Mega6

For each seed sequence, an initial alignment was performed in Uniprot using the sequences in the 50% sequence identity cluster (Clusterref_50) with the “Align” option. The raw multiple sequence alignment from Uniprot was loaded into Jalview (2.8.1) (Waterhouse et al., 2009 (link)) for editing, visualization and analysis. Masking was performed to remove ambiguous regions of the alignment, and the initial tree was generated with the Neighbor-joining method using the BLOSUM 62 substitution matrix. Additional sequences were added to the alignment using a combination of Blast searches (Altschul et al., 1990 (link)) of Uniprot, and joining of additional 50% sequence identity (ID) clusters to provide further evolutionary depth when necessary. The generated tree in Newick format was loaded into the evolutionary tree builder MEGA6 (Tamura et al., 2013 (link)) and the root chosen using an outgroup from the accepted species tree (Murphy and Eizirik, 2009 ). The figure for the final tree was created in MEGA6 and further manipulated in Illustrator 6 (Adobe Systems). The number of Cys residues in the CSD motif was mapped onto the phylograms manually.

A sequence alignment of the GH27 catalytic domains of 71 protein sequences (49 characterised and 22 uncharacterised proteins, identified by BLAST searching of GH27 enzymes with known activity and/or structure) was performed using the online Clustal Omega server [31 (link)]. Table B in S1 File provides details of the uncharacterised proteins included in the analysis. Full sequences of the catalytic domains of these proteins were used for an initial sequence alignment, which revealed the presence in CpArap27 of a number of loop insertions in conserved positions, comprising between 6 and 34 amino acids. For subsequent sequence alignments which were used to generate a phylogenetic tree, these loops were removed from the sequences, as in previous phylogenetic analyses of the family [21 (link)]. The software PhyML was used to produce a phylogenetic tree of the alignment results using the Blosum62 model of amino acid substitution, with bootstrapping of results (100 replicates) [32 (link)]. The software MEGA6 was used to view the tree [33 (link)] and Adobe Illustrator CS5 was used to produce the final figure.

To classify the position of D. citri ABCs within ABC classes (A-G), the amino acid sequences of NBDs of D. citri ABC transporters were used to resolve their phylogenetic relationships. When a protein had two NBDs, the N-terminal NBD was used. To analyse the evolutionary placement of ABC transporters in D. citri, comparison analyses among each subfamily of ABC transporters from D. citri, D. melanogaster, B. mori, T. castaneum, B. tabaci, and T. urticae were conducted, and the full-length protein sequences were subjected to phylogenetic analyses (Supplementary data). Sequences were aligned by the ClustalW alignment algorithm^{82 (link)}, and MEGA6 was used to construct the neighbor-joining trees with the Poisson model and 1,000 bootstrap replicates^{83 (link)}, the dendrograms were viewed in FigTree and edited in Adobe PhotoShop CS6.