Detection of alternative splicing in western corn rootworm (diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio iso-seq

Authors: ZHAO, IXIAO - University Of Missouri; ELSIK, CHRISTINE - University Of Missouri; Hibbard, Bruce; Shelby, Kent

Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2021
Publication Date: 7/7/2021
Citation: Zhao, I., Elsik, C.G., Hibbard, B.E., Shelby, K. 2021. Detection of alternative splicing in western corn rootworm (diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio iso-seq. Insect Molecular Biology. 30(4):436-445. https://doi.org/10.1111/imb.12709.
DOI: https://doi.org/10.1111/imb.12709

Interpretive Summary: Alternative splicing is a regulated process during gene expression that results in a single gene coding for multiple proteins. The phenomenon can also be observed in insect species that have gained resistance to pesticides and Bt toxins. The western corn rootworm is the most important pest of corn in the US Corn Belt and has evolved resistance to most management tactics targeting it. For the current project, we sequenced resistant and susceptible western corn rootworm which had fed on seedling maize with and without Bt toxins for 12 and 24 hours. Gene expression was evaluated for all treatments. Analysis showed that in Bt-resistant western corn rootworm, expression of one gene form was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the alternative gene form. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced forms of peritrophic matrix protein might be associated with Bt resistance.

Technical Abstract: Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date, the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24'h. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi-exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.