Table 1.
Overview of the raw sequence data.
Table 2.
Characterized trehalose as well as glucose/fructose transporters.
Table 3.
Number of assembled transcripts and average length after assembly and reassembly showing the usefulness of reassembling.
Figure 1.
Phylogenetic tree of the 68 chosen sugar transporters derived from P. cochleariae.
This circular phylogram shows the main 4 groups of chosen sugar transporters. Tree was calculated using MrBayes.
Figure 2.
Schematic model for the structure of the putative SLC2 transporters derived from P. cochleariae by means of PcSUT1.
All 4 groups show the known and conserved facilitated sugar transporter motifs, such as DRxGRR/K in the second loop, PESPR/K in the sixth loop, E/DRxGRR/K in loop 8, and PETK/RGK/R in the carboxy terminal [2], [15], [18], [76], [86]. Furthermore, there are conserved amino acids, such as E and R in loop 4 and 10 (red). Those are needed for the glucose transport activity. Conserved tyrosines (turquois), such as the PMY motif mentioned by Chen et al. [15], can be found in our sequences in TMD 4. Additionally, conserved glycines (yellowish) in TMD 1, 2, 4, 5, 7, 8, and 10 as well as in loop 2 and 7 are present, characteristic for the mammalian glucose transporter family. The purple branch exhibits a GWTAP motif in loop 1, a PFYV motif in loop 5, and a VILMNLH motif in TMD 10 (purple colored amino acids).
Figure 3.
Phylogenetic tree of 68 chosen SLC2 transporters derived from P. cochleariae and chosen sugar transporters that have been functionally annotated in various insects (see Table 2).
This tree was calculated by applying RAxML. The functionally characterized glucose/fructose transporters as well as trehalose transporters from insects are shaded in grey.
Figure 4.
Phylogenetic tree of the P. cochleariae sequences and homologous sequences derived from the tree of life calculated using RAxML.
Highlighted sequences regard to P. cochleariae and most similar sequences. Especially the green branch has to be subdivided into various subbranches, presenting all homologous sequences belonging to Metazoa. The tree significantly shows that the sugars (glucose) and trehalose transporters build up a huge tree in insects. Figure S5 shows the phylogeny of the selected organisms from the tree of life.
Figure 5.
The phylogenetic tree of the nine sequences derived from P. cochleariae belonging to the purple branch (see Figure 3 and 4, Figure S4) and homologous sequences derived from the whole tree of life, especially from Dendroctonus ponderosae (Dc) as well as from Tribolium castaneum (Tc), was calculated using RaxML.
Indicated in purple, it can be seen that the beetles’ sequences build up a separate branch.
Figure 6.
Distribution of mRNA levels of putative SLC2 transporters in various tissues of juvenile P. cochleariae by using quantitative real-time PCR.
Figure 7.
RNAi effects on transcript levels, amounts of defense secretions and chrysomelidial concentrations 10 days post RNAi induction in juvenile P. cochleariae.
A, Relative expression of chosen transporters in glandular tissue, normalized internally to Pcrpl6 and Pcrps3 and externally to gfp-control, n = 5. B, Amounts of secretions produced by individual larvae were weighted and normalized to the control treatments. n = 5. C, Secretions samples of RNAi induced larvae were analyzed using GC/MS; Amounts of chrysomelidial were normalized to internal standard (methylbenzoate), values were calculated against control. n = 5. Asterisks indicate level of significance (T-test, 2-tailed; p-value< = 0.05 = *, < = 0.01 = **, < = 0.001 = ***).
Table 4.
Differential expression analysis using DESeq package.
Figure 8.
Heatmap of the variance stabilization transformed data (vsd) of dsPcsut1-injected vs. dsgfp-injected samples.
Samples derived from glandular tissue. For this, the transcript counts of the sugar transporters of each sample after dsRNA-injection have been normalized to the effective library size and the variance over all samples has been stabilized by applying the DESeq package. For each heatmap, the 30 most abundant sugar transporter transcripts are shown. Dsgfp-injected samples are the same in each heatmap.
Figure 9.
Heatmap of the variance stabilization transformed data (vsd) of dsPcsut2-injected versus dsgfp-injected samples.
Samples derived from glandular tissue. For further explanation see Figure 8.
Figure 10.
Heatmap of the variance stabilization transformed data (vsd) of dsPcsut6-injected vs. dsgfp-injected samples.
Samples derived from glandular tissue. For further explanation see Figure 8.