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

Abstract

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. Insect pests are one of the main factors that reduce agricultural plant productivity. Global losses caused by these animals reach 220 billion dollars annually 1. In addition to the damage caused by their actions, problems caused by insects can be increased by the transmission of several plant pathogens 2. Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), stands out as the most important agricultural pest among citrus crops 3. ACP is a vector for the bacteria Candidatus Liberibacter asiaticus (Ca. Las), the causal agent of Huanglongbing (HLB). This disease affects all commercial citrus varieties by promoting plant decline and reducing fruit quality 4,5. In the last fifteen years, HLB has spread quickly across the American continent and the citrus production in the USA declined 58% 4. In Brazil, 34 million citrus trees have been removed since the first detection of HLB in 2004 6. So far, no efficient control for HLB has been found. Disease management consists of producing seedlings using Ca Las-free nursery stock, removal of infected trees and insecticide applications for vector control 7,8. However, the extensive application of insecticides could select for ACP-resistant populations 9-11. Diaphorina citri acquire Ca. Las via a circulative-persistent manner during feeding 12,13. The efficiency of Ca. Las transmission is affected by the duration of phloem ingestion by ACP 14. Studies have been developed to understand the interactions ...

Dissipation and Efficacy of Afidopyropen for Controlling Asian Citrus Psyllids Under Field Conditions

“…According to past toxicological studies of afidopyropen's effect on ACP, egg hatch and adult behavior was significantly reduced 24−72 h after exposure to 100 ng/μL. 45,46 Under ideal circumstances, a higher temporal resolution would be beneficial; however, more frequent ACP inspections were not possible for this entire study performed in collaboration with our commercial grove partners. ACP population percent changes were calculated using only trees that contained ACP before treatment.…”

The Hidden Secrets of Psylloidea: Biology, Behavior, Symbionts, and Ecology

“…These resources enable proteomic and transcriptome studies on specific tissues and the use of prediction pipelines to identify putative psyllid effectors secreted by salivary gland cells. De Souza Pacheco et al (31) used this approach to identify candidate effectors in transcriptomes from D. citri heads. They profiled 12 candidates using RT-PCR, and those highly expressed in head tissues were pursued for functional analysis through RNA interference (RNAi).…”

Spotlight on the Roles of Whitefly Effectors in Insect–Plant Interactions

“…The majority of whitefly effector proteins that have been described to date were detected by identifying transcripts encoding proteins with signal peptides that lack transmembrane domains (beyond the signal peptide) in transcriptome data (Su et al, 2019;Wang et al, 2019;Xu et al, 2019). This type of transcriptome data mining is commonly used to identify effectors from insect herbivores, including phloem-feeding insects (Bos et al, 2010;Hogenhout and Bos, 2011;Zhang et al, 2017;Pacheco et al, 2020). This mining tool is relatively easy to use and has led to the identification of many putative effectors.…”

“…Finally, the transcript levels of effector genes may also differ depending on microbes present in the insects, as observed in the citrus psyllid D. citri; several effector genes were differentially expressed in adults and nymphs following infection with Candidatus Liberibacter asiaticus (Ca. Las) (Pacheco et al, 2020) and it may be the case with B. tabaci. Therefore, more research needs to be conducted on this area.…”