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Publications - Stress and Develop Biology

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Publications

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.
Books and chapters

Eschen-Lippold, L.; Bauer, N.; Löhr, J.; Palm-Forster, M. A. T.; Lee, J.; Rapid Mutagenesis-Based Analysis of Phosphorylation Sites in Mitogen-Activated Protein Kinase Substrates (Komis, G. & Šamaj, J., eds.). Methods Mol. Biol. 1171, 183-192, (2014) ISBN: 978-1-4939-0922-3 DOI: 10.1007/978-1-4939-0922-3_15

In eukaryotes, mitogen-activated protein kinases (MAPKs) are one of the best studied pathways for posttranslational modification-mediated regulation of protein functions. Here, we describe a rapid in vitro method to screen potential protein phosphorylation sites targeted by MAPKs. The method is based on PCR-mediated mutagenesis together with a type IIs restriction digest. Screening for the successfully mutated clones is further facilitated through introduction of a second diagnostic restriction site. Besides time-saving, this reduces the cost for sequencing confirmation of the positive clones, which are used for subsequent recombinant protein production and kinase assay validation.
Publications

Palm-Forster, M. A. T.; Eschen-Lippold, L.; Lee, J.; A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates Anal. Biochem. 427, 127-129, (2012) DOI: 10.1016/j.ab.2012.05.015

Identification and characterization of protein phosphorylation sites often requires mass spectrometric analysis, which is not trivial or accessible to many laboratories. Here, a targeted strategy to mutagenize putative phosphorylation sites within mitogen-activated protein kinase (MAPK) substrates is described. This employs a combination of standard type II with type IIs restriction enzymes to rapidly create individual or multiple phosphorylation site mutant versions of kinase substrates with high efficiency, thereby reducing the cost for screening mutated clones.
Publications

Eschen-Lippold, L.; Bethke, G.; Palm-Forster, M. A. T.; Pecher, P.; Bauer, N.; Glazebrook, J.; Scheel, D.; Lee, J.; MPK11—a fourth elicitor-responsive mitogen-activated protein kinase in Arabidopsis thaliana Plant Signal Behav. 7, 1203-1205, (2012) DOI: 10.4161/psb.21323

Recognition of pathogen attack or elicitation with pathogen-associated molecular patterns (PAMPs) leads to defense signaling that includes activation of the three mitogen-activated protein kinases (MPKs), MPK3, MPK4 and MPK6 in Arabidopsis. Recently, we demonstrated the activation of a fourth MPK, MPK11, after treatment with flg22, a 22 amino acid PAMP derived from bacterial flagellin. Here, we extended the study by examining elicitation with two other PAMPs, elf18 (derived from bacterial elongation factor EF-Tu) and ch8 (N-acetylchitooctaose derived from fungal chitin). Both PAMPs led to rapid MPK11 transcript accumulation and increased MPK11 kinase activity, suggesting that multiple PAMPs (or stresses) can activate MPK11. However, probably due to functional redundancies, bacteria-induced phytoalexin accumulation does not absolutely require MPK11.
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