Ina de Souza Pacheco, Richard Redak, Linda Walling, and Peter Atkinson*
*University of CA – Riverside, Department of Entomology, Riverside, CA, 92521 (peter.atkinson@ucr.edu)

Pierce’s disease is a serious disease of California grapevines caused by a pathogenic bacterium, Xylella fastidiosa, which is transmitted by a xylem-feeding insect, Homalodisca vitripennis (the glassy-winged sharpshooter, GWSS). Current methods to control this invasive pest are expensive, relying on insecticides, quarantine, and eradication. By blocking the ability of GWSS to transmit X. fastidiosa, we can control the spread of Pierce’s disease in a sustainable manner. Our control strategy is feasible due to the powers of clustered, regularly-interspaced, short palindromic repeats (CRISPR)-based gene-editing technologies. CRISPR-based technologies will allow us to generate GWSS strains that are unable to acquire X. fastidiosa from infected plants or transmit X. fastidiosa to healthy grapevines. We have developed these technologies for GWSS. We have used CRISPR technologies to knock out a gene or to insert a gene into the GWSS genome. As proof of principle, we generated the first genetic mutants of GWSS using two eye pigmentation genes, white and cinnabar. We obtained mutants at high frequency, these mutant strains are robust, and we have maintained the strains in our laboratory for over 15 generations. We confirmed that the white and cinnabar mutations are specific to the target sites, which is critical for genetic control strategies. We have also demonstrated that we can integrate DNA fragments and genes into specific target sites in the white and cinnabar genes at high frequencies. This technology has allowed us to establish a platform for rapid screening of gene regulatory sequences in GWSS. We still need tissue-specific gene regulators for our genetic control strategy. With the gene-editing technology firmly established in GWSS, we are now identifying genes that can be used to block GWWS’ ability to acquire or transmit X. fastidiosa. Our goal is to make a Xylella-proof GWSS and end the threat of Pierces’ disease in California.

Funding Support: Our research was funded by the Pierce’s Disease/GWSS Board, California Department of Agriculture and the Animal and Plant Health Inspection Service of the United States Department of Agriculture.