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. 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.