Research ArticleECOLOGY

The genetic architecture of a host shift: An adaptive walk protected an aphid and its endosymbiont from plant chemical defenses

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Science Advances  06 May 2020:
Vol. 6, no. 19, eaba1070
DOI: 10.1126/sciadv.aba1070
  • Fig. 1 A large segmental duplication in M. p. nicotianae led to the amplification and overexpression of multiple genes.

    (A) Gene expression heat map showing genes consistently DE in 36 comparisons of M. p. nicotianae with M. persicae s.s. [6 M. p. nicotianae clones (Mn1 to Mn6) compared to 6 M. persicae s.s. clones (Mp1 to Mp6) are shown]; cell color indicates log2 fold change. Four of these genes localize to scaffold 16 [indicated by the blue lines linking (A) and (B)]. (B) Sliding window analysis of CNV between M. p. nicotianae and M. persicae across scaffold 16. In this representative plot, clone Mn3 was compared with clone Mp2; see data file S2 for the results of all 36 comparisons. (C) The region of elevated copy number includes some or all of the coding sequence of the genes, RPS11, Src42A (SRC), T-type calcium channel (CaCh), CYP6CY23 (CY23), CYP6CY4 (CY4), CYP6CY3 (CY3), pseudogene of unknown function (Un_Pro) and ADAMTS9, and has been tandemly duplicated as a series of direct repeats. (D) Precise determination of copy number of the region amplified by qPCR in all M. p. nicotianae clones compared to M. persicae clone Mp3. Error bars indicate 95% confidence limits (n = 4). (E) Localization of CYP6CY3 detected with tyramide-Cy3 (red, arrowheads) on metaphase chromosomes of Mp1 and Mn6 counterstained with 4′,6-diamidino-2-phenylindole (blue) by means of TSA-FISH. X, sex chromosome. Scale bar, 5 μm.

  • Fig. 2 An RPS11/ADAMTS9 chimeric gene is expressed in M. p. nicotianae.

    (A) The segmental duplication observed in M. p. nicotianae is predicted to create a chimeric gene fusing the promoter and first two exons of RPS11 with the last 23 exons of ADAMTS9. This would result in the loss of the ADAMTS9 signal peptide (SP) and much of the prodomain (Pro). Disintegrin-like domain (Dis), cysteine-rich domain (CR), thrombospondin type 1 repeat (TSR). (B) De novo assembly of RNA-seq data from all M. p. nicotianae clones assembled a chimeric contig (bottom sequence) comprising a fusion of the RPS11/ADAMTS9 gene that is not present in M. persicae (top sequence). Sequence from RPS11 is boxed in blue and from ADAMTS9 is boxed in red. Wt, wild-type. (C) Mapping M. p. nicotianae RNA-seq reads to the reference ADAMTS9 gene reveals chimeric reads, and these represent >90% of the reads mapping to this region (E). (D) Reverse transcription PCR verification that the chimeric gene is expressed only in M. p. nicotianae as predicted.

  • Fig. 3 Molecular, bioinformatic, and functional characterization of candidate genes within the segmental duplication.

    (A and B) qPCR analysis of copy number (A) and mRNA expression (B) of genes within the segmental duplication. In each case, data are shown as fold change between the six M. p. nicotianae clones and M. persicae clone Mp3. Error bars indicate 95% confidence limits (n = 4). (C) Nonsense mutations are observed in exon 1 of certain copies of Src42A (light blue, left), and in the T-type calcium channel gene (green, right) caused by an internal duplication (duplicated region indicated by white boxes). In each case, the wild-type sequence is shown on top, and those carrying mutations are shown underneath. (D) Metabolism of nicotine by recombinant CYP6CY3, CYP6CY4, and CYP6CY23. Recovery of the nicotine metabolite cotinine over time in the presence (blue lines) or absence of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate; black lines) is shown. Error bars display SD (n = 3). (E) Sensitivity of transgenic flies expressing ADAMTS9 or RPS11/ADAMTS9 to nicotine compared to a fly line of the same genetic background without a transgene (control). Sensitivity was measured by calculating lethal concentration 50 (LC50) values for each line. Error bars indicate 95% confidence limits (n = 5).

