Time course transcriptional profiles of the Arabidopsis thaliana response to in planta expression of the Pseudomonas syringae effector AvrRpt2 for all combinatorial genotypes of the alleles dde2-2, ein2-1, pad4-1, and sid2-2

Plant immunity protects plants from numerous potentially pathogenic microbes. The biological network that controls plant inducible immunity must function effectively even when network components are targeted and disabled by pathogen effectors. Network buffering could confer this resilience by allowing different parts of the network to compensate for loss of each other's functions. Networks rich in buffering rely on interactions within the network, but these mechanisms are difficult to study by simple genetic means. Through a network reconstitution strategy, where we disassemble and stepwise reassemble the plant immune network that mediates Effector-Triggered-Immunity, we have resolved systems-level regulatory mechanisms underlying the Arabidopsis transcriptome response to the ETI-eliciting Pseudomonas syringae effector AvrRpt2. To isolate ETI response from any other effects of the pathogen, AvrRpt2 was transgenically expressed in Arabidopsis cells under the control of the estradiol-inducible promoter system. The signaling sectors (subnetworks) interrogated for their roles and interactions in ETI through the network reconstitution were the jasmonate (JA), ethylene (ET), phytoalexin-deficient 4 (PAD4), and salicylate (SA) signaling sectors. Overall design: We profiled the Arabidopsis thaliana response to AvrRpt2 expression in the genetic backgrounds of the wild type (Col-0), as well as 4 single-gene mutants, 6 double-gene mutants, 4 triple-gene mutants, a quadruple-gene mutant for the null-mutant alleles dde2-2, ein2-1, pad4-1, and sid2-2, as well as a control genotype, rps2/rpm1, which has null mutations in the ETI receptors for AvrRpt2 (RPS2 is the major receptor while RPM1 is a minor receptor). The mutations dde2-2, ein2-1, pad4-1, and sid2-2 remove the JA, ET, PAD4, and SA signaling sectors, respectively. In these lines AvrRpt2 expression can be induced by estradiol (Ed) treatment (pER-AvrRpt2). We included another control line, in which an arbitrary reporter gene GUS (beta-glucuronidase) is induced upon estradiol treatment (pER-GUS) in the wild-type (Col-0) background. Profiles were collected through a time course 0, 1, 2, 3, 4, 5, and 6 hours after infiltration of 50µM estradiol in three biological replicates. Since most Arabidopsis cells undergo hypersensitive cell death (a typical ETI response) about 9 hours after estradiol-induction of AvrRpt2 expression, the time course of the transcriptome response was taken up to 6 hours after estradiol treatment. In addition, profiles from these 18 genotypes were collected through a time course 0, 2, and 5 hours after infiltration of water (mock) as a control treatment with three biological replicates. In each of these experiments, 3 leaves, leaf stages 7-9, of 31-32 days old plants were infiltrated with 50µM Ed or water and collected at the indicated timepoint. Thus each biological replicate contained the tissue of 4*3 = 12 leaves. These experiments resulted in 18 genotypes * 7 time points * 3 replicates = 378 libraries with Ed-treated tissues and 18 genotypes * 3 time points * 3 = 162 libraries with mock-treated tissues (total 540 libraries). These mRNA-Tag-Seq libraries were generated according to Rallapalli G, Kemen EM, Robert-Seilaniantz A, Segonzac C, Etherington GJ, Sohn KH, et al. EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics. 2014, BMC Genomics 15, 341. Internal barcodes—the first 4-8 bases of the sequence—were used to multiplex stranded mRNA Tag-Seq libraries for sets of 16 samples each (in most cases) into a single lane of an Illumina HiSeq 2000.

植物免疫可帮助植物抵御众多潜在致病微生物的侵染。控制植物诱导型免疫的生物网络，即便其组分会被病原菌效应因子靶向并失活，仍需维持正常功能。网络缓冲机制可通过允许网络不同组分相互代偿功能损失，赋予这种抗干扰韧性。富含缓冲机制的网络依赖内部交互作用，但这类机制难以通过简单遗传学手段开展研究。本研究通过网络重构策略——即拆解并逐步重组介导效应因子触发的免疫（Effector-Triggered Immunity, ETI）的植物免疫网络——解析了拟南芥转录组响应丁香假单胞菌（Pseudomonas syringae）效应因子AvrRpt2所引发的ETI的系统级调控机制。

为了将ETI响应与病原菌的其他效应分离开来，本研究在雌二醇诱导型启动子系统的调控下，在拟南芥细胞中转基因表达AvrRpt2。本研究通过网络重构策略，探究了茉莉酸（jasmonate, JA）、乙烯（ethylene, ET）、植保素缺陷4（phytoalexin-deficient 4, PAD4）以及水杨酸（salicylate, SA）信号模块（子网）在ETI中的作用与相互作用。

实验总体设计：
本研究针对野生型（Col-0）、4种单基因突变体、6种双基因突变体、4种三基因突变体、携带dde2-2、ein2-1、pad4-1与sid2-2四个敲除等位基因的四基因敲除突变体，以及对照基因型rps2/rpm1（该基因型在AvrRpt2的ETI受体RPS2（主受体）与RPM1（次受体）中均存在敲除突变）的遗传背景中，拟南芥对AvrRpt2表达的响应开展了转录组分析。dde2-2、ein2-1、pad4-1与sid2-2这四个突变分别会敲除JA、ET、PAD4与SA信号模块。在这些材料中，可通过雌二醇（Estradiol, Ed）处理诱导AvrRpt2的表达（对应载体为pER-AvrRpt2）。

本研究还设置了另一组对照材料：在野生型（Col-0）背景中，经雌二醇处理可诱导任意报告基因β-葡萄糖醛酸酶（beta-glucuronidase, GUS）表达的材料（对应载体为pER-GUS）。

本研究对50μM雌二醇浸润处理后的0、1、2、3、4、5、6小时共7个时间点的样本进行了转录组分析，每组设置3次生物学重复。由于在雌二醇诱导AvrRpt2表达约9小时后，多数拟南芥细胞会发生过敏性细胞死亡（典型ETI响应），因此本研究将转录组响应的采样时间范围限定在雌二醇处理后的6小时以内。

此外，本研究还对这18种基因型的材料在清水（mock）浸润处理后的0、2、5小时共3个时间点的样本进行了转录组分析，每组设置3次生物学重复作为对照。在每项实验中，均选取31~32日龄植株的7~9期叶片，每3片为一组，经50μM Ed或清水浸润后，在指定时间点收集样本。因此每份生物学重复样本包含4组×3片=12片叶片的组织。

本实验共获得378份Ed处理组的测序文库（18种基因型×7个时间点×3次重复）与162份清水处理组的测序文库（18种基因型×3个时间点×3次重复），总计540份测序文库。

本研究的mRNA-Tag-Seq文库构建参照Rallapalli等人2014年发表于《BMC Genomics》第15卷第341期的方法：EXPRSS：一种基于Illumina平台的高通量表达分析方法，用于揭示转录组动态变化。本研究利用序列前4~8个碱基作为内部条码，在多数情况下将每组16个样本的链特异性mRNA-Tag-Seq文库进行多重化测序，合并后加载至Illumina HiSeq 2000的单个测序泳道中。