Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional response of Arabidopsis thaliana to exogenous application of pipecolic acid

ABSTRACT: We have previously shown that the resistance increase observed after biological systemic acquired resistance (SAR) induction in plants can be mimicked by exogenous plant treatment with (pipecolic acid) Pip (Návarová et al., 2012; Bernsdorff et al., 2016). In order to investigate whether elevations of Pip are sufficient for a SAR-like transcriptional reprogramming in leaf tissue, we supplied individual Arabidopsis Col-0 and fmo1 mutant plants with doses of 10 µmol Pip and determined the transcriptional response in leaves one day later on the whole genome level by RNA-sequencing analyses in relation to untreated plants. Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil, vermiculite and sand (8:1:1) in a controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100 mol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night temperatures were set to 21°C and 18°C, respectively. Experiments were performed with 5- to 6-week-old, naive plants exhibiting a uniform appearance.Treatments with Pip were essentially performed as detailed in Návarová et al. (2012).10 ml of a 1 mM aqueous solution of Pip (equates to 10 µmol) were pipetted onto the soil of individually cultivated plants. 10 ml of water were applied in the control treatments. Three biologically independent, replicate experiments were performed. In each experiment, at least 4 full-grown leaves from 6 different plants were pooled 24 hours after the treatment with Pip or water for one biological replicate. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Jürgen Zeier

PROVIDER: E-MTAB-6243 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Previous studies from our laboratory have shown that the resistance increase observed after biological systemic acquired resistance (SAR) induction in plants can be mimicked by exogenous plant treatment with N-hydroxypipecolic acid (NHP, Hartmann et al., 2018, Schnake et al., 2020). Moreover, exogenous application of the NHP biosynthetic precursor pipecolic acid (Pip) induced a transcriptional response that was overlapping with the SAR transcriptional response and fully depended on the NHP synthase FMO1 (Hartmann et al., 2018; E-MTAB-6243). In order to investigate whether elevations of NHP lead to a SAR-like transcriptional reprogramming, we supplied individual Arabidopsis wildtype Col-0 plants, as well as sid2-1 and npr1-3 mutant plants, with doses of 10 µmol NHP and determined the transcriptional response in leaves 24 hours later on the whole genome level by RNA-sequencing analyses in relation to control-treated (H2O) plants. Col-0 plants were additionally treated with doses of 10 µmol Pip in order to directly compare the transcriptional responses of the foliage between Pip and NHP. Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil, vermiculite and sand (8:1:1) in a controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100 mol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night temperatures were set to 21°C and 18°C, respectively. Experiments were performed with 5-week-old, naive plants exhibiting a uniform appearance. Treatments with NHP and Pip were essentially performed as detailed in Hartmann et al. (2018, Cell 173, 456–469). In brief, 10 ml of a 1 mM aqueous solution of NHP or Pip (equates to a dose of 10 µmol) were pipetted onto the soil of individually cultivated plants. 10 ml of water applied in the way served as control treatments. In total, three biologically independent, replicate experiments were performed. In each experiment, 18 full-grown leaves from 6 different plants were pooled 24 hours after the respective treatments for one biological replicate. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained.

Project description:We investigated the relationships of the two immune-regulatory plant metabolites salicylic acid (SA) and pipecolic acid (Pip) in the establishment of plant systemic acquired resistance (SAR) in Arabidopsis thaliana induced by the bacterial pathogen Pseudomonas syringae. To characterize the transcriptional SAR response, we used wild-type Col-0 plants, SA-deficient sid2 plants and Pip-deficient ald1 plants and performed RNA-sequencing analyses (Bernsdorff et al., Plant Cell, 2016). SAR establishment in the wild-type is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by pre-activating multiple stages of defense signaling. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil, vermiculite and sand (8:1:1) in a controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100 mol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night temperatures were set to 21C and 18C, respectively. Experiments were performed with 5- to 6-week-old, naive plants exhibiting a uniform appearance. To activate SAR, plants were infiltrated between 10 AM and 12 AM into three lower (1) leaves with suspensions of the bacterial pathogen Pseudomonas syringae pv. maculicola (OD600 = 0.005). Infiltration with 10 mM MgCl2 served as the mock-control treatment. Upper (2) leaves were harvested 48 h after the primary treatment for the determination of systemic gene expression by RNA-seq analyses. Three biologically independent, replicate SAR induction experiments were performed with Col-0 and sid2 plants (experimental set 1), and three other biologically independent experiments with Col-0 and ald1 plants (experimental set 2). In each SAR experiment, at least 6 upper (2) leaves from 6 different plants pre-treated in 1 leaves with Psm (MgCl2) were pooled for one biological Psm- (mock-control) replicate. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained within each SAR set.

Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.

Project description:RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, understanding the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions focus on single RBPs, however there are many hundreds RBPs encoded in the human genome, and each cell type expresses a different catalog of these regulatory molecules, greatly limiting the ability of these single RBP approaches to capture the global landscape of RNA-protein interactions. We and others have developed approaches to globally catalog regions of mRNAs that are bound by proteins in an unbiased manner. Here we describe a detailed protocol for performing our RNase-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and un-bound regions are digested with RNases, leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and then RNP complexes are subject to RNase treatment. After high-throughput sequencing of remaining fragments, peak calling is performed to identify protein protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line and perform basic quality control, reproducibility and benchmarking analyses. Finally, we describe the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental processes will help to uncover the role of RBPs in post-transcriptional regulation. Protein interaction profile sequencing (PIP-seq) in HEK293T cells. Three replicates of formaldehyde cross-linked PIP-seq are described

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Transcriptional response of Arabidopsis thaliana to exogenous application of pipecolic acid

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