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Title: Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis

item JURAT-FUENTES, JUAN LUIS - University Of Tennessee
item KARUMBAHIAH, LOHITASH - University Of Georgia
item JAKKA, SIVA RAMA - University Of Tennessee
item NING, CHANGNING - Institute Of Plant Protection - China
item LIU, CHENXI - Institute Of Plant Protection - China
item WU, KONGMING - Institute Of Plant Protection - China
item JACKSON, JEREME - University Of Tennessee
item GOULD, FRED - North Carolina State University
item BLANCO, CARLOS - Animal And Plant Health Inspection Service (APHIS)
item Portilla, Maribel
item Perera, Omaththage
item ADANG, MICHAEL - University Of Georgia

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2011
Publication Date: 3/1/2011
Citation: Jurat-Fuentes, J., Karumbahiah, L., Jakka, S.K., Ning, C., Liu, C., Wu, K., Jackson, J., Gould, F., Blanco, C., Portilla, M., Perera, O.P., Adang, M.J. 2011. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis. PLoS One. 6:e17606.

Interpretive Summary: We previously reported identification of membrane-bound alkaline phosphatase (mALP) in tobacco budworm, Heliothis virescens, as binding targets for Bacillus thuringiensis crystalline toxin Cry1Ac. Proteomic and nucleic acid-based comparison of mALP expression levels were carried out between susceptible and Cry toxin-resistant strains of tobacco budworm, bollworm (Helicoverpa armigera), and fall armyworm (Spodoptera frugiperda). Reduced mALP expression levels were detected in the resistant strains of all three moth species. Potential use of mALP expression as a biomarker for resistance to Bt toxins is discussed.

Technical 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. Biomarkers for development of sensitive DNA-based methods to detect and monitor evolution of resistance to Bt toxins are currently needed. 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 is controlled at the transcriptional level. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived Cry1Fa-resistant strains of S. frugiperda. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest the potential use of alterations in mALP expression as an effective biomarker for resistance to Cry toxins in lepidopteran pests.