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Publikationen - Stress- und Entwicklungsbiologie

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Publikation

Strehmel, N.; Hoehenwarter, W.; Mönchgesang, S.; Majovsky, P.; Krüger, S.; Scheel, D.; Lee, J.; Stress-Related Mitogen-Activated Protein Kinases Stimulate the Accumulation of Small Molecules and Proteins in Arabidopsis thaliana Root Exudates Front. Plant Sci. 8, 1292, (2017) DOI: 10.3389/fpls.2017.01292

A delicate balance in cellular signaling is required for plants to respond to microorganisms or to changes in their environment. Mitogen-activated protein kinase (MAPK) cascades are one of the signaling modules that mediate transduction of extracellular microbial signals into appropriate cellular responses. Here, we employ a transgenic system that simulates activation of two pathogen/stress-responsive MAPKs to study release of metabolites and proteins into root exudates. The premise is based on our previous proteomics study that suggests upregulation of secretory processes in this transgenic system. An advantage of this experimental set-up is the direct focus on MAPK-regulated processes without the confounding complications of other signaling pathways activated by exposure to microbes or microbial molecules. Using non-targeted metabolomics and proteomics studies, we show that MAPK activation can indeed drive the appearance of dipeptides, defense-related metabolites and proteins in root apoplastic fluid. However, the relative levels of other compounds in the exudates were decreased. This points to a bidirectional control of metabolite and protein release into the apoplast. The putative roles for some of the identified apoplastic metabolites and proteins are discussed with respect to possible antimicrobial/defense or allelopathic properties. Overall, our findings demonstrate that sustained activation of MAPKs alters the composition of apoplastic root metabolites and proteins, presumably to influence the plant-microbe interactions in the rhizosphere. The reported metabolomics and proteomics data are available via Metabolights (Identifier: MTBLS441) and ProteomeXchange (Identifier: PXD006328), respectively.
Publikation

Palm-Forster, M. A. T.; Eschen-Lippold, L.; Uhrig, J.; Scheel, D.; Lee, J.; A novel family of proline/serine-rich proteins, which are phospho-targets of stress-related mitogen-activated protein kinases, differentially regulates growth and pathogen defense in Arabidopsis thaliana Plant Mol. Biol. 95, 123-140, (2017) DOI: 10.1007/s11103-017-0641-5

The molecular actions of mitogen-activated protein kinases (MAPKs) are ultimately accomplished by the substrate proteins where phosphorylation affects their molecular properties and function(s), but knowledge regarding plant MAPK substrates is currently still fragmentary. Here, we uncovered a previously uncharacterized protein family consisting of three proline/serine-rich proteins (PRPs) that are substrates of stress-related MAPKs. We demonstrated the importance of a MAPK docking domain necessary for protein–protein interaction with MAPKs and consequently also for phosphorylation. The main phosphorylated site was mapped to a residue conserved between all three proteins, which when mutated to a non-phosphorylatable form, differentially affected their protein stability. Together with their distinct gene expression patterns, this differential accumulation of the three proteins upon phosphorylation probably contributes to their distinct function(s). Transgenic over-expression of PRP, the founding member, led to plants with enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Older plants of the over-expressing lines have curly leaves and were generally smaller in stature. This growth phenotype was lost in plants expressing the phosphosite variant, suggesting a phosphorylation-dependent effect. Thus, this novel family of PRPs may be involved in MAPK regulation of plant development and / or pathogen resistance responses. As datamining associates PRP expression profiles with hypoxia or oxidative stress and PRP-overexpressing plants have elevated levels of reactive oxygen species, PRP may connect MAPK and oxidative stress signaling.
Publikation

Hempel, F.; Stenzel, I.; Heilmann, M.; Krishnamoorthy, P.; Menzel, W.; Golbik, R.; Helm, S.; Dobritzsch, D.; Baginsky, S.; Lee, J.; Hoehenwarter, W.; Heilmann, I.; MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol 4,5-Bisphosphate in an Apical Plasma Membrane Domain Plant Cell 29, 3030-3050, (2017) DOI: 10.1105/tpc.17.00543

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.
Publikation

Furlan, G.; Nakagami, H.; Eschen-Lippold, L.; Jiang, X.; Majovsky, P.; Kowarschik, K.; Hoehenwarter, W.; Lee, J.; Trujillo, M.; Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response Plant Cell 29, 726-745, (2017) DOI: 10.1105/tpc.16.00654

Crosstalk between posttranslational modifications, such as ubiquitination and phosphorylation, play key roles in controlling the duration and intensity of signaling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here, we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signaling. PUB22’s stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and, thus, autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo.
Publikation

Küster, N.; Rosahl, S.; Dräger, B.; Potato plants with genetically engineered tropane alkaloid precursors Planta 245, 355-365, (2017) DOI: 10.1007/s00425-016-2610-7

Main conclusionSolanum tuberosum tropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation.Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.
Publikation

Trempel, F.; Kajiura, H.; Ranf, S.; Grimmer, J.; Westphal, L.; Zipfel, C.; Scheel, D.; Fujiyama, K.; Lee, J.; Altered glycosylation of exported proteins, including surface immune receptors, compromises calcium and downstream signaling responses to microbe-associated molecular patterns in Arabidopsis thaliana BMC Plant Biol. 16, 31, (2016) DOI: 10.1186/s12870-016-0718-3

