Harnessing wolbachia: A gene drive approach to combat the citrus greening pathogen Candidatus liberibacter asiaticus

Wolbachia is an obligate intracellular endosymbiont estimated to be present in 40-60% of insect species. Its ability to spread through insect populations and affect pathogen transmission by altering vector competence has made this bacterium a possible tool for controlling vector-borne diseases. Considering this, we propose using Wolbachia as a maternally transmitted gene driver for managing transmission of the citrus pathogen, Candidatus Liberibacter asiaticus (CLas), a fastidious bacterium transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). This pathogen is responsible for citrus greening disease, the most devastating disease of citrus worldwide. Our preliminary studies demonstrate that CLas pathogen-blocking gut binding peptides (GBPs) disrupt CLas transmission by competing for pathogen binding sites in the insect gut. Here, we employ the CRISPR-Cas9 ribonucleoprotein (RNP) method for transgene insertion, using homology directed repair (HDR), into the D. citri Wolbachia isolate genome. Our goal is to develop a reliable method for delivery of pathogen interfering genes in the D. citri vector. Development of a Wolbachia transformation system for use as a gene driver to reduce CLas will be an important, environmentally friendly tool for management of citrus greening disease. Additionally, this technology will provide a framework to study the molecular basis of Wolbachia biology and host-endosymbiont interactions that have not been previously characterized.