Transcriptional regulation of Cry2Ab toxin receptor ABCA2 gene in insects involves GATAe and splicing of a 5' UTR intron

Bacillus thuringiensis (Bt) produces Cry toxins that are used to control insect pests worldwide. However, evolution of insect resistance threatens the sustainable application of these toxins. In some cases, Cry toxin resistance has been linked to mutations affecting toxin receptors expression. Previous work identified HaGATAe transcriptional factor (TF) to be involved in the expression of multiple Cry1 receptor genes. Also, it was reported that 5´untranslated region (UTR) could be involved in regulation of gene expression in eukaryotic cells. The ABCA2 protein functions as Cry2A toxin receptor in multiple lepidopteran species. Here, we investigated regulation of HaABCA2 expression in Helicoverpa armigera and in different insect cell lines. Transient expression of HaABCA2 gene resulted in susceptibility to Cry2Ab in Sf9 cells. Transient expression of HaGATAe transcriptional factor in Sf9 cells enhanced the expression of multiple larval midgut proteins including SfABCA2, increasing the susceptibility to activated Cry2Ab. The silencing of HaGATAe expression in H. armigera larvae by RNAi, resulted in lower expression of HaABCA2 which correlated with reduced susceptibility to Cry2Ab. The GATAe-binding site in the promoter of HaABCA2 gene was identified by systematic truncations, site directed mutagenesis and DNA Pull-down analysis. In addition, 5’ RACE analysis revealed that HaABCA2 transcripts in larval midgut cells had at least three different 5’ UTRs. Here we also show that the retention of an intron in one of these 5’ UTRs significantly inhibited the HaABCA2 expression. A short sequence after the start codon of translation of HaABCA2 was identified to be required for the intron removal. These findings provide new insight for mechanism of Cry2Ab resistance in H. armigera.