RNAi induced knockdown of a cadherin-like protein (EF531715) does not affect toxicity of Cry34/35Ab1 or Cry3Aa to Diabrotica virgifera virgifera larvae (Coleoptera: Chrysomelidae)

Abstract

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important maize pest throughout most of the U.S. Corn Belt. Bacillus thuringiensis (Bt) insecticidal proteins including modified Cry3Aa and Cry34/35Ab1 have been expressed in transgenic maize to protect against WCR feeding damage. To date, there is limited information regarding the WCR midgut target sites for these proteins. In this study, we examined whether a cadherin-like gene from Diabrotica virgifera virgifera (DvvCad; GenBank accession # EF531715) associated with WCR larval midgut tissue is necessary for Cry3Aa or Cry34/35Ab1 toxicity. Experiments were designed to examine the sensitivity of WCR to trypsin activated Cry3Aa and Cry34/35Ab1 after oral feeding of the DvvCad dsRNA to knockdown gene expression. Quantitative real-time PCR confirmed that DvvCad mRNA transcript levels were reduced in larvae treated with cadherin dsRNA. Relative cadherin expression by immunoblot analysis and nano-liquid chromatography - mass spectrometry (nanoLC-MS) of WCR neonate brush border membrane vesicle (BBMV) preparations exposed to DvvCad dsRNA confirmed reduced cadherin expression when compared to BBMV from untreated larvae. However, the larval mortality and growth inhibition of WCR neonates exposed to cadherin dsRNA for two days followed by feeding exposure to either Cry3Aa or Cry34/35Ab1 for four days was not significantly different to that observed in insects exposed to either Cry3Aa or Cry34/35Ab1 alone. In combination, these results suggest that cadherin is unlikely to be involved in the toxicity of Cry3Aa or Cry34/35Ab1 to WCR.

Knockdown of two Cadherin genes confers resistance to Cry2A and Cry1C in Chilo suppressalis

“…However, the affinity of cadherins for different Cry toxins varies in different insects. Fox example, some Cry toxins are not lethal to the Coleoptera or Lepidoptera 24, 25 .…”

Control of western corn rootwormviaRNAi traits in maize: lethal and sublethal effects ofSec23dsRNA

“…Non‐diapausing WCR eggs and newly emerged adults were purchased from Crop Characteristics, Inc. (Farmington, MN, USA). Eggs were washed from soil with water and surface‐sterilized with 10% formaldehyde for 10 min, rinsed with water, hatched on artificial diet, and held at 28 °C and 40% RH, at 16:8 (Light : Dark) photoperiod, as previously described . Newly emerged adults were placed in cages at arrival with untreated artificial diet modified from Branson and Jackson .…”

“…Eggs were washed from soil with water and surface-sterilized with 10% formaldehyde for 10 min, rinsed with water, hatched on artificial diet, and held at 28 ∘ C and 40% RH, at 16:8 (Light : Dark) photoperiod, as previously described. 36,37 Newly emerged adults were placed in cages at arrival with untreated artificial diet modified from Branson and Jackson. 38 The diet consisted of 6 g of the dry ingredients, 38 12.5 mL of water, 0.365 g of agar, 0.7 mL of glycerol and 27.5 mL of a solution of 47% propionic acid and 6% phosphoric acid to reduce microbial contamination.…”

Cry6Aa1, a Bacillus thuringiensis nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing

“…The Cry6Aa1 protein was expressed in P. fluorescens and the inclusion bodies were purified using the methods described previously (75). Approximately 100 mg of Cry6Aa1 inclusion bodies were thawed at 4°C and centrifuged at 31,000 ϫ g for 20 min at 4°C.…”

“…Cry3Aa toxin inclusion bodies were prepared as described previously using the P. fluorescens heterologous expression system (75). Inclusion bodies were solubilized in 20 ml of 0.1 M CAPS (pH 10) at room temperature for 2 h. The solubilized protein was centrifuged for 20 min at 31,000 ϫ g at 4°C.…”