Teh, O.-K.; Lee, C.-W.; Ditengou, F. A.; Klecker, T.; Furlan, G.; Zietz, M.; Hause, G.; Eschen-Lippold, L.; Hoehenwarter, W.; Lee, J.; Ott, T.; Trujillo, M. Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function BioRxiv (2018) DOI: 10.1101/266171
The exocyst is a conserved hetero-octameric complex mediating early
tethering during exocytosis. Its Exo70 subunit plays a critical role as a
spatiotemporal regulator by mediating numerous protein and lipid
interactions. However, a molecular understanding of the exocyst function
remains challenging. We show that Exo70B2 locates to dynamic foci at
the plasma membrane and transits through a BFA-sensitive compartment,
reflecting its canonical function in secretion. However, treatment with
the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH),
or the immunogenic peptide flg22, induced Exo70B2 transport into the
vacuole. We uncovered two ATG8-interacting motifs (AIMs) located in the
C-terminal domain (C-domain) of Exo70B2 that mediate its recruitment
into the vacuole. Moreover, we also show that Exo70B2 is phosphorylated
near the AIMs and mimicking phosphorylation enhanced ATG8 interaction.
Finally, Exo70B2 phosphonull lines were hypersensitive to BTH and more
resistant to avirulent bacteria which induce SA production. Our results
suggests a molecular mechanism in which phosphorylation of Exo70B2 by
MPK3 functions in a feed-back system linking cellular signalling to the
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.
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.
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.
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.
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.