Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea’s defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.越来越多的研究表明，多细胞生物往往携带有能帮助宿主抵御天敌的微生物共生体，但目前对于绝大多数防御性共生体系的作用机制，我们仍知之甚少。螺旋体属细菌（Spiroplasma）是陆生节肢动物的广泛共生类群，其部分菌株能够保护多种果蝇属（Drosophila）昆虫免受寄生蜂和线虫的侵害。近期研究发现，一种由螺旋体编码的核糖体失活蛋白（ribosome-inactivating protein, RIP）——与肠出血性大肠埃希氏菌（enterohemorrhagic Escherichia coli）中的志贺样毒素具有同源性——在林地果蝇新拟果蝇（Drosophila neotestacea）抵御致命寄生线虫的过程中发挥了关键作用。本研究旨在验证RIP介导的防御作用是否具有普遍性，我们以黑腹果蝇（Drosophila melanogaster）和新拟果蝇为研究对象，探究螺旋体RIP是否同样在抵御寄生蜂的过程中发挥功能。我们的研究结果为RIP的核心防御作用提供了强有力的证据：幼虫内寄生蜂异足旋小蜂（Leptopilina heterotoma）和布氏旋小蜂（Leptopilina boulardi）的核糖体RNA（ribosomal RNA, rRNA）呈现出典型的RIP活性特征。在携带螺旋体的宿主体内，寄生蜂的核糖体在28S rRNA的α-栓菌素/蓖麻毒素环（α-sarcin/ricin loop）区域出现了大量位点特异性脱嘌呤现象，且该脱嘌呤过程发生在寄生蜂卵在果蝇幼虫体内孵化后不久。有趣的是，我们发现蛹外寄生蜂隆背赘须金小蜂（Pachycrepoideus vindemmiae）能够逃脱螺旋体的防御，其核糖体未出现高水平的脱嘌呤现象。此外，我们的实验显示，果蝇自身的核糖体几乎未出现被RIP靶向的迹象。最后，我们发现新拟果蝇的防御性螺旋体基因组编码了一套多样化的RIP基因家族，且各基因的丰度存在显著差异。本研究表明，防御性共生体对不同天敌的特异性可能由毒素组的演化所驱动，而毒素多样性或许在塑造宿主-共生体-天敌三者互作关系中发挥了重要作用。