A Phase-Separated Nuclear GBPL Circuit Controls Immunity in Plants

Liquid-liquid phase separation (LLPS) has emerged as a central paradigm for understanding how membrane-less organelles compartmentalize diverse cellular activities in eukaryotes. Here, we identified a new superfamily of plant Guanylate Binding Protein-Like GTPases (GBPLs) that assemble LLPS-driven condensates within the nucleus to protect against infection and autoimmunity. In Arabidopsis thaliana, two family members - GBPL1 and GBPL3 - undergo phase transition behavior to control transcriptional responses as part of an allosteric switch triggered by exposure to biotic stress. GBPL1, a pseudoGTPase, sequesters catalytically-active GBPL3 under basal conditions but is displaced by LLPS when GBPL3 enters the nucleus following immune cues to drive formation of unique membrane-less organelles termed GDACs (GBPL Defense-Activated Condensates) that we visualized by in situ cryo-electron tomography. Within these mesoscale GDAC structures, native GBPL3 directly bound defense gene promoters and recruited specific transcriptional coactivators of the Mediator complex plus RNA Pol II machinery to massively reprogram host gene expression for disease resistance. Together, our study identifies a new GBPL circuit that reinforces the biological importance of phase-separated condensates, in this case, as indispensable players in plant defense. Overall design: RNA-seq analysis of WT, gbpl1-1, gbpl3-5, and GBPL3-OX arabidopsis seedlings. Four genotypes each contain 2 biological replicates resulting in a total of 8 samples.

液-液相分离（Liquid-liquid phase separation, LLPS）已成为阐释真核生物中无膜细胞器（membrane-less organelles）如何分区调控多种细胞活动的核心范式。本研究鉴定得到一类全新的植物鸟苷酸结合蛋白样GTP酶（Guanylate Binding Protein-Like GTPases, GBPLs）超家族，该家族蛋白可在细胞核内组装由LLPS驱动的凝聚体，以抵御病原体侵染与自身免疫反应。
在拟南芥（Arabidopsis thaliana）中，该家族的两个成员GBPL1与GBPL3会发生相变行为，作为生物胁迫触发的变构开关的一部分来调控转录应答。作为假GTP酶（pseudoGTPase）的GBPL1在基础条件下会螯合具有催化活性的GBPL3，但当免疫信号触发后GBPL3进入细胞核时，LLPS会使GBPL1解离，进而驱动独特的无膜细胞器GDACs（GBPL Defense-Activated Condensates，GBPL防御激活凝聚体）的形成，本研究通过原位冷冻电子断层扫描技术（in situ cryo-electron tomography）对该结构进行了可视化观测。
在这些介观尺度的GDAC结构中，天然状态的GBPL3可直接结合防御基因启动子，并招募中介体复合物（Mediator complex）与RNA聚合酶II（RNA Pol II）系统的特异性转录共激活因子，从而大规模重编程宿主基因表达以增强抗病性。
综上，本研究鉴定出一条全新的GBPL信号通路，进一步证实了相分离凝聚体的生物学重要性——在本研究场景中，这类凝聚体是植物防御反应中不可或缺的关键参与者。
实验设计：对野生型（Wild Type, WT）、gbpl1-1、gbpl3-5以及GBPL3过表达（GBPL3-OX）的拟南芥幼苗进行RNA测序（RNA-seq）分析。共设置4种基因型，每种基因型包含2个生物学重复，总计8个样本。