Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis.
ABSTRACT The crystal proteins of Bacillus thuringiensis are widely used in transgenic crops and commercially available insecticides. Manduca sexta, the tobacco hornworm, is the model insect for B. thuringiensis studies. Although brush border vesicles prepared from larval M. sexta midgut have been used in numerous mode-of-action studies of B. thuringiensis toxins, their protein components are mostly unknown. Vesicles prepared from the brush border of M. sexta midgut were analyzed using one- and two-dimensional gel electrophoresis to establish a midgut brush border proteome. Sub-proteomes were also established for B. thuringiensis Cry1Ac binding proteins and glycosylphosphatidyl inositol (GPI) anchored proteins. Peptide mass fingerprints were generated for several spots identified as Cry1Ac binding proteins and GPI-anchored proteins and these fingerprints were used for database searches. Results generally did not produce matches to M. sexta proteins, but did match proteins of other Lepidoptera. Actin and alkaline phosphatase were identified as novel proteins that bind Cry1Ac in addition to the previously reported aminopeptidase N. Aminopeptidase N was the only GPI-anchored protein identified. Actin, aminopeptidase N, and membrane alkaline phosphatase were confirmed as accurate protein identifications through western blots.
Article: Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches.[show abstract] [hide abstract]
ABSTRACT: Bacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis). Several Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut. By combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.BMC Genomics 06/2012; 13:248. · 4.07 Impact Factor
Article: Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.[show abstract] [hide abstract]
ABSTRACT: Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.PLoS ONE 01/2011; 6(3):e17606. · 4.09 Impact Factor
Article: Exploring the midgut transcriptome and brush border membrane vesicle proteome of the rice stem borer, Chilo suppressalis (Walker).[show abstract] [hide abstract]
ABSTRACT: The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac.PLoS ONE 01/2012; 7(5):e38151. · 4.09 Impact Factor