| Monochamus alternatus Hope is the main vector of the pinewood nematode,which causes huge economic losses in many countries.Therefore,controlling M.alternatus populations is one of the most important measures to prevent the spread of the pinewood nematode.Bacillus thuringiensis（Bt）is a widely used microbial pesticide,which can express many different kinds of crystal proteins that have biological activity to different pests.Cry3Aa toxin exhibit specific toxicity against a number of coleopteran larvae.However,due to its low toxicity to M.alternatus,Cry3A toxin is not useful for controlling M.alternatus larvae.During Cry3Aa toxin mechanism of action,the proteolytic activation and receptor binding of midgut protease are the important steps that can affect the toxicity of Cry3Aa toxin.Therefore,in order to determine the reasons related to the low toxicity of Cry3Aa toxin to M.alternatus larvae,in this study,the proteolytic activation and receptor binding of Cry3Aa toxin in the midgut of M.alternatus larvae were studied.To overcome the proteolytic activation barriers and over-digestion of Cry3Aa toxin,the main midgut protease of M.alternatus larvae was quantified,as well as its specific cleavage sites.This information provided a theoretical basis for the modification of Cry3Aa sites.Finally,based on the specific cleavage sites of the major protease,molecular modification of Cry3Aa toxins were carried out to promote an efficient proteolytic activation of Cry3Aa toxin in the midgut of M.alternatus larvae,to improve its insecticidal activity.The results are as follows:The proteolytic activation of Cry3Aa toxin by midgut protease and the physiological p H of M.alternatus larvae gut was determined.The result showed that the p H value was between 5-6,indicating that the gut of the M.alternatus larvae has a weak acidic environment.The in vitro proteolytic activation of Cry3Aa toxin by midgut protease of M.alternatus larvae were determined by western blot.Treatment with M.alternatus lumen fractions resulted in complete digestion of GST-Cry3Aa protoxin,and no 55 k Da active Cry3Aa band was observed at mass ratios（proteases:Cry）of 5:1,2:1,or 1:1.When the lumen fractions decreased（at mass ratios of 1:2,1:5,1:10,and 1:20）,GST-Cry3Aa protoxin was partly activated and a 55 k Da band was observed.By contrast,most GST-Cry3Aa protoxin was not activated into the 55 k Da Cry3Aa toxin when it was incubated with membrane proteases.Incubation with the midgut proteases and GST-Cry3Aa protoxin at a mass ratio of 1:10,only a small amount of 55 k Da activated fragment was formed at 19°C,24.8°C,29.1°C,33.2°C,and 37°C.Furthermore,Cry3Aa protoxin was not activated completely even with a longer incubation time.As the p H increased,the yield of the 55 k Da activated fragment increased gradually,but activation was not complete.Protease from different larvae instars,the 73 k Da Cry3Aa protoxin was completely digested by proteases of the third-instar larvae and partially activated into the 55 k Da Cry3Aa toxin by proteases from the first-,second-,and fourth-instar larvae.The results indicated that insufficient processing and over-digestion of Cry3Aa protoxin in the midgut of M.alternatus larvae led to low toxicity.The binding recognition of Cry3Aa receptors in the brush border membrane vesicles（BBMV）of M.alternatus larval midgut were demonstrated.The results of binding assay and ELISA showed that Cry3Aa toxin was able to bind to BBMV of M.alternatus（Kd=247 n M）.Ligand blot assays showed that Cry3Aa protein exhibited a specific binding pattern（～100,～60,～50,～36,～35,and～30-k Da）.Moreover,the transcriptome of the midgut from M.alternatus larvae exhibited a total of 73 B.thuringiensis receptor unigenes,including 9 ALPs,13 APNs,41 cadherins,and 10 ABC transporters.To determine the proteins that potentially can bind to Cry3Aa,a pull-down assay and mass spectrometry were performed and lead to the identification of a 107 k Da aminopeptidase N（APN）.The GST-APN was purified,and the western blot and ELISA results showed that Cry3Aa can bind to APN with high affinity（Kd=57 n M）.The results indicated that Cry3Aa toxin can bind with the receptor on