Background The unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host's UPR to facilitate infection. Results We developed a high-throughput screening assay to identify proteins that interfere with UPR signaling in planta. A set of 35 genes from a library of secreted proteins from the maize pathogen Ustilago maydis were transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress in Nicotiana benthamiana plants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method. Conclusions We have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

中文翻译：

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一种高通量筛选方法，用于鉴定参与植物内质网未折叠蛋白反应的蛋白质。

背景未折叠蛋白反应（UPR）是真核生物中高度保守的过程，在适应和发育中起着至关重要的作用。虽然已经对该途径最普遍的成分进行了表征，但目前的工作重点是识别和表征在特定条件下发挥作用的其他 UPR 因素，例如发育变化、非生物线索和生物相互作用。考虑到蛋白质分泌在植物病原体相互作用中的核心作用，最近还关注了解病原体如何操纵宿主的 UPR 以促进感染。结果 我们开发了一种高通量筛选试验来鉴定干扰植物中 UPR 信号传导的蛋白质。来自玉米病原体 Ustilago maydis 的分泌蛋白库中的一组 35 个基因与报告构建体瞬时共表达，该构建体在本氏烟草植物中 UPR 胁迫下上调增强的黄色荧光蛋白 (eYFP) 表达。在 UPR 应力诱导后，将叶盘置于 96 孔板中并测量 eYFP 表达。这使我们能够鉴定出一种先前未描述的抑制植物 UPR 信号传导的真菌蛋白，然后使用经典但更费力的 qRT-PCR 方法证实了这一点。结论 我们已经建立了一种快速可靠的基于荧光的方法来鉴定植物中与 UPR 胁迫有关的异源表达蛋白。