Biochemical And Molecular Mechanisms Of Resistance To Toxin Cry1Ac In The Diamondback Moth, Plutella Xylostella

The widespread use of chemical pesticides has played a very important role in agriculture and food security. Due to the development of pesticide resistance, pest prevention and control is becoming more and more difficult. Bacillus thuringiensis (Bt) based its crystal protein (ICPs) are highly friendly for humans and non-target insects and are widely used control diamondback moth and other Lepidoptera. Diamondback moth (Plutella xylostella) is a worldwide pest of cruciferous vegetables. As early as 1990, cases of resistance to Bt formulations were documented in open-field populations of diamondback moth. But, the precise mechanism of diamondback moth resistance to Bt toxin Cry1Ac is poorly understood. A Cry1Ac-resistant strain (SZBT) was selected in the laboratory in order to investigate resistance mechanisms. Resistance allele frequency of three field populations was detected with an F1 screen (single pair crossing between field-derived insects and SZBT insects). Using proteomic analysis, Cry1Ac binding proteins in midgut membranes from both CrylAc-resistant and susceptible strains of P. xylostella were compared and identified. An aminopeptidase N (PxAPN2) with reduced expression in the resistant strain was identified. PxAPN2 was demonstrated as a functional receptor of Cry1Ac by using RNAi technique. Understanding of biochemical and molecular mechanisms of CrylAc resistance is crucial for designing rational resistance management strategy in P. xylostella.1. Genetic complementation test for CrylAc resistance and detection of CrylAc resistance allele frequency in field populations of P. xylostellaBacillus thuringiensis (Bt) formulations have been widely used in China against the diamondback moth, P. xylostella, and different levels of resistance to Cry1Ac have been evolved in field populations of P. xylostella. Because Cry1Ac resistance is generally incompletely recessive in P. xylostella, detection of resistance allele frequency is very important for resistance management. A resistant strain (SZBT) selected under laboratory developed about 2000-fold resistance to Cry1Ac. Cry1Ac resistance in the SZBT strain was characterized as "Mole 1" resistance. Allelic complementation tests showed that Cry1Ac resistance in the SZBT strain and the Cry1Ac-R strain (originated from Florida of the USA) share a common locus. An F1 screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of CrylAc resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province. Cry1Ac resistance levels in Guangzhou. Huizhou and Yuxi populations were 73-,117- and 13-fold respectively, which is correlated with the resistance frequencies detected by thescreen.2. Idientification of CrylAc binding proteins in midgut membranes from P. xylostellaMidgut membranes of 4th instar larvae from the susceptible strain ROTH, the resistant strain SZBT and the reference strain SZ were separated by the traditional SDS-PAGE and blotted with biotin-labeled Cry1A toxins (Cry1Aa, Cry1Ab and Cry1Ac). About ten major Cry1Ac-binding bands were revealed in the ligand blotting. Pattern and darkness of the toxin-binding protein bands were similar between the resistant SZBT and the susceptible ROTH strains, except a 110 kDa protein band was significantly lighter in the resistant SZBT strain than in the ROTH strain. It is suggested that reduced expression of this 110 kDa Cry1Ac-binding protein may be associated with Cry1Ac resistance in the SZBT strain.Two-dimensional electrophoresis (2-DE) combined with ligand blotting assay were employed to separate and identify Cry1Ac-bingding proteins from both the resistant SZBT and the susceptible ROTH strains, and peptidase mass fingerprints were generated for several spots identified as Cry1Ac- binding proteins. The results showed that, in addition to the reported receptor aminopeptidase (PxAPN2), the storage protein, actin, glucosinolate sulfatase, V-ATPase subunit B were also identified as novel binding proteins in P. xylostella. As found in the 1-DE experiments, an 110 kDa Cry1Ac-binding protein was significantly reduced in the resistant SZBT strain. This 110 kDa protein was identified as an aminopeptidase N (PxAPN2). Aminopeptidase N is an important class of Bt toxin receptors in lepidopteran insects. Lower expression of PxAPN2 may relate to Cry1Ac resistance in the SZBT strain.3. Effect of RNAi-mediated gene silencing of PxAPN2 on Cry1Ac toxicity to the susceptible P. xylostella PxAPN2 gene encoding the 110 kDa toxin binding protein was cloned from both SZBT and ROTH strains using RT-PCR. Amino acid substitutions at several sites of PxAPN2 were present in both strains. Using quantitative real-time PCR, mRNA expression levels of PxAPN2 were compared between the resistant and the susceptible strains, and it showed that expression of PxAPN2 in the fourth instar larval midgut from the resistant SZBT strain was reduced to 33.9% of the susceptible ROTH strain.In order to verify the role of PxAPN2 on Cry1Ac resistance, PxAPN2 was down-regulated through RNAi to see whether it can make the susceptible strain obtain CrylAc resistance. Expression of PxAPN2 was reduced by as high as 53% when fed with PxAPN2 dsRNA. The third instar larvae from the ROTH fed with dsRNA of PxAPN2 obtained 4- to 14-fold resistance to Cry1Ac compare with the control larvae. This demonstrated that PxAPN2 is a functional receptor of Cry1Ac, and lower expression of PxAPN2 can result in resistance to Cry1Ac in P. xylostella.In the present study, biochemical and molecular mechanisms of resistance to CrylAc in the SZBT strain were extensively characterized through a combination of proteomic analysis, ligand blotting, gene cloning and RNA interference. PxAPN2 was confirmed as a functional receptor for CrylAc, and down-regulation of this receptor is associated with a high level resistance to CrylAc in the SZBT strain. PxAPN2 locus does not locate in the same linkage group as the CrylAc resistance gene. Therefore, lower mRNA expression of PxAPN2 may relate to changes of cis-acting factors, and lower expression of PxAPN2 protein may also be caused by post-transcriptional regulation in the resistant SZBT strain of P. xvlostella.