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.
Table 1.
Summary of transcriptome and proteome data and differential expression.
Table 2.
Wolbachia proteins differentially expressed in CLas+ ACP guts.
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.
Table 3.
ACP proteins with antiparallel patterns of regulation between the transcriptome and proteome.
Table 4.
Digestive proteins and transcripts detected in the ACP gut.
Proteins annotated as proteases or lipases are included here from both the RNAseq and proteome dataset.
Table 5.
Mitochondrial proteins differentially expressed in the ACP gut.
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.