Transcriptome Analysis of Asian Citrus Psyllid RNAi Induced Mortality By Quantitative Real-Time PCR and Next Generation Sequencing

The Asian Citrus Psyllid (Diaphorina citri Kuwayama) is an invasive Homopteran that has crippled citrus production in the state of Florida with the spread of Huanglongbing "Citrus Greening" disease, causing discolored bitter fruit. The disease is associated with the bacterium ‘Candidatus’ Liberibacter and is rapidly spreading in other citrus producing states. The RNAi screening of more than 50 psyllid genes was made possible by a competitive Innocentive Challenge in which experts from diverse fields were awarded by the Citrus Research and Development Foundation (CRDF) for choosing the most lethal psyllid gene to target. Seven of the fifty genes tested resulted in significantly high psyllid mortality and four showed a dose response when oral uptake was administered by artificial diets containing gene targeting dsRNA fragments. The Citrus Tristeza Virus (CTV) was then engineered to express dsRNAs with sequence specificity for one of these genes and was graft inosculated onto Citrus Macrophylla producing plants with dsRNA enriched leaves (paratransgenic control) comparable in concentration to that of the artificial diets. Transcriptome analysis was performed using qRT-PCR and Ion Torrent RNA Seq to quantify the expression profile of psyllid gut tissue and identify “off-target effects”.