ABCB transporters in a leaf beetle respond to sequestered plant toxins

Abstract

Phytophagous insects can tolerate and detoxify toxic compounds present in their host plants and have evolved intricate adaptations to this end. Some insects even sequester the toxins for their defence. This necessitates specific mechanisms, especially carrier proteins that regulate uptake and transport to specific storage sites or protect sensitive tissues from noxious compounds. We identified three ATP-binding cassette subfamily B (ABCB) transporters from the transcriptome of the cardenolide-sequestering leaf beetle Chrysochus auratus and analysed their functional role in the sequestration process. These were heterologously expressed and tested for their ability to interact with various potential substrates: verapamil (standard ABCB substrate), the cardenolides digoxin (commonly used), cymarin (present in the species's host plant) and calotropin (present in the ancestral host plants). Verapamil stimulated all three ABCBs and each was activated by at least one cardenolide, however, they differed as to which they were activated by. While the expression of the most versatile transporter fits with a protective role in the blood-brain barrier, the one specific for cymarin shows an extreme abundance in the elytra, coinciding with the location of the defensive glands. Our data thus suggest a key role of ABCBs in the transport network needed for cardenolide sequestration.

Keywords: ABCB transporters; Chrysochus; cardenolides; multidrug resistance; sequestration; tissue-specific expression.

Figure 1.

Maximum-likelihood tree (LG + Γ + I) of ABC transporter amino acid sequences (cf. electronic supplementary material, table S1); Chrysochus sequences marked with stars (colours according to figure 2); numbers indicate bootstrap proportions in 1000 replicates. (Online version in colour.)

Figure 2.

Three-dimensional structural superposition of Ca_ABCB proteins (a). Horizontal sections through the Ca_ABCB transporters' periplasmic area affording a view of the potentially important residues discussed in the text for Ca_ABCB1 (b), Ca_ABCB2 (c), Ca_ABCB3 (d). Amino acids identical to the human_ABCB1 are shown in orange, yellow indicates dissimilar residues. Val982 highlighted in red is essential for the transport of convallatoxin [31]. (Online version in colour.)

Figure 3.

Ca_ABCB mediated ATP hydrolysis (nmol pi mg⁻¹ protein min⁻¹) in the presence of (a) digoxin, (b) calotropin or (c) cymarin (host plant of C. auratus). Mean ± s.d. and a dose–response fit of the data are shown as solid lines, broken lines indicate data where no dose–response curves could be fitted. (d) For statistical comparisons, exponential curves were fitted to all data by nonlinear mixed effect models and the slopes of the resulting curves compared. Different letters indicate statistically significant differences (p < 0.05) between the slopes. (Online version in colour.)

Figure 4.

Quantification of Ca_ABCB transporters expression levels in eight adult tissues of C. auratus. Bars represent the copies of Ca_ABCB transcripts per µg RNA. All data are means of three biological replicates ±s.d. Different letters indicate statistically significant differences (p < 0.05) between expression levels of each transporter across tissues (ANOVA, with post hoc Tukey tests). (Online version in colour.)