Intraspecific variation and molecular regulation of a multipartite defensive symbiosis

共生微生物可以通过产生抗菌化合物来保护动物宿主免受病原体的侵害，但在实验室和野外条件下形成防御化学的选择压力以及共生建立背后的分子因素仍然知之甚少。虽然我们的第一个资助阶段的项目旨在揭示在受控的实验室条件下形成抗菌防御鸡尾酒的选择压力，但目前的提案旨在表征自然界中共生体产生的化学防御的变化（定性和定量），并确定与昆虫宿主建立关联所必需的共生体基因。将Lagria甲虫- burkholderia共生作为一个实验和遗传上可处理的系统，我们的目标是：(1)表征两个Lagria物种的微生物群落和防御化学的自然种内和种间变化；(2)揭示共生生物在植物致病性和昆虫共生之间转换的双重生活方式的分子因素。为此，我们将在野外采集两种瓢虫，并通过高通量测序对其微生物群落特征进行表征，同时通过HPLC-MS/MS分析共生体产生的防御化学成分。研究结果将为了解瓢虫个体、种群和物种之间的防御化学和微生物群落特征的自然变化提供有价值的见解。对于目标2，我们将通过生成靶向突变体并评估其在甲虫中的定殖能力，来测试先前转座子插入测序实验中获得的候选共生定殖因子。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来阐明感染植物的候选定殖因子。研究结果将有助于深入了解防御性共生关系建立的分子因素，以及昆虫共生与植物致病性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.