BackgroundCalcium, as a second messenger, transduces extracellular signals into cellular reactions. A rise in cytosolic calcium concentration is one of the first plant responses after exposure to microbe-associated molecular patterns (MAMPs). We reported previously the isolation of Arabidopsis thaliana mutants with a “changed calcium elevation” (cce) response to flg22, a 22-amino-acid MAMP derived from bacterial flagellin.ResultsHere, we characterized the cce2 mutant and its weaker allelic mutant, cce3. Besides flg22, the mutants respond with a reduced calcium elevation to several other MAMPs and a plant endogenous peptide that is proteolytically processed from pre-pro-proteins during wounding. Downstream defense-related events such flg22-induced mitogen-activated protein kinase activation, accumulation of reactive oxygen species and growth arrest are also attenuated in cce2/cce3. By genetic mapping, next-generation sequencing and allelism assay, CCE2/CCE3 was identified to be ALG3 (Asparagine-linked glycosylation 3). This encodes the α-1,3-mannosyltransferase responsible for the first step of core oligosaccharide Glc3Man9GlcNAc2 glycan assembly on the endoplasmic reticulum (ER) luminal side. Complementation assays and glycan analysis in yeast alg3 mutant confirmed the reduced enzymatic function of the proteins encoded by the cce2/cce3 alleles – leading to accumulation of M5ER, the immature five mannose-containing oligosaccharide structure found in the ER. Proper protein glycosylation is required for ER/Golgi processing and trafficking of membrane proteins to the plasma membrane. Endoglycosidase H-insensitivity of flg22 receptor, FLS2, in the cce2/cce3 mutants suggests altered glycan structures in the receptor.ConclusionProper glycosylation of MAMP receptors (or other exported proteins) is required for optimal responses to MAMPs and is important for immune signaling of host plants.
Publikation

Sheikh, A. H.; Eschen-Lippold, L.; Pecher, P.; Hoehenwarter, W.; Sinha, A. K.; Scheel, D.; Lee, J.; Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana Front. Plant Sci. 7, 61, (2016) DOI: 10.3389/fpls.2016.00061

Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense.
Publikation

Ranf, S.; Scheel, D.; Lee, J.; Challenges in the identification of microbe-associated molecular patterns in plant and animal innate immunity: a case study with bacterial lipopolysaccharide Mol. Plant Pathol. 17, 1165-1169, (2016) DOI: 10.1111/mpp.12452

Immunity against pathogen infection depends on a host's ability to sense invading pathogens and to rapidly trigger defence reactions that block pathogen proliferation. Both plants and animals detect conserved structural motifs of microbe‐specific compounds, so‐called microbe‐associated molecular patterns (MAMPs), through germline‐encoded immune sensors, which are accordingly termed pattern recognition receptors (PRRs) (Akira et al., 2006; Boller and Felix, 2009). Activated PRRs initiate signal transduction and trigger innate immune responses. MAMPs are generally derived from elements essential for microbial fitness and are conserved across species, thus enabling the host to detect a range of potential pathogens. In mammals, innate immune sensing of MAMPs is not only crucial for basal immune responses but is also tightly connected with and required for a subsequent adaptive, antibody‐mediated immunity (Akira et al., 2006; Janeway and Medzhitov, 2002). Plants, lacking an adaptive immune system, have apparently evolved a greater capacity to detect a broader repertoire of MAMPs. Different plant species possess distinct sets of highly specific PRRs, but the downstream signalling pathways are rather conserved and converge on common signalling steps. This allows the transfer of PRRs, even to different plant families, whilst maintaining their functionality and specificity (Zipfel, 2014). This also enables researchers to use well‐studied, genetically amenable model systems for the identification of MAMPs and their respective PRRs. Several examples of interfamily PRR transfer have demonstrated that the introduction of novel PRRs into plant species can confer relevant levels of resistance to otherwise susceptible plants (e.g. Afroz et al., 2011; Hao et al., 2015; Lacombe et al., 2010; Mendes et al., 2010; Schoonbeek et al., 2015; Tripathi et al., 2014). Hence, MAMP sensing by PRRs has great potential for the engineering of disease resistance in crop plants. In recent years, it has therefore become a major task to identify and isolate MAMPs from a range of microorganisms, and their respective PRRs, to study their role in innate immunity and their application potential.
Publikation

Penselin, D.; Münsterkötter, M.; Kirsten, S.; Felder, M.; Taudien, S.; Platzer, M.; Ashelford, K.; Paskiewicz, K. H.; Harrison, R. J.; Hughes, D. J.; Wolf, T.; Shelest, E.; Graap, J.; Hoffmann, J.; Wenzel, C.; Wöltje, N.; King, K. M.; Fitt, B. D. L.; Güldener, U.; Avrova, A.; Knogge, W.; Comparative genomics to explore phylogenetic relationship, cryptic sexual potential and host specificity of Rhynchosporium species on grasses BMC Genomics 17, 953, (2016) DOI: 10.1186/s12864-016-3299-5

BackgroundThe Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts.ResultsThe genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type.ConclusionBoth cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition.
Publikation

Mönchgesang, S.; Strehmel, N.; Trutschel, D.; Westphal, L.; Neumann, S.; Scheel, D.; Plant-to-Plant Variability in Root Metabolite Profiles of 19 Arabidopsis thaliana Accessions Is Substance-Class-Dependent Int. J. Mol. Sci. 17, 1565, (2016) DOI: 10.3390/ijms17091565

Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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