DISSERTATION RESEARCH: The Genetic Basis of Cytoplasmic Incompatibility

共生细菌在自然界中非常普遍，近年来，人们对它们操纵动物进化的方式的兴趣迅速上升。发现的范围从影响营养、免疫和繁殖的细菌到导致一个物种分裂为两个物种的细菌。围绕着这些适应性的进化之谜--细菌是如何进化以促进这些复杂的动物过程的？ 参与这些相互作用的主要细菌之一，Wolbachia pipientis，在全世界约40%的昆虫物种和其他无脊椎动物中存在。先前的研究表明，沃尔巴克氏体改变动物繁殖的能力通过刺激新昆虫物种、繁殖策略和后代生产的进化来影响动物种群。尽管动物-沃尔巴克氏体共生体的这些进化结果，我们还不知道沃尔巴克氏体基因进化控制它们的动物宿主。本研究首次对Wolbachia的两个可能参与共生的基因进行了深入的分析，细胞质不相容性（CI）是Wolbachia改变宿主性比的主要机制。CI大大降低生育率的未感染的女性在交叉沃尔巴克氏体感染的男性，并使感染的女性在人口中的明显优势。初步工作发现两个Wolbachia基因，具有保守的蛋白质和DNA结合结构域，参与CI。为了研究它们的相互作用，每个基因的标记版本将在无沃尔巴克氏体的果蝇中表达。然后将利用免疫荧光显微镜来确定这些基因在未感染的男性与未感染的女性杂交的表达是否会导致发育中的胚胎出现明显的CI缺陷。接下来，解剖幼虫睾丸后，使用市售抗体对标记蛋白进行免疫沉淀，以分离任何结合的宿主因子。将制备用于质谱分析的样品，以确定可能结合的宿主蛋白。使用染色质免疫沉淀的额外样本，加上下一代测序，将确定宿主DNA的哪些区域是靶向的。最后，候选果蝇通路将通过遗传突变体和RNAi敲除的组合来验证它们在CI中的作用。

Symbiotic bacteria are extremely widespread in nature and interest in the way that they manipulate animal evolution has risen rapidly in recent years. Discoveries have ranged from bacteria that affect nutrition, immunity, and reproduction to bacteria that cause the splitting of one species into two. An evolutionary enigma surrounds these adaptations - how do bacteria evolve to contribute to these complex animal processes? One of the preeminent bacteria involved in these interactions, Wolbachia pipientis, occurs worldwide in ~40% of all insect species and other invertebrates. Previous work has shown that Wolbachia's ability to alter animal reproduction affects animal populations by spurring the evolution of new insect species, reproductive strategies, and offspring production. Despite these evolutionary outcomes of animal-Wolbachia symbioses, we do not yet know the Wolbachia genes that evolved to control their animal hosts. This study begins the first in-depth analysis of two Wolbachia genes that are likely involved in the symbiosis.Cytoplasmic incompatibility (CI) is Wolbachia's main alteration of host sex ratios. CI drastically decreases fertility of uninfected females in crosses to Wolbachia-infected males and gives infected females a distinct advantage within the population. Preliminary work discovered two Wolbachia genes, with conserved protein and DNA binding domains, putatively involved in CI. To study their interactions, tagged versions of each gene will be expressed in Wolbachia-free Drosophila melanogaster. Immunofluorescent microscopy will then be utilized to determine whether expression of these genes in uninfected males crossed with uninfected females causes explicit CI defects in developing embryos. Next, after dissecting the larval testes, immunoprecipitation of the tagged proteins using commercially available antibodies will be used to isolate any bound host factors. Samples will be prepared for mass spectrometry to determine what host proteins may be bound. Additional samples using chromatin immunoprecipitation, coupled with next generation sequencing, will determine what regions of host DNA are targeted. Finally, candidate Drosophila pathways will be verified for their role in CI through a combination of genetic mutants and RNAi knockdown.