Aphid BCR4 Structure and Activity Uncover a New Defensin Peptide Superfamily

Abstract

Aphids (Hemiptera: Aphidoidea) are among the most detrimental insects for agricultural plants, and their management is a great challenge in agronomical research. A new class of proteins, called Bacteriocyte-specific Cysteine-Rich (BCR) peptides, provides an alternative to chemical insecticides for pest control. BCRs were initially identified in the pea aphid Acyrthosiphon pisum. They are small disulfide bond-rich proteins expressed exclusively in aphid bacteriocytes, the insect cells that host intracellular symbiotic bacteria. Here, we show that one of the A. pisum BCRs, BCR4, displays prominent insecticidal activity against the pea aphid, impairing insect survival and nymphal growth, providing evidence for its potential use as a new biopesticide. Our comparative genomics and phylogenetic analyses indicate that BCRs are restricted to the aphid lineage. The 3D structure of BCR4 reveals that this peptide belongs to an as-yet-unknown structural class of peptides and defines a new superfamily of defensins.

Keywords: 3D structure; aphid BCR; bioinsecticidal peptide; defensin peptide; symbiosis.

Figure 1

Multiple sequence alignment of Acyrthosiphon pisum BCRs. The number and the spacing between these cysteine residues allow to distinguish four BCR sub-families: BCR1-2-4-5, BCR3, BCR8 (containing all six cysteines) and BCR6 (eight cysteines). The signal peptides predicted by SignalP 6.0 server [23] of BCR sequences are highlighted in yellow. The fully conserved residues are framed and highlighted in red.

Figure 2

Chemical synthesis of BCR4. (A) Schematic representation of the BCR4 peptide chemical through native chemical ligation (NCL) of two peptide segments BCR4[1-20] in blue and BCR4[21-50] in red, followed by oxidative folding to form the three disulfide bridges. (B) RP-HPLC chromatogram and ESI-HRMS mass spectrum of the purified folded peptide.

Figure 3

(A) Survival curves of the aphid Acyrthosiphon pisum reared on artificial diets containing different concentrations of BCR4 peptide. Mean values of Lethal Time 50 (LT50), in days, are indicated above each curve. (B) Mass (mg) of 7-day-old pea aphid Acyrthosiphon pisum subjected to BCR4 treatment. Concentrations labeled with different letters are significantly different (p < 0.05). The surviving aphid sample sizes of BCR4 at 35 and 40 µM were too small to be statistically meaningful.

Figure 3

(A) Survival curves of the aphid Acyrthosiphon pisum reared on artificial diets containing different concentrations of BCR4 peptide. Mean values of Lethal Time 50 (LT50), in days, are indicated above each curve. (B) Mass (mg) of 7-day-old pea aphid Acyrthosiphon pisum subjected to BCR4 treatment. Concentrations labeled with different letters are significantly different (p < 0.05). The surviving aphid sample sizes of BCR4 at 35 and 40 µM were too small to be statistically meaningful.

Figure 4

Phylogenetic trees of BCR proteins. Maximum likelihood phylogenetic tree reconstruction was performed using PhyML [37] with an LG 4-rate class model. Branch-support values were calculated using the bootstrap method, with 1000 replicates. Poorly supported branches (<50%) were collapsed using TreeCollapseCL4 [38]. The sequences used for the phylogenetic analysis are listed in Supplementary Materials Table S2. Sequences labeled in red reflect those identified by the Shigenobu and Stern [15] study in the pea aphid (A. pisum genome V3.0). Abbreviations: Acra, Aphis craccivora; Agly, Aphis glycines; Agos, Aphis gossypii; Akon, Acyrthosiphon kondoi; Apis, Acyrthosiphon pisum; Cced, Cinara cedri; Dnox, Diuraphis noxia; Dvit. Daktulosphaira vitifoliae; Masc, Myzus ascalonicus; Mcer, Myzus cerasi; Mper, Myzus persicae; Msac, Melanaphis sacchari; Rmai, Rhopalosiphum maidis; Rpad, Rhopalosiphum padi; Save, Sitobion avenae; Sgra, Schizaphis graminum; Tcit, Toxoptera citricida.

Figure 5

Partitioning of BCR peptides in aphid taxonomic groups. Phylogeny of the 14 members of the aphid group whose genome is annotated and available in databases (among the 19 with sequenced genomes). Their distributions into subfamilies and tribes are indicated in red and black, respectively (adapted from Calevro et al. [4]). Colored dots near each species name indicate whether a member of each BCR subfamily is present in the genome.

Figure 6

(A) Primary structure of BCR4 peptide. (B) Three-dimensional structure of BCR4. (C) Topology diagram of BCR4. α-helix, β-sheet, and random coil are represented in red, cyan and black, respectively. Disulfide bonds are colored in yellow.

Figure 7

Disulfide connectivities in BCR4 peptide and in CSαβ and β-defensin groups. α-helices are indicated by rectangle, β-strands are represented by arrows, and disulfides in black lines.