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

根与它们所处的生物和非生物环境（根际）相互作用，并通过质和量调节根分泌的物质来作出反应。这些渗出物使用化学语言在生物和非生物成分之间进行交流。强大的DNA测序平台和鉴定化学成分的分析工具现在可用于分析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从根和根际获取和分离渗出物的能力的限制。现有的渗出液取样工具体积庞大，需要大量的土壤，并且显著地改变了土壤结构。这种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过程。该项目解决了开发下一代根分泌物采样工具所面临的关键设计、制造、集成和操作问题。该研究将为探索植物与根际微生物和宏观生物之间的化学交换提供急需的工具。根系分泌物是微生物组聚集和植物病虫害/病原体结果的关键驱动因素。根系分泌物的动态和环境响应特性说明了开发在现实环境中起作用的采样工具的重要性，而不是目前仅限于人工水培和聚合物嵌入系统的工具。将开发的采样器将通过实现在现实条件下生长的根上的微生物和渗出物的连续、空间分辨采样，显著影响根际微生物组研究的步伐。面对与气候变化相关的不确定性，这种增强的能力将满足社会对增加全球人口的农业生产力的需求，从而制定新的战略来影响农业生产力的提高。这项研究将通过在霍华德休斯医学研究所的本科生暑期实习项目中接待至少两名代表性不足的学生，为四名本科高年级学生提供研究机会，并为高中科学Bound项目提供实践研讨会，让学生参与技术转移的努力，同时强调植物微生物对农业和全球粮食安全的贡献，从而加强跨学科STEM劳动力的发展。该项目将采用先进的技术收集根际代谢物和微生物的高时空分辨率数据。这一目标将通过开发一个模块化工具包来实现，用于在土壤基质中生长的根的根际渗出物的局部采样。该工具包将包括：(i)单个站点渗出物采样器，它将作为更复杂的模块化采样系统的构建块；（ii）能够以高空间分辨率从关键位置提取渗出物的分布式渗出物采样器；（iii）棘状柔性渗出液采样器，在根-土界面处提供构象拟合，从而最大限度地在这一关键界面取样；（iv）平行梯度采样器从根部径向向外放置，提供对渗出物径向梯度的访问。这些采样器将独特地与微流控分选器相结合，以便从收集的渗出液中自动分离和分离微生物，同时分析微生物和可溶性渗出液。此外，这些采样器将集成微型张力计，这将允许在采样点监测当地土壤潜在条件，并触发自动开始采样。渲染这样一个“智能”设备将提高采样的时间分辨率。验证这些集成装置的效用将涉及安装它们，收集和分类样品，以及分析根际和基因指定的玉米根之间的相互作用，这些根是在有微生物和没有微生物的非生物条件下生长的。多学科研究汇集了微系统设计与构建、微生物组学和代谢组学等方面的专业知识，以解决提出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.