Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut

Abstract

Huanglongbing, or citrus greening disease, is an economically devastating bacterial disease of citrus. It is associated with infection by the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid (ACP). For insect transmission to occur, CLas must be ingested during feeding on infected phloem sap and cross the gut barrier to gain entry into the insect vector. To investigate the effects of CLas exposure at the gut-pathogen interface, we performed RNAseq and mass spectrometry-based proteomics to analyze the transcriptome and proteome, respectively, of ACP gut tissue. CLas exposure resulted in changes in pathways involving the TCA cycle, iron metabolism, insecticide resistance and the insect's immune system. We identified 83 long non-coding RNAs that are responsive to CLas, two of which appear to be specific to the ACP. Proteomics analysis also enabled us to determine that Wolbachia, a symbiont of the ACP, undergoes proteome regulation when CLas is present. Fluorescent in situ hybridization (FISH) confirmed that Wolbachia and CLas inhabit the same ACP gut cells, but do not co-localize within those cells. Wolbachia cells are prevalent throughout the gut epithelial cell cytoplasm, and Wolbachia titer is more variable in the guts of CLas exposed insects. CLas is detected on the luminal membrane, in puncta within the gut epithelial cell cytoplasm, along actin filaments in the gut visceral muscles, and rarely, in association with gut cell nuclei. Our study provides a snapshot of how the psyllid gut copes with CLas exposure and provides information on pathways and proteins for targeted disruption of CLas-vector interactions at the gut interface.

Fig 1. Principle components analysis (PCA) showing variations among replicates of RNA sequencing data.

Four different replicates for each sample, either CLas exposed (CLas+) or non-exposed (CLas-, psyllids reared on healthy citrus plants) are plotted on the PCA plot. Approximately 60% of the variation is seen in principle component (PC) 1, which can be explained by differences in the CLas status of the samples (either + or -). Variation in PC2 is much less, 8% and can be ascribed to expression variation among CLas+ samples.

Fig 2. Single gut quantitative PCR showing Wolbachia copy number values in excised psyllid guts.

Quantitative PCR was performed on DNA extracted from single, excised guts to determine if the titer of Wolbachia is altered in CLas-exposed (CLas+) as opposed to CLas-unexposed (CLas-) guts. The resulting copy numbers shown above are not statistically different using error bars reflecting the standard error of these calculated values. Black bars indicate the mean copy number, and the height of the boxes reflects the spread of copy number values.

Fig 3. Schematic diagram of the citric acid cycle showing the trend of regulation in the proteome upon CLas exposure.

This image was taken from https://en.wikipedia.org/wiki/File:Citric_acid_cycle_with_aconitate_2.svg and adapted to indicate up- and down-regulation shown by proteomic and transcriptomic data using the creative commons license http://creativecommons.org/licenses/by-sa/3.0/deed.en. Blue shapes reflect the whole body ACP proteome data from [14], red shapes reflect the ACP gut proteome, and maroon shapes reflect the gut transcriptome. Arrows indicate the trend of up- or down-regulation, and ovals indicate that these enzymes were detected but were not differentially expressed.

Fig 4. Fluorescent in situ hybridization to visualize microbial interactions in ACP guts.

Dissected guts were reacted with probes specific to CLas (green) and Wolbachia (red) and imaged using confocal microscopy. Nuclei are stained using DAPI and visualized in blue. (A) Light imaging allowed for identification of regions of the gut, scale bar = 250μm, mt = Malpighian tubule, hg = hindgut, mg—midgut, fc = filter chamber. (B) Confocal micrographs of CLas- guts show no CLas signal and patchy distribution of Wolbachia (B, C, D), scale bars = 250μm, 50μm and 25μm, respectively. (E-H) Confocal microscopy of CLas+ guts shows a wider distribution of CLas in the gut and a patchy distribution of Wolbachia in the gut, with Wolbachia signal concentrated in the filter chamber, midgut, and Malpighian tubules, scale bar = 250μm.

Fig 5. High magnification images of CLas and Wolbachia in CLas exposed guts visualized using fluorescence in situ hybridization (FISH) and confocal microscopy.

CLas signal (Cy3) is in green, Wolbachia (Cy5) in red, and DAPI counterstaining of nuclei is in blue. (A-D) Optical cross-section of the gut visualizing CLas localization in along the brush border membrane of the gut lumen. Rarely, CLas can be seen co-localized with the DAPI signal, indicating nuclear association. Wolbachia signal does not overlap with CLas signal, see overlay (D), although the two bacteria are frequently observed within the same cell. (E-H) CLas can also be observed in puncta within cells and there is no overlap with Wolbachia in this distribution either. (I-L) At the basal surface of the gut, CLas is frequently observed along the actin cytoskeleton of the gut-associated muscles. Wolbachia signal is never detected in these filaments. Scale bars = A-D 25μm, E-H 25μm and I-L 75μm.