced by B.bassiana,and identified 190 immune-related transcripts.Finally,we selected one peptidoglycan recognition protein(PGRP1)and two C-type lectins(IML-1 and CTL-S4)to study their functions and possible functioning mechanism in the innate immune response of Asian com borer.The overall results are as follows:1.Five types of circulating hemocytes in the hemolymph of Asian corn borer larvae,including granulocytes,oenocytoids,plasmatocytes,prophemocytes,and spherulocytes were identified.The relative number of total free hemocytes per larva decreased significantly 0.5 h,24 h,and 36 h after the injection of B.bassiana conidia.Upon conidia challenge 0.5 h post injection,both phagocytosis and nodulation to conidia by hemocytes were obviously observed in the collected hemolymph from Asian corn borer larvae.2.The transcriptomes of Asian corn borer larvae induced by B.bassiana were sequenced and analyzed,and 190 immune-related transcripts were identified.Among those,45 transcripts encode potential signal recognition related proteins,including 10 putative PGRP transcripts and 14 possible CTL transcripts.3.A detailed bioinformatics analysis was performed on the sequences of 14 CTL transcripts,and their key residues,feature motifs,and three-dimensional structural models were predicted.Combined with the above analysis about putative PGRP transcipts,one peptidoglycan recognition protein(PGRP1)and two C-type lectins(IML-1 and CTL-S4)were selected for the further functional studies.4.The full-length cDNA sequences of PGRP1,IML-1,and CTL-S4 were cloned and their spatial and temporal expression patterns at different developmental stages and in different tissues were examined.Their expression patterns in the larvae upon the challenges with Escherichia coli,Micrococcus luteus or B.bassiana were also checked.In addition,recombinant proteins of PGRP1,IML-1,and CTL-S4 were produced using a prokaryotic expression system,and a large amount of recombinant proteins with high purity were obtained.5.Recombinant PGRP1,IML-1,and CTL-S4 could bind to E.coli,M.luteus,B.bassiana,and Pichia pastoris.Further studies revealed that IML-1 could bind to lipopolysaccharide(LPS),peptidoglycans(PGNs)and curdlan,while CTL-S4 could only bind to peptidoglycan.Recombinant PGRP1 could agglutinate E.coli,M.luteus and B.bassiana;while recombinant IML-1 and CTL-S4 could only agglutinate E.coli and M.luteus in the presence of Ca2+.6.The addition of recombinant PGRP1,IML-1 or CTL-S4 to plasma significantly increased plasma phenoloxidase(PO)activity and IEARase activity in Asian corn borer plasma.7.The Q-Sepharose Fast Flow(QFF)beads coated with recombinant IML-1 and CTL-S4 protein could enhance the encapsulation and melanization of beads by hemocytes.In summary,O.furnacalis PGRP1,IML-1 and CTL-S4 serve as PRPs to recognize and bind to microorganisms and initiate the melanization of hemolymph.Furthermore,IML-1 and CTL-S4 were also involved in the cellular immune response and promote the encapsulation and melanization of hemocytes.