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Publications - Stress and Develop Biology

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Publications

Sopeña-Torres, S.; Jordá, L.; Sánchez-Rodríguez, C.; Miedes, E.; Escudero, V.; Swami, S.; López, G.; Piślewska-Bednarek, M.; Lassowskat, I.; Lee, J.; Gu, Y.; Haigis, S.; Alexander, D.; Pattathil, S.; Muñoz-Barrios, A.; Bednarek, P.; Somerville, S.; Schulze-Lefert, P.; Hahn, M. G.; Scheel, D.; Molina, A.; YODA MAP3K kinase regulates plant immune responses conferring broad-spectrum disease resistance New Phytol. 218, 661-680, (2018) DOI: 10.1111/nph.15007

Mitogen‐activated protein kinases (MAPKs) cascades play essential roles in plants by transducing developmental cues and environmental signals into cellular responses. Among the latter are microbe‐associated molecular patterns perceived by pattern recognition receptors (PRRs), which trigger immunity.We found that YODA (YDA) – a MAPK kinase kinase regulating several Arabidopsis developmental processes, like stomatal patterning – also modulates immune responses. Resistance to pathogens is compromised in yda alleles, whereas plants expressing the constitutively active YDA (CA‐YDA) protein show broad‐spectrum resistance to fungi, bacteria, and oomycetes with different colonization modes. YDA functions in the same pathway as ERECTA (ER) Receptor‐Like Kinase, regulating both immunity and stomatal patterning.ER‐YDA‐mediated immune responses act in parallel to canonical disease resistance pathways regulated by phytohormones and PRRs. CA‐YDA plants exhibit altered cell‐wall integrity and constitutively express defense‐associated genes, including some encoding putative small secreted peptides and PRRs whose impairment resulted in enhanced susceptibility phenotypes. CA‐YDA plants show strong reprogramming of their phosphoproteome, which contains protein targets distinct from described MAPKs substrates.Our results suggest that, in addition to stomata development, the ER‐YDA pathway regulates an immune surveillance system conferring broad‐spectrum disease resistance that is distinct from the canonical pathways mediated by described PRRs and defense Hormones.
Books and chapters

Lassowskat, I.; Hoehenwarter, W.; Lee, J.; Scheel, D.; Phosphoprotein Enrichment Combined with Phosphopeptide Enrichment to Identify Putative Phosphoproteins During Defense Response in Arabidopsis thaliana (Duque, P., ed.). Methods Mol. Biol. 1398, 373-383, (2016) ISBN: 978-1-4939-3356-3 DOI: 10.1007/978-1-4939-3356-3_30

Phosphoprotein/peptide enrichment is an important technique to elucidate signaling components of defense responses with mass spectrometry. Normally, proteins can be detected easily by shotgun experiments but the low abundance of phosphoproteins hinders their detection. Here, we describe a combination of prefractionation with desalting, phosphoprotein and phosphopeptide enrichment to effectively accumulate phosphorylated proteins from leaf tissue of stressed Arabidopsis plants.
Publications

Lee, J.; Eschen-Lippold, L.; Lassowskat, I.; Böttcher, C.; Scheel, D.; Cellular reprogramming through mitogen-activated protein kinases Front. Plant Sci. 6, 940, (2015) DOI: 10.3389/fpls.2015.00940

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.
Publications

Carrasco, J. L.; Castelló, M. J.; Naumann, K.; Lassowskat, I.; Navarrete-Gómez, M.; Scheel, D.; Vera, P.; Arabidopsis Protein Phosphatase DBP1 Nucleates a Protein Network with a Role in Regulating Plant Defense PLOS ONE 9, e90734, (2014) DOI: 10.1371/journal.pone.0090734

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.
Publications

Majovsky, P.; Naumann, C.; Lee, C.-W.; Lassowskat, I.; Trujillo, M.; Dissmeyer, N.; Hoehenwarter, W.; Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer J. Proteome Res. 13, 4246-4258, (2014) DOI: 10.1021/pr500164j

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor.
Publications

Lassowskat, I.; Böttcher, C.; Eschen-Lippold, L.; Scheel, D.; Lee, J.; Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana Front. Plant Sci. 5, 554, (2014) DOI: 10.3389/fpls.2014.00554

Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).
Publications

Lassowskat, I.; Naumann, K.; Lee, J.; Scheel, D.; PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants Proteomes 1, 254-274, (2013) DOI: 10.3390/proteomes1030254

Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events.
Publications

Widjaja, I.; Lassowskat, I.; Bethke, G.; Eschen-Lippold, L.; Long, H.-H.; Naumann, K.; Dangl, J. L.; Scheel, D.; Lee, J.; A protein phosphatase 2C, responsive to the bacterial effector AvrRpm1 but not to the AvrB effector, regulates defense responses in Arabidopsis Plant J. 61, 249-258, (2010) DOI: 10.1111/j.1365-313X.2009.04047.x

Using a proteomics approach, a PP2C‐type phosphatase (renamed PIA1, for PP2C induced by AvrRpm1) was identified that accumulates following infection by Pseudomonas syringae expressing the type III effector AvrRpm1, and subsequent activation of the corresponding plant NB‐LRR disease resistance protein RPM1. No accumulation of PIA1 protein was seen following infection with P. syringae expressing AvrB, another type III effector that also activates RPM1, although PIA transcripts were observed. Accordingly, mutation of PIA1 resulted in enhanced RPM1 function in response to P. syringae pathover tomato (Pto) DC3000 (avrRpm1) but not to Pto DC3000 (avrB). Thus, PIA1 is a protein marker that distinguishes AvrRpm1‐ and AvrB‐dependent activation of RPM1. AvrRpm1‐induced expression of the pathogenesis‐related genes PR1, PR2 and PR3, and salicylic acid accumulation were reduced in two pia1 mutants. By contrast, expression of other defense‐related genes, including PR5 and PDF1.2 (plant defensin), was elevated in unchallenged pia1 mutants. Hence, PIA1 is required for AvrRpm1‐induced responses, and confers dual (both positive and negative) regulation of defense gene expression.
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