  • Fig. 4 Overexpression of CYP6CY3 in the bacteriocyte and the gut of M. p. nicotianae protects this aphid subspecies and its obligate endosymbiont Buchnera aphidicola from nicotine.

    (A to D) Immunohistochemical localization of CYP6CY3 (green signal) in the bacteriocytes of M. persicae [(A) and (B)] and M. p. nicotianae (C) and (D). Nucleic acids stained with To-PRO 3-Iodide (red signal). (E) Expression of CYP6CY3 and CYP6CY4 in the carcass (CAR), gut, bacteriocyte (BAC), and head of the M. persicae clone Mp1 and M. p. nicotianae clone Mn6 as determined by qPCR. Error bars indicate 95% confidence limits (n = 4). (F) Titer of B. aphidicola in two clones of M. persicae (blue bars) and two clones of M. p. nicotianae (red bars) after feeding on a diet with [2 or 8 parts per million (ppm)] or without nicotine (control). Error bars indicate 95% confidence limits (n = 4). Significant differences (P < 0.05) in expression between the different treatments and the control are denoted using letters above bars as determined by one-way analysis of variance (ANOVA) with post hoc Tukey honestly significant difference.

  • Fig. 5 Schematic of the evolution of the molecular innovations in M. p. nicotianae that provide protection from toxic nicotine.

    Nicotine (1) is taken up via the gut during feeding and causes toxicity to M. persicae via its action at the nicotinic acetylcholine receptor (nAChR) (2) and on the obligate endosymbiont B. aphidicola in aphid bacteriocytes (3). In M. p. nicotianae, chromosomal rearrangements result in the increased expression of CYP6CY3 and CYP6CY4, which detoxify nicotine. In the case of CYP6CY3, expression is significantly enhanced in bacteriocytes (4) and reprogrammed to include the aphid gut (5) providing two lines of defense that protect M. p. nicotianae and its symbiont from this secondary metabolite. CNS, central nervous system.

  • Fig. 6 Further amplification of CYP6CY3 is associated with the insertion of transposable elements that are highly expressed in the aphid gut.

    (A) Copy number of CYP6CY3, CYP6CY4, and CYP6CY23 in M. p. nicotianae clones Mn1–6 compared to M. persicae clone Mp3 as determined by qPCR. Error bars indicate 95% confidence intervals (n = 4). (B) Schematic of the CYP6CY3 amplicon obtained from BAC sequencing. The insertion sites of a Tc1/Mariner (Tc1/Mar), hAT, and TTAA3 transposable element are illustrated. (C) Colocalization of the BAC clone bearing a copy of CYP6CY3 on scaffold 15, with scaffold 16 on metaphase chromosomes of Mp1 and Mn6. The scaffold 16 probe was detected with tyramide–fluorescein isothiocyanate (green, arrows), and the BAC was directly labeled with Cy3 (red, arrowheads). Note that only one scaffold 16 locus was detected; this was due to a limitation of TSA-FISH to provide balanced signals, rather than its absence in the homologous chromosome. X, sex chromosome. Scale bar, 5 μm. (D) Additional long single-molecule sequencing identified copies of CYP6CY3 at the scaffold 16 locus in combination with the hAT element or in combination with the TTAA3, HAT, and Mutator-like (MULE) elements. (E) Features of the four transposons found in association with CYP6CY3. ORF, open reading frame; aa, amino acid. (F) Expression of hAT, TTAA3, Tc1/Mariner, and MULE in carcass, gut, or head tissue of M. persicae clone Mp1 and M. p. nicotianae clone Mn6. Error bars indicate 95% confidence limits (n = 4).

Supplementary Materials

  • Supplementary Materials

    The genetic architecture of a host shift: An adaptive walk protected an aphid and its endosymbiont from plant chemical defenses

    Kumar Saurabh Singh, Bartlomiej J. Troczka, Ana Duarte, Vasileia Balabanidou, Nasser Trissi, Leonela Z. Carabajal Paladino, Petr Nguyen, Christoph T. Zimmer, Kyriaki M. Papapostolou, Emma Randall, Bettina Lueke, Frantisek Marec, Emanuele Mazzoni, Martin S. Williamson, Alex Hayward, Ralf Nauen, John Vontas, Chris Bass

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    • Figs. S1 to S7
    • Tables S1 to S3
    • Legends for data files S1 and S2
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