Project description:Citrus greening disease, also known as Huanglongbing (HLB), is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ‘Candidatus Liberibacter asiaticus’ (CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues, including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects, and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.

Project description:The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (CLas), which is associated with citrus greening disease. Variability in CLas titer in insects collected from infected plants has been attributed in part to the host plant from which the insects were collected. CLas accumulates to high titers in infected Citrus macrophylla, and in D. citri feeding on the infected plants of this species. In contrast, in the citrus relative Murraya paniculata, CLas titers remain low in infected plants and in D. citri exposed to infected plants. In this study, top-down and bottom-up proteomics methods were used to investigate the impact of these different host plants on D. citri protein expression. Difference in gel electrophoresis (DIGE) was used to identify protein spots on two-dimensional gels that were larger in one of three insect sample classes compared to the other two: D. citri continuously reared on C. macrophylla, D. citri reared continuously on M. paniculata, and D. citri transferred to M. paniculata for five days feeding after continuous rearing on C. macrophylla. Peptide mass spectrometry was used to identify and quantify proteins in target spots upregulated in each sample class. Shotgun proteomics was used to identify and quantify proteins from analysis of tryptic peptide samples prepared from whole insects from four sample classes: the reciprocal host switch condition (D. citri transferred to C. macrophylla for five days feeding after continuous rearing on M. paniculata) in addition to the three sample classes used in DIGE analysis. Integration of the results of both analyses reveals proteins identified by separate experimental workflows to be upregulated in insects adapted to each host plant, and in insects adapting to a novel host plant. A peptidoglycan-degrading protein involved in the immune response to bacterial pathogens was found to be upregulated in M. paniculata-reared D. citri. In the absence of CLas infection, host plant factors specific to M. paniculata may prime the antibacterial immune response in D. citri. Understanding the insect proteins involved in the adaptation of D. citri to host plants with variation in their susceptibility to CLas will inform the development of control strategies aimed at stopping the spread of citrus greening disease.

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Psyllid metagenome

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