Gene silencing of Diaphorina citri candidate effectors promotes changes in feeding behaviors

Insect effectors are mainly secreted by salivary glands, modulate plant physiology and favor the establishment and transmission of pathogens. Feeding is the principal vehicle of transmission of Candidatus Liberibacter asiaticus (Ca. Las) by the Asian citrus psyllid (ACP), Diaphorina citri. This study aimed to predict putative ACP effectors that may act on the Huanglongbing (HLB) pathosystem. Bioinformatics analysis led to the identification of 131 candidate effectors. Gene expression investigations were performed to select genes that were overexpressed in the ACP head and modulated by Ca. Las. To evaluate the actions of candidate effectors on D. citri feeding, six effectors were selected for gene silencing bioassays. Double-stranded RNAs (dsRNAs) of the target genes were delivered to D. citri adults via artificial diets for five days. RNAi silencing caused a reduction in the ACP lifespan and decreased the salivary sheath size and honeydew production. Moreover, after dsRNA delivery of the target genes using artificial diet, the feeding behaviors of the insects were evaluated on young leaves from citrus seedlings. These analyses proved that knockdown of D. citri effectors also interfered with ACP feeding abilities in planta, causing a decrease in honeydew production and reducing ACP survival. Electrical penetration graph (EPG) analysis confirmed the actions of the effectors on D. citri feeding behaviors. These results indicate that gene silencing of D. citri effectors may cause changes in D. citri feeding behaviors and could potentially be used for ACP control.