title: A ribosome-inactivating protein in a Drosophila defensive symbiont.
abstract: Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense.
authors.affiliation: Department of Biology, University of Victoria, Victoria, BC, Canada V8W 2Y2; phin.hamilton@gmail.com stevep@uvic.ca.
authors.forename: Phineas T
authors.initials: PT
authors.lastname: Hamilton
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8P 5C2;
authors.forename: Fangni
authors.initials: F
authors.lastname: Peng
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8P 5C2;
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Boulanger
authors.affiliation: Department of Biology, University of Victoria, Victoria, BC, Canada V8W 2Y2; Integrated Microbial Biodiversity Program, Canadian Institute for Advanced Research, Toronto, ON, Canada M5G 1Z8 phin.hamilton@gmail.com stevep@uvic.ca.
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Perlman
mesh_terms: D000818:Animals; D004330:Drosophila / Q000378:metabolism* / Q000382:microbiology*; D004722:Endoribonucleases / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification; D054788:Ribosome Inactivating Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012276:Ricin / Q000737:chemistry; D017423:Sequence Analysis, RNA; D013149:Spiroplasma / Q000502:physiology*; D013559:Symbiosis
chemical_list: D005656:Fungal Proteins; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; C014801:alpha-sarcin; D012276:Ricin; D004722:Endoribonucleases; D054788:Ribosome Inactivating Proteins
keywords: Shiga toxin; Spiroplasma; male-killing; nematode; symbiosis