The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium.

The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.

Composition and abundance of midgut surface proteins in the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is the vector of Candidatus Liberibacter asiaticus (CLas), the presumed causative agent of citrus greening disease. For successful transmission, CLas must cross the gut barrier, requiring interaction with proteins on the midgut epithelium. We compared the relative abundance of gut surface proteins for both adult and nymph D. citri, as nymphs are particularly susceptible to CLas infection. To enrich for gut surface proteins, brush border membrane vesicles were prepared from dissected guts, and proteins identified from triplicate samples run on a timsTOF mass spectrometer. A total of 1516 and 1219 proteins were identified from D. citri adults and nymphs respectively. Based on bioinformatics analysis software and manual curation, 112 adult and 87 nymph proteins were predicted to localize to the surface of the microvilli and were further categorized into integral membrane and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteins exploited by insect pathogens such as aminopeptidase, alkaline phosphatase, cadherin, ABC transporters, and carboxypeptidase were among the most abundant proteins on the gut surface. In addition to providing insights into hemipteran gut physiology, the D. citri gut surface proteome will inform novel approaches to interfere with CLas interaction with the psyllid gut to prevent the spread of citrus greening. BIOLOGICAL SIGNIFICANCE: The Asian citrus psyllid (ACP), D. citri is one of the most serious pests of citrus worldwide. ACP transmits the pathogenic bacterium that causes citrus greening or huanglongbing (HLB), which has resulted in severe economic losses in global citriculture. The putative causative agent of this disease, the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), is vectored by the Asian citrus psyllid, D. citri, in a persistent and circulative manner. CLas must interact with gut surface proteins in order to enter midgut epithelial cells. However, the specific proteins exploited by CLas have yet to be identified. The characterization of the most abundant proteins on the surface of the D. citri gut provides insight into candidate receptors for CLas and other pathogens of D. citri. We hypothesize that pathogens of D. citri exploit the most abundant proteins on the surface of the gut for entry into the host insect. Importantly, the abundant gut surface proteins will provide the basis for novel approaches to disrupt CLas-D. citri interactions, with the goal of preventing further economic loss to the citrus industry.