Knockdown of an ATP-binding cassette transporter in resistant western corn rootworm larvae partially reverses resistance to eCry3.1Ab protein

Abstract

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, has evolved resistance to nearly every management tactic utilized in the field. This study investigated the resistance mechanisms in a WCR strain resistant to the Bacillus thuringiensis (Bt) protein eCry3.1Ab using dsRNA to knockdown WCR midgut genes previously documented to be associated with the resistance. ATP-binding cassette transporter (ABCC4), aminopeptidase-N, cadherin, and cathepsin-B were previously found to be differentially expressed in eCry3.1Ab-resistant WCR larvae when compared to susceptible larvae after feeding on maize expressing eCry3.1Ab and its near-isoline. Here we compared the susceptibility of resistant and susceptible WCR larvae to eCry3.1Ab protein in presence or absence of dsRNA targeting the above genes using 10-day diet overlay toxicity assays. Combining ABCC4 dsRNA with eCry3.1Ab protein increased susceptibility to Bt protein in WCR-resistant larvae, but the other three genes had no such effect. Among 65 ABC transport genes identified, several were expressed differently in resistant or susceptible WCR larvae, fed on eCry3.1Ab-expressing maize versus its isoline, that may be involved in Bt resistance. Our findings provide strong evidence that ABCC4 is indirectly involved in WCR resistance to eCry3.1Ab protein by enhancing the effects of Bt-induced toxicity.

Keywords: Bacillus thuringiensis; Diabrotica virgifera virgifera; Corn rootworm; Insect resistance management; RNAi; Resistance mechanisms.

Fig. 1

Relative gene expression after knockdown of ABCC4 transporter, aminopeptidase-N, cadherin, and cathepsin-B using dsRNAs in eCry3.1Ab Diabrotica virgifera virgifera resistant and susceptible neonates that fed for three consecutive days on 1 µg of dsRNA/cm², overlaid on artificial diet. Means (± SEM) generated from six replicates (8–10 larvae per replicate). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2

Percent mortality (A), percent of 2nd instar (B), and larval dry weight (C) in Diabrotica virgifera virgifera resistant and susceptible strains after exposure to eCry3.1Ab protein (24.3 µg/cm²) alone, to ABCC4 dsRNA alone (1.0 µg/cm²), or mixed with 1.0 µg/cm² of ABCC4 dsRNA, as well as to nuclease free water (NFW), 10 mM carbonate/bicarbonate buffer, and green fluorescent protein (GFP) dsRNA (1.0 µg/cm²) as controls, in 10-day diet overlay toxicity assays. Data represent means (± SEM) of ten replicates (8 larvae per replicate). Bars followed by different upper-case letters (resistant strain) or lower-case letters (susceptible strain), within each strain were significantly different (P < 0.05).

Fig. 3

Relative ATP-binding cassette (ABC) gene expression in eCry3.1Ab resistant or susceptible Diabrotica virgifera virgifera neonate larvae after feeding on eCry3.1Ab expressing or isoline maize seedlings, as determined by RNASeq analyses. The letter following ‘ABC’ in gene names represents the ABC transporter family, and the numbers reflect gene transcript contig IDs reported previously. For heatmap visualization, read count data were rounded to integers before applying a normalizing transformation to each transcript count (log2 (x + 0.5)).