Intraspecific variation and molecular regulation of a multipartite defensive symbiosis

共生微生物可以通过产生抗微生物化合物来保护动物宿主免受病原体的侵害，但在实验室和田间条件下形成防御化学的选择压力以及共生建立的分子因素仍然知之甚少。虽然我们第一个资助阶段的项目旨在解开在受控实验室条件下形成抗菌防御剂鸡尾酒的选择压力，但目前的提案旨在表征自然界中由共生菌产生的化学防御物的变异(定性和定量)，并鉴定与其昆虫宿主建立联系所必需的共生体基因。我们的目标是：(1)研究两个拉格里亚甲虫-伯克霍尔德里亚(Lagria)甲虫-伯克霍尔德里亚(Burkholderia)共生系统中微生物群落和防御化学在种内和种间的自然变异；(2)揭示共生体在植物致病和昆虫互惠之间切换的双重生活方式背后的分子因素。为此，我们将在野外采集两种田鼠的甲虫，并通过高通量测序来表征它们的微生物群落，同时通过高效液相-质谱仪/质谱仪分析共生体产生的防御化学，结果将为研究甲虫个体、种群和物种之间的防御化学和微生物群落的自然变异提供有价值的见解。对于目标2，我们将通过产生目标突变体并评估它们在甲虫中的定植能力来测试之前的转座子插入测序实验中的候选共生菌定植因子。由于甲虫共生体也可以机会性地感染甲虫的食物植物，我们将进行转座子-序列实验，以阐明感染该植物的候选定植因素。这些结果将深入了解防御性共生建立的分子因素，以及昆虫互惠和植物致病之间的生活方式转换。

Symbiotic microorganisms can protect animal hosts against pathogens by producing antimicrobial compounds, but the selective pressures shaping the defensive chemistry under lab and field conditions and the molecular factors underlying symbiosis establishment remain poorly understood. While our project of the first funding phase was targeted towards unraveling the selective pressures shaping antimicrobial defense cocktails under controlled laboratory conditions, the current proposal aims to characterize the variation in a symbiont-produced chemical defense in nature (both qualitatively and quantitatively), and to identify the symbiont genes that are necessary for establishing the association with its insect host. Using the Lagria beetle-Burkholderia symbiosis as an experimentally and genetically tractable system, we aim to (1) characterize the natural intra- and interspecific variation in microbial communities and defensive chemistry in two Lagria species and (2) unravel the molecular factors underlying the symbionts’ dual lifestyle, switching between plant pathogenicity and insect mutualism. To this end, we will collect beetles of two Lagria species in the field and characterize their microbial community profiles by high-throughput sequencing, while at the same time analyzing the symbiont-produced defensive chemistry via HPLC-MS/MS. The results will provide valuable insights into the natural variation in defensive chemistry and microbial community profiles between individuals, populations, and species of Lagria beetles. For objective 2, we will test candidate symbiont colonization factors from a previous transposon-insertion sequencing experiment by generating targeted mutants and assessing their colonization ability in the beetle. As the beetle symbionts can also opportunistically infect the beetle’s food plant, we will perform a transposon-seq experiment to elucidate candidate colonization factors for infecting the plant. The results will yield insights into the molecular factors underlying defensive symbiosis establishment as well as the life-style switch between insect mutualism and plant pathogenicity.