PAPM EAGER: Microfluidic Root Exudate Sampler with High Spatio-Temporal Sampling Resolution

根与其所处的生物和非生物环境(根际)相互作用，并通过定性和定量地调节根系分泌的物质而对其作出反应。这些分泌物使用化学语言在生物和非生物成分之间进行交流。现在有强大的DNA测序平台和分析工具来识别化学成分，可以用来描述根际和根成分之间的这些相互作用。这些分析策略的综合应用受到获取和分离来自根和根际的分泌物的能力的限制。现有的渗出物采样工具体积庞大，需要大量的土壤，并显著改变土壤结构。这种对分泌物取样的困难减缓了将植物遗传决定因素与根际微生物组、基因组和代谢特征联系起来的进程。该项目解决了在开发下一代根系分泌物采样工具时面临的关键设计、制造、集成和操作问题。这项研究将开发急需的工具，以探索植物与根际微生物和宏观生物之间的化学交换。根分泌物是微生物组组装和植物-病虫害/病原菌结果的关键驱动因素。根分泌物的动态和环境响应性说明了开发在现实世界中起作用的采样工具的重要性，而不是目前仅限于主要用于人工水培和聚合物嵌入系统的采样工具。将开发的采样器将通过对在现实世界条件下生长的根上的微生物和分泌物进行连续的、空间分辨率的采样，从而显著影响根际微生物群的研究速度。这种增强的能力将满足在气候变化相关不确定性的情况下为不断增加的全球人口提高农业生产力的社会需要，从而制定新的战略来影响农业生产力的提高。这项研究将通过招待至少两名霍华德·休斯医学院本科生暑期实习项目的代表不足的学生，为四名本科生提供研究机会，并为高中理科项目提供动手研讨会，让学生参与技术转移努力，同时强调植物微生物对农业和全球粮食安全的贡献，来促进跨学科STEM劳动力的发展。该项目将阐述收集高时空分辨率的根际代谢物和微生物数据的先进技术。这一目标将通过开发一个模块工具包来实现，该工具包用于对种植在土壤基质中的根际渗出物进行局部采样。这个工具包将包括：(I)单部位渗出物采样器，它将作为更复杂的模块化采样系统的组成部分；(Ii)分布式渗出物采样器，能够以高空间分辨率从关键位置提取渗出物；(Iii)脊状柔性渗出物采样器，在根-土壤界面提供构象拟合，将在这一关键界面上最大限度地采样；(Iv)平行的梯度采样器，从根部径向向外放置，提供渗出物的径向梯度。这些采样器将独特地与微流控分类器相结合，以实现从收集的渗出物中自动分离和分离微生物，以便同时分析微生物和可溶性渗出物。此外，这些采样器将集成微型张力计，可以监测采样点的当地土壤潜力状况，并触发采样的自动开始。渲染这样的“智能”设备将提高采样的时间分辨率。验证这些集成设备的效用将包括安装它们、收集和分类样本，以及分析根际和基因特定玉米根之间的相互作用，这些玉米根生长在有和没有微生物的灵知生物条件下。这项多学科研究吸收了从微系统设计和建设、微生物组学和代谢组学在内的各种专业知识，以解决拟议的目标并实现具体目标。

Roots interact with and respond to the biotic and abiotic environment in which they live (the rhizosphere) by qualitatively and quantitatively modulating material exuded by roots. These exudates use the language of chemistry to communicate between and among the biotic and abiotic components. Powerful DNA sequencing platforms and analytical tools for identifying chemical components are now available for profiling these interactions among the rhizosphere and the root components. The integrated application of these analytical strategies is limited by the ability to access and isolate exudates from roots and the rhizosphere. Existing exudate sampling tools are bulky, require large amounts of soil, and significantly alter the soil structure. This difficulty of sampling exudates has slowed the process of linking plant genetic determinants to rhizosphere microbiome genomic and metabolic features. This project addresses key design, fabrication, integration, and operation problems faced in developing next-generation root exudate sampling tools. The research will develop greatly-needed tools for probing the chemical exchange between plants and the micro- and macro-organisms in the rhizosphere. The root exudates are critical drivers of microbiome assembly and plant-pest/pathogen outcomes. The dynamic and environmentally responsive nature of root exudates illustrates the importance of developing sampling tools that are functional in a real-world situation, rather than the current tools that are limited to use in primarily artificial hydroponic and polymer-embedded systems. The samplers that will be developed will significantly impact the pace of research on rhizosphere microbiome by enabling continuous, spatially-resolved sampling of the microbes and exudates on roots grown in real-world conditions. This enhanced capability will meet societal needs to increase agricultural productivity for an increasing global population in the face of the uncertainties associated with climate-change, and thus develop new strategies to impact gains in agricultural productivity. This research will enhance interdisciplinary STEM workforce development by hosting at least two under-represented students in an undergraduate Howard Hughes Medical Institute summer internship program, providing research opportunities to four undergraduate senior students, and providing hands-on workshops to a high school Science Bound program to engage students in tech-transfer endeavors, while highlighting plant-microbe contributions to agriculture and global food security. This project will elaborate advanced technology for gathering high spatiotemporal resolution data of metabolites and microbes in the rhizosphere. This objective will be met by developing a modular toolkit for the localized sampling of rhizosphere exudates from roots grown in soil matrices. This toolkit will consist of (i) a single site exudate sampler, which will serve as a building block of more complex modular sampling systems; (ii) distributed exudate samplers able to extract exudates from key locations with high spatial resolution; (iii) spine-like flexible exudate samplers, providing conformational fitting at the root-soil interface, which will maximize sampling at this crucial interface; and (iv) parallel gradient samplers positioned radially outward from a root, providing access to radial gradients of exudates. These samplers will be uniquely coupled with microfluidic sorters to enable automated separation and isolation of microbes from the collected exudates for simultaneous analysis of both the microbes and the soluble exudates. Furthermore, these samplers will integrate miniature tensiometers, which will allow monitoring of local soil potential condition at the sampling sites, and trigger the automatic start of sampling. Rendering such a "smart" device will improve temporal resolution of sampling. Validating the utility of these integrated devices will involve installing them, collecting and sorting samples, and analyzing the interactions between the rhizosphere and genetically specified maize roots, grown under gnotobiotic conditions with and without microbes. The multidisciplinary research has drawn expertise ranging from microsystems design and construction, microbiome and metabolomics, to address the proposed goal and deliver on the specific aims.