Resistance Mechanisms Of Bacillus Thuringiensis Cry1Ac Toxin By Trypsin And Chymotrypsin Family Genes In Plutella Xylostella(L.)

Bacillus thuringiensis(Bt),can form Cry-like insecticidal crystal toxins during its sporulation,which are harmless to biological organisms.It is widely used in the control of agricultural pests through the use of Bt leaf sprays and genetically modified crops.However,the rapid resistance evolution in the field caused by the large-scale commercial application of Bt toxins has hindered the sustainable use of this technology.In addition,the current limited knowledge of insect Bt Cry toxin resistance mechanisms severely hinders the management of Bt pest resistance and the development and application of new Bt insecticidal crystal proteins.Therefore,in order to ensure the safety and economic benefits of Bt biopesticides,it is necessary to clarify the molecular mechanism of insect resistance to Bt Cry toxins.Diamondback moth(Plutella xylostella)(L.)is a globally distributed important pest that mainly harms cruciferous crops.It can quickly develop resistance to various pesticides and can cause about $4 billion worldwide serious economic losses annually.Therefore,Plutella xylostella(P.xylostella),as the first pest to be reported developing resistant to Bt spraying agents in the field,has become a model insect to investigate the complex molecular mechanisms and theoretical basis of Bt resistance.The mechanism of Bt resistance in different insect populations of different insects is very complex,a number of pests have been reported to be related to or unrelated to Bt resistance in recent years.And in the established classic model and the newly proposed dual model of Bt Cry toxin,the activation of protoxin by midgut protease is an essential key step,and the incorrect activation process of protoxin may cause insects to produce Bt resistance.So,what is the relationship between these factors and the resistance strains of P.xylostella in our laboratory? Based on previous research,this paper selects Bt Cry1 Ac susceptible(DBM1Ac-S)and different resistance levels of P.xylostella strains as experimental materials.And various genomics,molecular biology,biochemistry,genetics and bioinformatics experimental techniques and platforms were used to carry out experiments and verification analysis to reveal the relationship between midgut trypsin or chymotrypsin and Bt Cry1 Ac resistance in diamondback moth.Our results demonstrated that high-level field-evolved Cry1 Ac resistance in P.xylostella is independent of altered Cry1 Ac protoxin activation mediated by midgut proteases and the conclusion supports classic toxin modes of action.In the SZ-R resistant strain of P.xylostella selected in the laboratory,the down-regulation of the new midgut trypsin gene PxTryp?SPc1 involved in toxin activation was associated with Bt Cry1 Ac resistance.The main contents and consequences are as follows:1? Continuous rearing of susceptible P.xylostella in the laboratory and resistance selectionA large-scale artificial isolation and breeding of P.xylostella was carried out in the laboratory's specialized insect breeding room,took advantage of a good insect breeding environment and the cabbage radish seedling method explored in the early stage of our laboratory.At the same time,the Bt resistance selection method of P.xylostella larvae which has been standardized by our laboratory has been used for screening to maintain and ensure the resistance and stability of the resistant strain,providing sufficient and good experimental materials for the subsequent functional experiments.2?The relationship between trypsin or chymotrypsin-mediated toxin activation and Bt Cry1 Ac resistance in P.xylostellaHerein,we evaluated the Cry1 Ac protoxin activation in susceptible and its near-isogenic high-level field-evolved Cry1Ac-resistant P.xylostella.Bioassay results indicated that resistant larvae developed high resistance to both Cry1 Ac protoxin and trypsin-activated toxin,but Cry1 Ac protoxin is not more potent than activated toxin.Subsequently,enzyme activity detection showed that caseinolytic,trypsin and chymotrypsin protease activities did not significantly alter between susceptible and resistant strains.Furthermore,there was no notable effect on the susceptibility of susceptible and resistant larvae to Cry1 Ac protoxin when treated with either trypsin inhibitor N?-Tosyl-L-lysine chloromethyl ketone(TLCK)or chymotrypsin inhibitor N-p-Tosyl-L-phenylalanine chloromethyl ketone(TPCK).The results showed that the larvae of susceptible and resistant populations had no significant effect on the sensitivity of Cry1 Ac toxin after the corresponding protease inhibitors were added.Furthermore,the protoxin activation and incubation time or the midgut fluid concentration has little effect on activation process between the susceptible and resistant strain.These experimental results indicate that the resistance of P.xylostella NIL-R resistant population to Bt Cry1 Ac was independent of midgut protease-mediated alteration of protoxin activation.3?The annotation of P.xylostella trypsin and chymotrypsin family genesThe bioinformatics methods and technologies were used to analyze the genome data of P.xylostella.After identification and cloning,21 trypsin genes and 8 chymotrypsin genes were successfully obtained,which are the basic genes of midgut protease in Lepidoptera diamondback moth.Then,using the transcriptome data to analyze the expression levels of these midgut protease genes,the differential genes were filtered at the overall level.At the same time,phylogenetic analysis about trypsin or chymotrypsin proteins in other insects of different orders,we found that these genes are evolutionarily conserved among different species.4?Study on the relationship between the midgut trypsin gene PxTryp?SPc1 and Bt Cry1 Ac resistance in diamondback mothIn addition,we identified down-regulation of a trypsin-like serine protease gene PxTryp?SPc1 previously in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant P.xylostella strain SZ-R.(1)The results of protease activity measurement and toxin activation experiment showed that decreased PxTryp?SPc1gene expression significantly reduced caseinolytic and trypsin protease activities affecting Bt Cry1 Ac protoxin activation.(2)The bioassay results indicated that in the SZ-R strain,higher resistance of P.xylostella larvae to Cry1 Ac protoxin than activated toxin and Cry1 Ac protoxin is not as effective as activated toxin.(3)In order to continue the subsequent functional verification,the full-length c DNA sequence of PxTryp?SPc1 gene was cloned,and we found that it was mainly expressed in midgut tissue in all larval instars,stating its potential importance.(4)We confirmed that the PxTryp?SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1 Ac protoxin.(5)Analysis by genetic linkage experiment demonstated that down-regulation of the PxTryp?SPc1 gene was genetically linked to resistance to Cry1 Ac in the SZ-R strain.(6)Finally,RNAi-mediated silencing of PxTryp?SPc1 gene expression decreased larval susceptibility to Cry1 Ac protoxin in the susceptible DBM1Ac-S strain,supporting that low expression of PxTryp?SPc1 gene is involved in Cry1 Ac resistance in P.xylostella.In summary,these above findings provide new insights into the mechanisms of Bt resistance and help to better understand the role of midgut proteases in insect resistance to Bt toxins,also having important guidances for the detection and integrated management of insect resistance to Bt crops/biopesticides and the development and sustainable application of new Bt crops or new toxin proteins.