Single-cell profiling of complex plant responses to Pseudomonas syringae infection [bulk RNA-seq]

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We exposed Arabidopsis to Pseudomonas syringae or mock treatment and profiled the transcriptomes of 11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from Pseudomonas and mock inoculated leaves identified five distinct pathogen responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection revealed a continuum of disease progression from an immune to susceptible state. Confocal imaging of promoter reporter lines for pathogen responsive cell clusters visualized immune activated cell clusters surrounding substomatal cavities colonized by bacteria, suggesting immune clusters are sites of early pathogen invasion. Susceptibility cell clusters exhibited more general localization and were highly induced at later stages of infection. Overall, our work uncovers cellular heterogeneity within an infected leaf and provides unique insight into plant differential response to infection at a single-cell level. Overall design: 4-week old Arabidopsis thaliana leaves syringe-infiltrated with Pseudomonas syringae pv. tomato DC3000 or a mock treatment for 24 hours. After treatment, cells (Protoplasts) were isolated from leaf tissue using a tape-sandwich method to enrich for the mesophyll layer. In parallel, whole leaf tissue was also isolated. RNA was then extracted from protoplast preparations and whole leaf tissue, and processed for RNA sequencing. Three biological replicates for mock- and DC3000-treated whole leaves were prepared, and one replicate of protoplasts derived from mock- and DC3000-treated leaves were also processed.植物对病原菌侵染的应答在单叶内存在异质性，但目前该异质性尚未得到充分解析。我们将拟南芥（Arabidopsis thaliana）分别暴露于丁香假单胞菌（Pseudomonas syringae）侵染与Mock对照处理，采用单细胞RNA测序（single-cell RNA sequencing）对11000个单个细胞的转录组进行了谱图分析。对丁香假单胞菌接种与Mock接种叶片的细胞群进行整合分析，共鉴定出5个独特的病原菌应答细胞簇，其转录应答范围覆盖从免疫状态到感病状态的连续谱系。病原菌侵染过程中的拟时间（pseudotime）分析揭示了病害从免疫状态向感病状态进展的连续动态过程。对病原菌应答细胞簇的启动子报告株系（promoter reporter lines）进行共聚焦成像（confocal imaging），观察到免疫激活的细胞簇环绕在被细菌定殖的气孔下腔（substomatal cavities）周围，表明免疫细胞簇是病原菌早期侵染的位点。感病细胞簇则呈现更为广泛的定位分布，并在侵染后期被显著诱导激活。综上，本研究揭示了侵染叶片内的细胞异质性，并为解析植物在单细胞水平上的差异化侵染应答提供了全新视角。 实验设计：将4周龄的拟南芥（Arabidopsis thaliana）叶片通过针筒浸润接种丁香假单胞菌番茄致病变种（Pseudomonas syringae pv. tomato）DC3000，或进行Mock对照处理，处理时长为24小时。处理结束后，采用胶带三明治法从叶片组织中分离细胞（原生质体，Protoplasts），以富集叶肉层细胞。同时，我们还收集了完整叶片组织。随后从原生质体制备物与完整叶片组织中提取RNA，并进行RNA测序（RNA sequencing）建库测序。Mock处理与DC3000处理的完整叶片样本均设置3个生物学重复（biological replicates），同时对Mock处理与DC3000处理叶片来源的原生质体样本各设置1个生物学重复并进行测序分析。