the midgut BBMV.These results further demonstrated that the insufficient processing and over-digestion are the main causes of the low toxicity of Cry3Aa to M.alternatus larvae.To overcome the proteolytic activation barriers and over-digestion of the Cry3Aa toxin,the main midgut protease of M.alternatus larvae and its specific cleavage sites were determined.First,the transcriptome data showed that a total of 159 protease-related unigenes were identified in the M.alternatus transcriptome.Serine proteases were most represented（153,96.22%）,followed by metalloproteases（2,1.26%）,cysteine proteases（2,1.26%）,and aspartic proteases（2,1.26%）.Among the serine proteases,trypsin proteases（32,20.92%）and chymotrypsin proteases（39,25.49%）were the main midgut proteases.The results of fluorescence substrates enzymatic activity showed that AEBSF inhibited protease activity significantly when compared to the control,followed by E-64 and EDTA.Pepstatin-A did not inhibit protease activity.These results indicated that serine proteases were the main proteases in M.alternatus larval midgut.To provide information and theoretical basis for the design of molecular modification Cry3Aa toxin sites,the multiplex substrate profiling by mass spectrometry（MSP-MS）was used to obtain the specific cleavage sites map of midgut proteases.The results showed that the main protease in midgut of M.alternatus larvae was trypsin and its cleavage sites were arginine（R）and lysine（K）at P1 position.The majority of cleavages were in the interior of the sequence,which indicated a strong endopeptidase activity.Therefore,the targeted modification of R and K on the surface of Cry3Aa toxin can effectively overcome the problems of insufficient processing and over-digestion.Finally,in this study,molecular modification was used to improve the toxicity of Cry3Aa toxin.The four peptides of arginine and lysine（FMR,FMRP,FCKY and FKMW）were inserted into the loop region betweenα-helix 3 andα-helix 4（amino acid 155-157）of the domain I of Cry3Aa toxin to promote the effective enzymatic digestion of the toxin.The results showed that the digestion processes of the Cry3Aa-FMRP and Cry3Aa-FCKY protoxins were similar to that of native Cry3Aa protoxin,and 70k Da protoxins were still observed.In contrast,the Cry3Aa-FKMW toxin showed over-digestion by the midgut protease.The Cry3Aa-FMR protoxin was over-digested by the lumen fractions and insufficiently hydrolyzed by the membrane fractions.Moreover,the LC50 of Cry3Aa,Cry3Aa-FMRP,and Cry3Aa-FKMW protoxins were 116.8,105.6,and 167.9μg/m L,respectively.The toxicity of Cry3Aa-FMR and Cry3Aa-FCKY protoxins was so low that it was not possible to calculate the LC50.These results suggested that adding trypsin cleavage sites to increase the proteolytic activation of Cry3Aa protoxin did not significantly improve Cry3Aa toxicity to M.alternatus larvae,and the over-digestion of the toxin greatly affected the insecticidal activity of Cry3Aa.To overcome the over-digestion of Cry3Aa toxin,the trypsin and chymotrypsin cleavage sites（lysine（K）,tyrosine（Y）,and phenylalanine（F））located on the non-functional region of Cry3Aa protein were mutated into alanine（A）to avoid toxin over-digestion by the midgut protease.We obtained three modified Cry3Aa toxins,including Cry3Aa-T（K was mutated into A）,Cry3Aa-C（Y and F were mutated into A）,and Cry3Aa-T-C（K,Y,and F were mutated into A）.The results showed that the yield of the 55 k Da active fragments were increased of the Cry3Aa-T,Cry3Aa-C,and Cry3Aa-T-C protoxins compared to native Cry3Aa protoxin,and Cry3Aa-T produced the most 55 k Da activated fragment.Besides,compared to native Cry3Aa protoxin(LC50=116.8μg/m L),the toxicity of Cry3Aa-T protoxin(LC50=12.3μg/m L)and the toxicity of Cry3Aa-C(LC50=59.6μg/m L)and Cry3Aa-T-C(LC50=91.5μg/m L)all increased.Among them,the Cry3Aa-T toxin showed a 9.5-fold increase in toxicity to M.alternatus larvae.This strategy may be suitable for managing the resistance of transgenic plants to other pests,including some of the most important pests in agriculture.This study provides an effective strategy for increasing the toxicity of Cry3Aa toxin against M.alternatus. |
