41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

Abstract not available

We previously reported the identification of an alkaline phosphatase (HvALP) that bound Cry1Ac toxin in brush border membrane vesicles (BBMV) from Heliothis virescens. Lower alkaline phosphatase-specific activity and HvALP protein levels in Cry1Ac-resistant larvae suggested a functional role for this protein in the Cry1Ac intoxication process. In this work we report the cloning of three isoforms of membrane bound alkaline phosphatase (mALP) from H. virescens larval midgut. These three isoforms share high sequence identity, although they also present distinctive features. Analysis of expression confirmed that although alkaline phosphatases are present in a number of larval tissues, all three mALP clones are only expressed in the foregut and midgut epithelium. Heterologously expressed mALP isoforms were used to study interactions with Cry1Ac toxin and characterize the role of HvALP in Cry1Ac intoxication.

Cry1A toxins synthesized by the bacterium Bacillus thuringiensis target mature cells in the midgut of Lepidopteran larvae. In Heliothis virescens, Cry1Ac toxin causes mature midgut cell lysis, compromising epithelial integrity. To overcome this injury, midgut stem cells undergo quick cell divisions and differentiation to replace damaged mature cells. This process has been proposed to result in lower susceptibility and resistance to Cry1A toxins in some insect strains. In this work we utilize a combined genomic and proteomic approach to study this regenerative mechanism. Based on previous reports, our current hypothesis is that this process is regulated by growth factors and cytokines synthesized by dying mature midgut cells. We treated primary midgut cell cultures from H. virescens larvae with Cry1Ac or Cry3Aa (inactive against H. virescens), and isolated the proteins secreted by the dying cells. This secretome was then compared among treatments using proteomics and a bioactivity assay with stem cells to identify the growth factors involved in the regenerative process. This proteomic approach is coupled to a microarray analysis of changes in expression of putative growth factors in whole midgut from H. virescens larvae upon feeding on Cry1Ac toxin.

A new Bacillus subtilis isolate showed high anti-fungal activities (more than 80% control efficacy) against several plant diseases such as rice blast (Magnaporthe grisea), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans) and wheat leaf rust (Puccinia recondita). We tried to confer an insecticidal activity to this B. subtilis isolate for constructing a recombinant strain which has dual functions, anti-fungal and insecticidal activity. The insecticidal cry1Ac gene of B. thuringiensis was constructed under its own promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K-1Ac). The plasmid, pHT1K-1Ac was introduced into B. subtilis isolate by electroporation and the transformant was confirmed by PCR with cry1Ac specific primers. B. subtilis transformant produced a parasporal inclusion in the cells as in B. thuringiensis and the size of that protein was appox. 130 kDa. The insecticidal activity of the transformant was checked against lepidopteran pest. This result suggests that this recombinant B. subtilis strain shows the possibility of controlling harmful insect pests as well as plant fungal diseases simultaneously at one crop or on industrial downstream, the culture broth and harvested cells can be used as individual biological control agents separately for integrated crop protection.

A microarray containing 15,202 probes developed from Heliothis virescens expressed sequence tags (ESTs) were hybridized with labeled complementary RNA (cRNA) from midgut tissues of fourth instar larvae fed on a diet containing 1 ppm Cry1Ac for 0.5, 2, 6, and 24 hours. Larvae fed on a diet without Cry1Ac were used to obtain control cRNA. Expression levels of several aminopeptidases were investigated using normalized data.