Identification And Functional Characteration Of Hyphantria Cunea Aminopeptidase N In Cry1Ab35 Intoxication

The fall webworm,Hyphantria cunea（Drury）（Lepidoptera:Arctiidae）is an important quarantine pest worldwide anda major invasive pest in China.It has caused serious damage to forests,agricultural crops,and ecological constructionin China due to its wide host range,strong reproductive capacity,and rapid propagation speed.According to the No.3 and No.4 Announcements of the State Forestry Administration in 2018,the total occurrence range of H.cuneain China have involved 572 county-level administrative regions in 11 provinces.Bacillus thuringiensis（Bt）is one of the most comprehensive studied insecticidal microorganisms and offers a number of advantages over synthetic pesticides,such as environmental benefits,high specificity to target insects and convenience to use.In spite that Bt based bioinsecticides and transgenic plants has been successfully used globally to control some key pests,Bt strains or Cry toxins exhibiting high toxicity towards H.cunea are rarely reported.In our study,five Bt strains with high toxicity against H.cunea larvae were obtained.The 50%lethal concentrations(LC₅₀s)of these strains against H.cunea day 3 first instar larvae ranged from 0.084to 0.268 mg/mL spores and crystals mixture,and the strain GS36exhibited the highest toxicity with a LC₅₀0 of 0.084 mg/mL.These strains contained a variety of cry genes and all harboured cry1Aandcry2A typegenes.They produced bipyramidal shapes in different sizes or spherical crystals.The crystals showed two major protein bands of approximately 130 kDa and 60 kDa in SDS-PAGE analysis.A novel cry1Ab gene（Accession number KT01852882）was identified from GS36strainand designated cry1Ab35 by the Bt Delta-endotoxin Nomenclature Committee.Thecry1Ab35genecontained a 3495 bp open reading frame which encoded 1181 amino acids.The predicted molecular weight of Cry1Ab35 protein was 133.6kDa and its isoelectric point（pI）was 4.68.It was expressed as a 140 kDa recombinant protein in B.thuringiensis engineered HD1Ab35 strainand yielded a 60 kDa fragment upon 1/20（w/w）trypsin activation.The Cry1Ab35 toxin exhibited high toxicity against H.cuneaday 3 first instar larvae and the LC₅₀ was 4.34μg/mL.It also showed moderate toxicity againstHelicoverpa armigera andSpodoptera exiguaneonates and the LC₅₀s were24.31μg/mL and 41.87μg/mL respectively.Anenhancin-like gene（Accession number MG012232）was obtained from GS36strain.It contained a 3495 bp open reading frame and encoded 1182 amino acids.The deduced Cry1Ab35 protein was 133.6kDa and its isoelectric point was 4.68.It wasexpressedas a 84 kDa protein in E.coli BL21（DE3）cells.The Enhancin protein extracted from TnGV was 90 kDa in mass.Bioassay results showed that when 20μg/mL Enhancin or 100μg/mL Enhancin-like protein was added,the Cry1Ab35 toxicity was enhanced by 4.93-fold and 4.21-fold respectivily.Moreover,when1.0×10⁵ PIBs/mL HcNPVwas added,an 11.16-fold enhancement of Cry1Ab35 toxicity was detected.In controls,insecticidal activity was not observed when larvae were fed with three synergistic factors alone.These results represented significant synergistic effects on Cry1Ab35 toxin by the three synergistic factors.Furthermore,five novel aminopeptidase N isoforms have been identified and cloned from the midgut of H.cunea by ligand blot and mass spectrometryanalyses.According to the results of phylogenetic analysis the five genes were named hcapn2（KY949254）,hcapn3（KY949256）,hcapn5（KY949257）,hcapn6（KY949258）and hcapn8（KY949259）,and contained a open reading frameof 2811,3072,2817,2877 and 2796 bprespectively.The deduced HcAPN2,HcAPN3,HcAPN5,HcAPN6,HcAPN8 proteinconsisted of936,999,938,958 and 930amino acidsand their predicted molecular weight were105,114,106,110 and 106 kDarespectively.Along with the HcAPN1（KP053647）and HcAPN4（KJ013598）protein had been published in GenBank,seven putative HcAPN proteins presented APNs typical motifs in homologous positions:a signal peptide in the N-terminus,the GAMEN gluzincin aminopeptidase motif,the zinc-binding motif HEXXH（X）₁₈E,a GPI anchor signal sequence at the C-terminal except for HcAPN1 and HcAPN6protein,and containing multiple N-and O-glycosylation sites.Seven hcapn genes were highly expressed throughout all H.cunea larval developmental stages and were highly abundant in the midgut and hindgut tissues.Theywere up-regulated significantly at the 12 h to 24 h time point after Cry1Ab35 toxin treatment.HcAPN1,HcAPN2,HcAPN3,HcAPN4,HcAPN5,HcAPN6 and HcAPN8wereexpressed as a 130,110,120,115,120,130and120kDarecombinantprotein in infected Sf9 cells respectively and were detected their interaction with Cry1Ab35 toxin by ligand blot assays.Furthermore,RNAi against hcapngeneswas employed to illustrate the functional role for HcAPNs in Cry1Ab35 toxicity againstH.cunealarvae using injection of double-strand RNA（dsRNA）.After injection of dsRNA for 3 days,the transcript levels of hcapn1,hcapn2,hcapn3,hcapn4,hcapn5,hcapn6 andhcapn8gene in larvae were reduced by 91.9%,72.0%,87.2%,64.0%,68.3%,64.0%and 67.1%respectively.Then ddH₂O and dsRNA-egfp/-hcapns injected larvae were reared on Cry1Ab35-overlaid diets for 5 days,the70.8%,74.5%,51.4%,41.7%,49.6%,61.1%,50.3%,62.5%and63.9%larvae correctedmortalities were observed.ANOVA analysis revealed that the Cry1Ab35-induced correctedmortalities indsRNA-hcapn1/-hcapn2/-hcapn3/-hcapn5injected larvae were significantlylowerthanthoseinjectedwithddH₂Oand dsRNA-egfp/-hcapn4/-hcapn6/-hcapn8.Theseworksuggestthat HcAPN1,HcAPN2,HcAPN3 andHcAPN5protein in H.cunealarvae are putative functionalreceptors involved in Cry1Ab35 susceptibility.