Inaiara Pacheco, Jason Stajich, Rick Redak, Linda Walling, and Peter Atkinson*
*University of California, Department of Entomology, 900 University Avenue, Riverside, CA, 92521 (peter.atkinson@ucr.edu)

Pierce’s Disease (PD) is a serious disease of California grapevines caused by a pathogenic bacterium, Xylella fastidiosa, which is transmitted by the xylem-feeding insect, Homalodisca vitripennis, commonly known as the glassy-winged sharpshooter.  We propose to eliminate PD by generating glassy-winged sharpshooter lines that are unable to transmit X. fastidiosa to grapevine though editing of the glassy-winged sharpshooter genome. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene-editing technologies now make this possible provided that we can efficiently introduce the CRISPR macromolecules into chromosomes of glassy-winged sharpshooter to generate genetic lines. We developed a delivery system that achieves this goal. As proof of principle, we generated, at high frequencies, the first genetic mutants of glassy-winged sharpshooter using two eye pigmentation genes, white and cinnabar. These strains are robust, and we have maintained them in our laboratory for over 10 generations. We confirmed that these mutations were specific to the target sites, which is critical for genetic control strategies. We have also demonstrated that we can integrate DNA fragments into specific target sites in the white and cinnabar genes with high frequencies. We have used this technology to establish a platform for the rapid screening of gene regulatory sequences in glassy-winged sharpshooter. Our results show that we can perform gene-editing technology in this important pest insect, enabling us to inactivate specific gene targets and to introduce transmission-blocking genes into specific genetic sites in the glassy-winged sharpshooter genome. We have developed the foundational genetic tools required for the generation and testing of genetic strains of glassy-winged sharpshooter for the control of PD.

Funding Support: Pierce’s Disease/GWSS Board, Californian Department of Agriculture and the Animal and Plant Health Inspection Service of the United States Department of Agriculture.