DISSERTATION RESEARCH: The Genetic Basis of Cytoplasmic Incompatibility

共生细菌在自然界中极其广泛，近年来人们对它们操纵动物进化的方式的兴趣迅速上升。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，这些发现应有尽有。围绕着这些适应的进化之谜--细菌如何进化为这些复杂的动物过程做出贡献？参与这些相互作用的优势细菌之一，沃尔巴克氏菌，存在于全世界约40%的昆虫和其他无脊椎动物中。先前的研究表明，沃尔巴克氏菌改变动物繁殖的能力通过刺激新昆虫物种的进化、繁殖策略和后代生产来影响动物种群。尽管动物-沃尔巴克氏菌共生的这些进化结果，我们还不知道沃尔巴克氏菌基因进化到控制它们的动物宿主。这项研究首次对两个可能参与共生的沃尔巴克氏菌基因进行了深入分析。细胞质不亲和性(CI)是沃尔巴克氏菌寄主性别比的主要变化。CI极大地降低了未感染的雌性与感染沃尔巴克氏菌的雄性杂交的生育力，并使受感染的雌性在种群中具有明显的优势。初步工作发现了两个沃尔巴克氏菌基因，具有保守的蛋白质和DNA结合域，推测与CI有关。为了研究它们之间的相互作用，每个基因的标记版本将在不含沃尔巴克氏菌的黑腹果蝇中表达。然后将利用免疫荧光显微镜来确定这些基因在未感染的雄性和未感染的雌性杂交中的表达是否会导致发育中的胚胎明显的CI缺陷。下一步，在解剖幼虫睾丸后，将使用商用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。样品将被准备用于质谱分析，以确定哪些宿主蛋白可能结合。使用染色质免疫沉淀的额外样本，结合下一代测序，将确定宿主DNA的哪些区域为目标。最后，候选果蝇途径将通过基因突变和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.