Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
Energy Processes and Materials Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
Appl Environ Microbiol. 2024 Oct 23;90(10):e0102624. doi: 10.1128/aem.01026-24. Epub 2024 Sep 9.
Interactions between plants and soil microbial communities that benefit plant growth and enhance nutrient acquisition are driven by the selective release of metabolites from plant roots, or root exudation. To investigate these plant-microbe interactions, we developed a photoaffinity probe based on sorgoleone (rgoleone iazirine lkyne for hotoffinity abeling, SoDA-PAL), a hydrophobic secondary metabolite and allelochemical produced in root exudates. We applied SoDA-PAL to the identification of sorgoleone-binding proteins in SO1, a potential plant growth-promoting microbe isolated from sorghum rhizosphere soil. Competitive photoaffinity labeling of whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins, including putative transporters, transcriptional regulators, and a subset of proteins with predicted enzymatic functions. We performed computational protein modeling and docking with sorgoleone to prioritize candidates for experimental validation and then confirmed binding of sorgoleone to four of these proteins : the α/β fold hydrolase SrgB (OH685_09420), a fumarylacetoacetase (OH685_02300), a lysophospholipase (OH685_14215), and an unannotated hypothetical protein (OH685_18625). Our application of this specialized sorgoleone-based probe coupled with structural bioinformatics streamlines the identification of microbial proteins involved in metabolite recognition, metabolism, and toxicity, widening our understanding of the range of cellular pathways that can be affected by a plant secondary metabolite.IMPORTANCEHere, we demonstrate that a photoaffinity-based chemical probe modeled after sorgoleone, an important secondary metabolite released by sorghum roots, can be used to identify microbial proteins that directly interact with sorgoleone. We applied this probe to the sorghum-associated bacterium and showed that probe labeling is dose-dependent and sensitive to competition with purified sorgoleone. Coupling the probe with proteomics and computational analysis facilitated the identification of putative sorgoleone binders, including a protein implicated in a conserved pathway essential for sorgoleone catabolism. We anticipate that discoveries seeded by this workflow will expand our understanding of the molecular mechanisms by which specific metabolites in root exudates shape the sorghum rhizosphere microbiome.
植物与土壤微生物群落之间的相互作用，有利于植物生长并增强养分获取，这是由植物根系选择性释放代谢物（即根分泌物）驱动的。为了研究这些植物-微生物相互作用，我们开发了一种基于 sorgoleone（rgoleone iazirine lkyne for hotoffinity abeling，SoDA-PAL）的光亲和探针，这是一种疏水次生代谢物和在根分泌物中产生的化感物质。我们将 SoDA-PAL 应用于从高粱根际土壤中分离出的潜在植物促生菌 SO1 中 sorgoleone 结合蛋白的鉴定。用 SoDA-PAL 对全细胞裂解物进行竞争性光亲和标记，鉴定出 137 个统计学上富集的蛋白质，包括推测的转运蛋白、转录调节因子和一组具有预测酶功能的蛋白质。我们进行了蛋白质的计算机建模和对接，与 sorgoleone 一起确定了用于实验验证的候选蛋白，并随后确认了 sorgoleone 与其中四种蛋白的结合：α/β折叠水解酶 SrgB（OH685_09420）、富马酰乙酰乙酸酶（OH685_02300）、溶血磷脂酶（OH685_14215）和一个未注释的假设蛋白（OH685_18625）。我们应用这种专门的基于 sorgoleone 的探针结合结构生物信息学，简化了参与代谢物识别、代谢和毒性的微生物蛋白的鉴定，扩大了我们对可被植物次生代谢物影响的细胞途径范围的理解。
在这里，我们证明了一种基于 sorgoleone 的光亲和探针可用于鉴定与高粱根释放的重要次生代谢物 sorgoleone 直接相互作用的微生物蛋白。我们将该探针应用于与高粱相关的细菌，并表明探针标记是剂量依赖性的，并且对与纯化的 sorgoleone 的竞争敏感。将探针与蛋白质组学和计算分析相结合，有助于鉴定推定的 sorgoleone 结合蛋白，包括一种与 sorgoleone 代谢至关重要的保守途径相关的蛋白。我们预计，该工作流程产生的发现将扩展我们对特定根分泌物代谢物塑造高粱根际微生物组的分子机制的理解。
