Single cell transcriptome analysis of Arabidopsis roots inoculated by Plasmodiophora brassicae indicates a role for brassinosteroids in clubroot formation.. Arabidopsis thaliana

The clubroot disease caused by the obligate biotrophic protist Plasmodiophora brassicae on host plants of the Brassicaceae family is characterized by enhanced cell division and cell expansion. Since a typical root section of an infected plant always includes different stages of the pathogen as well as uninfected cells, we were interested to investigate specific developmental stages of the pathogen and their effect on host transcriptional changes. We extended previous microarray studies on whole roots by using Laser Microdissection and Pressure Catapulting (LMPC) to isolate individual cells harboring defined developmental stages of the pathogen. In addition, we compared the central cylinder of infected to contol plants. We were especially interested to elucidate the stage-specific hormonal network. The upregulation of genes involved in auxin and cytokinin metabolism and signaling was confirmed. In addition, we found evidence that brassinosteroid (BR) synthesis and signal perception was in many cases upregulated in enlarged cells and the central cylinder. This was confirmed by qPCR and mutant analysis of the BR receptor mutant bri1-6, which exhibited less severe gall formation than the respective wild type. Our results identify novel hormone pathways involved in clubroot development. Using this method of single cell preparation combined with transcriptome analysis has been very useful to elucidate the regulation of gall growth by this obligate biotropic pathogen in a cell- and stage-specific manner. Overall design: Transcription profiling was performed in isolated Arabidopsis thaliana root cells harboring different developmental stages of Plasmodiophora brassicae at two time points after inoculation (dai) (14 and 21 dai), as well as in infected central cylinder tissue from roots at 14 dai (days after inoculation). Control samples were taken from uninfected roots. Host cells were dissected from paraffin embedded roots using Laser Microdissection and Pressure Catapulting (LMPC). 8 samples have been analyzed.

由专性生物营养原生生物芸苔根肿菌（Plasmodiophora brassicae）引发的十字花科（Brassicaceae）寄主植物根肿病，其典型特征为细胞分裂与细胞扩增异常增强。受侵染植株的典型根组织切片中，总会同时存在病原菌的不同发育阶段与未受侵染的寄主细胞，因此本研究旨在探究病原菌的特定发育阶段及其对寄主转录调控变化的影响。相较于此前针对全根开展的基因芯片研究，我们通过激光显微切割与压力弹射术（Laser Microdissection and Pressure Catapulting, LMPC）分离得到携带明确病原菌发育阶段的单个细胞，以此拓展了相关研究范畴。此外，我们还对比了受侵染植株与对照植株的根部中柱组织。本研究的重点之一是阐明阶段特异性的激素调控网络。本研究证实了生长素与细胞分裂素代谢及信号通路相关基因的上调表达。同时，我们发现油菜素甾醇（brassinosteroid, BR）的合成与信号感知过程，在多数膨大细胞及根部中柱组织中均呈现上调趋势。这一结论通过qPCR实验以及BR受体突变体bri1-6的遗传分析得到验证，该突变体的根肿症状相较于其野生型对照更为轻微。本研究明确了参与根肿病发育过程的新型激素调控通路。采用单细胞制备结合转录组分析的实验策略，能够以细胞类型与发育阶段特异性的方式，阐明该专性生物营养病原菌调控根肿生长的分子机制，应用成效显著。整体实验设计：我们对两个接种后时间点（14天接种后、21天接种后，dai）下，携带芸苔根肿菌不同发育阶段的拟南芥（Arabidopsis thaliana）根部分离细胞进行了转录谱分析；同时也对14天接种后受侵染的根部中柱组织开展了转录谱分析。对照样本取自未受侵染的拟南芥根部。寄主细胞通过激光显微切割与压力弹射术（LMPC）从石蜡包埋的根部组织中分离得到。本研究共完成8个样本的转录谱分析。