| Institution: | 1. Instituto de Agrobiotecnología, CSIC-UPNA, Gobierno de Navarra, Campus Arrosadía, 31192 Mutilva, Navarra, Spain;2. Laboratorio de Entomología Agrícola y Patología de Insectos, Departamento de Producción Agraria, Universidad Pública de Navarra, 31006 Pamplona, Spain;3. Departamento de Genética, Facultad de CC. Biológicas, Universitat de València, 46100 Valencia, Spain;1. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;2. CSIRO, Black Mountain Laboratories, Canberra, Australian Capital Territory, Australia;1. Departamento de Genética, Facultad de CC. Biológicas, Universidad de Valencia, Burjassot, Spain;1. School of Life Sciences, Central China Normal University, Wuhan 430079, PR China;2. State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China |
| Abstract: | Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity. |
