Fig 1.
Multiple alignments of newly identified LmDEFs with other insect CSαβ defensins.
Residues conserved in >50% of proteins are shaded. The numbers to the right refer to the position of the last residue of each line. Signal peptides are underlined. Bars indicate gaps to optimize the alignments. The six conserved cysteine residues involved in disulfide bridges are gray shaded. Possible activation peptide cleavage sites are marked with a triangle; enzymatic processing sites (e.g. -KR↓) to release the mature peptides are double underlined. The latter was predicted and/or determined based on various bioinformatical tools (PeptideCutter prediction, the cleaver package, and others) and similarity searches with mature insect defensins from GenBank and reported in the literature. The secondary elements (loop, α-helix, and β-sheet) of insect defensins are indicated as follow: β1, β2, β3, and β4 are regions for potential β turns; A1 and A2 are regions for potential β strands; H is a region for potential α-helix; C1, C2, and C3 are the potential disulfide linkages; S are the regions for bends; T are regions for hydrogen-bonded turns of CSαβ defensins (Following Dassanayake et al. [10]). Accession numbers for the selected insect defensins: AmelDEF, Apis mellifera: [GeneBank: NP_001011616.2]; NvitDEF, Nasonia vitripennis: [GeneBank: NP_001159944.1]; PhcapDEF, Pediculus humanus corporis: [GeneBank: XP_002432619.1]; PaptDEF, Pyrrhocoris apterus: [GeneBank: AGI17576.1]; RproDEF, Rhodnius prolixus [GeneBank: AAO74626.1]; DmelDEF, Drosophila melanogaster [GeneBank: AAO72500.1]; AgamDEF, Anopheles gambiae [GeneBank: ABB00983.1]; AcupDEF, Anomala cuprea [GeneBank: BAD77967.1]; OrhiDEF, Oryctes rhinoceros [GeneBank: BAA36401.1]; BmorDEF, Bombyx mori [GeneBank: BAG48202.1]; SexiDEF, Spodoptera exigua [GeneBank: AEW24427.1].
Table 1.
Identity and similarity values among the four putative locust defensins.
Table 2.
Analysis of some predicted domains and motifs of the putative LmDEFs.
Fig 2.
a, Representation of the homology-derived solution structure of LmDEFs (a1-a4). α-helix (red), strand or β-sheet (blue), and coil (gray); the images were optimized with the Protein Picture Generator v1.21. Secondary structure elements were predicted with ProFunc server at EMBL-EBI; key: Helix-Strand: purple; helices labelled H1, H2,…and strands by their sheets A, B,…Motifs: β beta, turn γ gamma turn, and beta hairpin (red arch). Disulfide bond (1–1, 2–2, 3–3). b, Electrostatic potential distribution on the peptide surfaces (b1-b4). Positive potential is shown in blue, and negative potential is in red; the contouring value of the potential is in kT/e; the bar at the bottom (-5 to +5). The images were drawn using PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA).
Fig 3.
Structural homology of LmDEFs to known antiprotozoal defensin.
a, Alignment of LmDEF1, -3, and -5 with the antiprotozoal Phlebotomus duboscqi defensin (PhdDEF: Boulanger et al. [11]). Percentage identity of LmDEFs to PhdDEF are shown at the end of each individual sequence; a denote the % identity with PhdDEF full peptide excluding the signal peptide, while b is denoting that % with the PhDEF mature active peptide. The amino acid residues are colored according to their physicochemical properties (red: small+ hydrophobic [incl. aromatic -Y]; blue: acidic; magenta: basic–H; green: hydroxyl + sulfhydryl + amine + G). The symbols under the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. The boxes indicate the six conserved cysteines; the conserved disulfide bridges are shown by # above these boxes (1–1; 2–2; 3–3). The active peptide cleavage site in PhdDEF is marked with a triangle; while the prodefensin is marked by a line. b, Three-dimensional in silico structure of “active” PhdDEF based on PDB entry 1icaA (defensin A of Protophormia terraenovae) as a template. The homology modeling was carried out with RaptorX; 40(100%) residues were modeled (p-value 9.45e-05). c, The secondary structure elements of PhdDEF predicted by ProFunc server for the purpose of comparison between it and LmDEFs (reported in Fig 2). It comprises 1 sheet, 1 beta hairpin, 1 beta bulge, 2 strands, 1 helix, 4 beta turns, and 3 disulfides.
Fig 4.
The relationship between LmDEFs with other insect defensin was inferred using the Neighbor-Joining method. Ixodes ricinus defensin 2 (tick) was used as the out-group. LmDEFs are marked with solid squares. Accession numbers are written next to insect species. Only representatives for defensin-2 were used. More closely related insect defensins within this group were removed to facilitate phylogenetic analysis and representation; therefore, only different lineages of the defensin2 family were shown. Bootstrap (1000 replicates) values are indicated for each root. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 26 amino acid sequences that were aligned by Kalign. All ambiguous positions were removed for each sequence pair. The evolutionary analysis was conducted with MEGA6.
Fig 5.
Tissue specificity and developmental expression patterns of LmDEFs.
a, spatiotemporal expression of LmDEFs in N. locustae infected fat body and salivary gland cells. b, same, but with M. anisopliae. M, DNA ladder; h, healthy insect; 1i, 3i, 5i,7i, 10i, and 15i the RNA of tissues collected on the 1st, 3rd, 5th, 7th, 10th, and 15th days after inoculation with pathogens; L.m. actin, locust actin gene. N.l., Nosema spores and M.a, Metarhizium hyphae as positive controls with RT-PCR. The locust actin gene was used as a control for the integrity of the cDNA templates. Amplification products were analyzed on agarose gels and visualized by UV illumination after ethidium bromide staining. All tissues were dissected from gregarious locusts.
Fig 6.
Real-time quantitative PCR profile of LmDEF transcripts following N. locustae infection in the fat body (FB) and salivary glands (SG) in relation to time.
Transcript accumulation values were normalized to the constitutively expressed β-actin gene and expressed as a function of the reference condition according to the 2−ΔΔCT method. The bars indicate the relative changes in RNA levels compared with the average expression of each gene under non-infected control conditions. The error bars indicate the standard errors of the means. The results are the means of at least three independent experiments. The statistical annotations, including the letters indicating significance, among treatments are given in S4 Fig.
Fig 7.
The expression pattern of LmDEFs in the fat body and salivary glands in locusts infected with Nosema on the 1st, 3rd, 10th, and 15th days post-infection in comparison to control at each time.
PCR products, using qRT-PCR primers (S2 Table), were ran on 1.2% agarose gels and visualized by ethidium bromide staining. M, DNA-ladder; numbers preceding characters are gene numbers in healthy locust (h), or infected locust (i); A-actin gene.