Project description:Expression profiles of 22 reference Arabidopsis immunity mutants were collected using the Arabidopsis Pathoarray 464_001 (GPL3638) in order to build a network model predicting the Arabidopsis immune signaling network. Biological signaling processes may be mediated by complex networks in which network components and network sectors interact with each other in complex ways. Studies of complex networks benefit from approaches in which the roles of individual components are considered in the context of the network. The plant immune signaling network, which controls inducible responses to pathogen attack, is such a complex network. Here, we demonstrate that use of mRNA profiling to collect and analyze detailed descriptions of changes in the network state resulting from specific network perturbations is a powerful and economical strategy to elucidate regulatory relationships among the components of a complex signaling network. Specifically, we studied the Arabidopsis immune signaling network upon challenge with a strain of the bacterial pathogen Pseudomonas syringae expressing the effector protein AvrRpt2 (Pto DC3000 AvrRpt2). This bacterial strain feeds multiple inputs into the signaling network, allowing many parts of the network to be activated at once. mRNA profiles of 22 Arabidopsis immunity mutants and wild type were collected 6 hours after inoculation with the Pto DC3000 AvrRpt2 and used as detailed descriptions of the network states resulting from specific genetic perturbations. Regulatory relationships among the genes corresponding to the mutations were inferred by recursively applying a non-linear dimensionality reduction procedure to the mRNA profile data. The resulting network model accurately predicted 22 of 23 regulatory relationships reported in the literature, suggesting that predictions of novel regulatory relationships are also accurate. The network model revealed two striking features: (i) the components of the network are highly interconnected; (ii) negative regulatory relationships are common between signaling sectors. One case of a novel negative regulatory relationship, between the early microbe-associated molecular pattern (MAMP)-activated sector and the salicylic acid (SA)-mediated sector, was further validated. We propose that prevalent negative regulatory relationships among the signaling sectors make the plant immune signaling network a "sector-switching" network, which effectively balances two apparently conflicting demands, robustness against pathogenic perturbations and moderation of negative impacts of immune responses on plant fitness. Keywords: Responses of reference Arabidopsis immunity mutants to Pseudomonas syringae pv. tomato DC3000 carrying avrRpt2 This experiment consists of two (group00) or three (group01-04) biological replicates of each genotype (total 25 genotypes [3 are multiple mutants, which were removed for the network modeling, but were used for normalization]). For each genotype, two leaves per plant were pooled from three pots to prepare total RNA.

Project description:Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirement. First, the internal physiological state itself could feedback to regulated gene expression. Second, the expected physiological state could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis appears to be particularly important, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative importance of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in chemostat-grown cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmental changes presented a major selection force during yeast evolution. Keywords: Saccharomyces_Cerevisiae, Stress response, ADH1 deletion, time courses, chemostat Experiment 1: ADH1 deletion cells, which grow faster on glycerol rather then glucose medium, were grown in both glucose and glycerol medium. The cells expression on glycerol is compared to their expression glucose (5 microarrays). Experiment 2: Time course of ADH1 deletion cells upon growth on glycerol and on glucose (7 microarrays). Experiment 3: Response of steady state grown cells to environmental perturbations. Cells were grown in glucose or histidine limited chemostats and reached steady state. The cells were then subjected to perturbations such as DTT, heat shock, NaCl, Clotrimazole, H2O2, and addition of limiting factors (histidine and glucose, respectively). Overall we have examined 10 timecourses with 83 microarrays.

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