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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 post-translational modifications such as ubiquitination and phosphorylation play key roles in controlling the duration and intensity of signalling 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 signalling. 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

Ziegler, J., Schmidt, S., Strehmel, N., Scheel, D. & Abel, S. Arabidopsis transporter ABCG37/PDR9 contributes primarily highly oxygenated coumarins to root exudation.  Scientific Rep 7, 3704, (2017) DOI: 10.1038/s41598-017-03250-6

The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins.
Publikation

Blüher, D., Laha, D., Thieme, S., Hofer, A., Eschen-Lippold, L., Masch, A., Balcke, G., Pavlovic, I., Nagel, O., Schonsky, A., Hinkelmann, R., Wörner, J., Parvin, N., Greiner, R., Weber, S., Tissier, A., Schutkowski, M., Lee, J., Jessen, H., Schaaf, G. & Bonas, U. A 1-phytase type III effector interferes with plant hormone signaling. Nature Commun. 8(1), 2159, (2017) DOI: 10.1038/s41467-017-02195-8

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.
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

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

Strehmel, N., Hoehenwarter, W., Mönchgesang, S., Majovsky, P., Krüger, S., Scheel, D. & Lee, J. Stress-reated 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

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

Background

Calcium, 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.

Results

Here, 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.

Bücher und Buchkapitel

Trempel, F., Ranf, S., Scheel, D. & Lee, J. Quantitative analysis of microbe-associated molecular pattern (MAMP)-induced Ca2+ transients in plants. In: Environmental Responses in Plants (Paula Duque). Meth. Mol. Biol. 1398, 331-344, (2016) ISBN: 978-1-4939-3354-9 (Print) 978-1-4939-3356-3 (Online) DOI: 10.1007/978-1-4939-3356-3_27

Ca2+ is a secondary messenger involved in early signaling events triggered in response to a plethora of biotic and abiotic stimuli. In plants, environmental cues that induce cytosolic Ca2+ elevation include touch, reactive oxygen species, cold shock, and salt or osmotic stress. Furthermore, Ca2+ signaling has been implicated in early stages of plant–microbe interactions of both symbiotic and antagonistic nature. A long-standing hypothesis is that there is information encoded in the Ca2+ signals (so-called Ca2+ signatures) to enable plants to differentiate between these stimuli and to trigger the appropriate cellular response. Qualitative and quantitative measurements of Ca2+ signals are therefore needed to dissect the responses of plants to their environment. Luminescence produced by the Ca2+ probe aequorin upon Ca2+ binding is a widely used method for the detection of Ca2+ transients and other changes in Ca2+ concentrations in cells or organelles of plant cells. In this chapter, using microbe-associated molecular patterns (MAMPs), such as the bacterial-derived flg22 or elf18 peptides as stimuli, a protocol for the quantitative measurements of Ca2+ fluxes in apoaequorin-expressing seedlings of Arabidopsis thaliana in 96-well format is described.

Bücher und Buchkapitel

Lassowskat, I., Hoehenwarter, W., Lee, J. & Scheel, D. Phosphoprotein Enrichment Combined with Phosphopeptide Enrichment to Identify Putative Phosphoproteins During Defense Response in Arabidopsis thaliana. In: Environmental Responses in Plants Meth. Mol. Biol 1398, 373-383, (2016) ISBN: 978-1-4939-3354-9 (Print) 978-1-4939-3356-3 (Online) 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.

Publikation

Eschen-Lippold, L., Jiang, X., Elmore, J. M., Mackey, D., Shan, L., Coaker, G., Scheel, D. & Lee, J. Bacterial AvrRpt2-like cysteine proteases block activation of the Arabidopsis mitogen-activated protein kinases, MPK4 and MPK11. Plant Physiol 171, 2223-2238, (2016) DOI: ​10.​1104/​pp.​16.​00336

To establish infection, pathogens deliver effectors into host cells to target immune signalling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defence regulator, RIN4, and interference with plant auxin signalling. We show now that AvrRpt2 specifically suppresses flagellin-induced phosphorylation of Arabidopsis MPK4 and MPK11, but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defence genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called NOI family, as well as from auxin signalling. Thus, this selective suppression of specific MAPKs is independent of the previously known AvrRpt2 targets and represents potentially a novel virulence function of AvrRpt2.

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