Use of Crowdsourcing Challenge to Develop RNAi Based Biological Control of the Citrus Greening Vector, Diaphornia citri.

Citrus Greening, or Huanglongbing (HLB), is a disease of citrus causing significant reductions in citrus production worldwide. The phloem-limited bacteria that cause this disease, Candidatus Liberibacter sp., are transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama. We are reporting the result of a screen of forty-three D. Citri genes, selected by a crowdsourcing challenge, for susceptibility to RNAi silencing by introducing cognate dsRNAs (ranging in size from 200 to 300 bps) into a D. citri artificial diet for oral uptake. Three dsRNA sequences, each targeting different psyllid genes, were identified to cause significantly high mortality in adult D. Citri. All three genes encode proteins with established functional roles in some form of cellular redox metabolism. These dsRNA sequences were cloned into the amplicon of the a citrus phloem-limited virus (citrus tristeza virus, CTV T36), which was then used as a vector delivery system for synthetic dsRNAs within CTV inoculated (paratrangenic) plants. Virus Induced Gene Silencing (VIGS) using paratransgenic citrus leaves targeting the same gene as the corresponding artificial diet resulted in 30% higher mortality. RNASeq transcriptome analysis using next-generation sequencing also identified other genes that encode proteins that are involved in a variety of different redox reactions that are functionally related (by GO annotation) to to the target gene.