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Publications - Stress and Develop Biology

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Publications

Sheikh, A. H.; Fraz Hussain, R. M.; Tabassum, N.; Badmi, R.; Marillonnet, S.; Scheel, D.; Lee, J.; Sinha, A.; Possible role of WRKY transcription factors in regulating immunity in Oryza sativa ssp. indica Physiol. Mol. Plant Pathol. 114, 101623, (2021) DOI: 10.1016/j.pmpp.2021.101623

Plants have developed a robust transcription machinery to combat potential pathogenic organisms. One of the hallmarks of early immune responses is the activation of the WRKY transcription factors post infection. Specific WRKYs proteins from Arabidopsis are known substrates of MAPK pathway to mediate the flg22 elicited early immunity. In the current study, using the Golden Gate cloning strategy, we aim to clone the entire WRKY transcription factor family from Oryza sativa ssp. indica consisting of more than 100 members and study their MAPK interaction and subsequent role in PTI. Using a reporter LUC assay in protoplasts we investigated the early defense responses in a few interesting OsWRKY candidates. Interestingly, we observed stringent regulation of WRKY expression in cells and their transcriptional expression only under specific stress responses. The phenomenon of gene expression regulation by intron retention (IR) was prevalently observed in rice WRKY transcripts. We could show the role of WRKY8, 24, and 77 in early defense responses. It was observed that WRKY24 enhanced the expression of early defense response marker genes like NHL10 while WRKY8 and WRKY77 supressed their expression. This study highlights the complicated mechanism by which OsWRKYs expression is possibly regulated and the distinctive roles of some individual members in plant immunity. At the same time this study serves as a cautionary warning for plant researchers to be mindful of the intron retention mechanism while cloning OsWRKYs.
Publications

Jiang, X.; Hoehenwarter, W.; Scheel, D.; Lee, J.; Phosphorylation of the CAMTA3 Transcription Factor Triggers Its Destabilization and Nuclear Export Plant Physiol. 184, 1056-1071, (2020) DOI: 10.1104/pp.20.00795

The Arabidopsis (Arabidopsis thaliana) calmodulin-binding transcription activator3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced or general stress-responsive genes in plants. Post-transcriptional or posttranslational mechanisms have been proposed to control CAMTA3 functions in different stress responses. Here, we show that treatment with the bacterial flg22 elicitor induces CAMTA3 phosphorylation, which is accompanied by its destabilization and nuclear export. Two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, directly phosphorylate CAMTA3, with the phospho-sites contributing to CAMTA3 degradation and suppression of downstream target gene expression. However, the flg22-induced nuclear export and phospho-mobility shift can still be observed for the CAMTA3 phospho-null variant of the MAPK-modified sites, suggesting additional flg22-responsive kinases might be involved. Taken together, we propose that flg22-induced CAMTA3 depletion facilitates de-repression of downstream defense target genes, which involves phosphorylation, increased protein turnover, and nucleo-cytoplasmic trafficking.
Publications

Trempel, F.; Eschen‐Lippold, L.; Bauer, N.; Ranf, S.; Westphal, L.; Scheel, D.; Lee, J.; A mutation in Asparagine‐Linked Glycosylation 12 (ALG12) leads to receptor misglycosylation and attenuated responses to multiple microbial elicitors FEBS Lett. 594, 2440-2451, (2020) DOI: 10.1002/1873-3468.13850

Changes in cellular calcium levels are one of the earliest signalling events in plants exposed to pathogens or other exogenous factors. In a genetic screen, we identified an Arabidopsis thaliana ‘changed calcium elevation 1 ’ (cce1 ) mutant with attenuated calcium response to the bacterial flagellin flg22 peptide and several other elicitors. Whole genome re‐sequencing revealed a mutation in ALG12 (Asparagine‐Linked Glycosylation 12 ) that encodes the mannosyltransferase responsible for adding the eighth mannose residue in an α‐1,6 linkage to the dolichol‐PP‐oligosaccharide N ‐glycosylation glycan tree precursors. While properly targeted to the plasma membrane, misglycosylation of several receptors in the cce1 background suggests that N ‐glycosylation is required for proper functioning of client proteins.
Publications

Tabassum, N.; Eschen-Lippold, L.; Athmer, B.; Baruah, M.; Brode, M.; Maldonado-Bonilla, L. D.; Hoehenwarter, W.; Hause, G.; Scheel, D.; Lee, J.; Phosphorylation‐dependent control of an RNA granule‐localized protein that fine‐tunes defence gene expression at a post‐transcriptional level Plant J. 101, 1023-1039, (2020) DOI: 10.1111/tpj.14573

Mitogen‐activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen‐associated molecular patterns (PAMPs, e.g. bacterial flg22 peptide). The Tandem Zinc Finger Protein 9 (TZF9) is an RNA‐binding protein that is phosphorylated by two PAMP‐responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA‐binding activity, in vivo flg22‐induced rapid disappearance of TZF9‐labelled processing body‐like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome‐associated mRNA) in the tzf9 mutant, with more mRNAs associated to ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)‐binding protein 2 (PAB2), a hallmark constituent of stress granules – a site for stress‐induced translational stalling/arrest. TZF9 even promotes stress granule assembly in the absence of stress. Hence, MAPKs may control defence gene expression post‐transcriptionally through release from translation arrest within TZF9‐PAB2‐containing RNA granules or perturbing PAB2 functions in translation control (e.g. in the mRNA closed‐loop model of translation).
Publications

Dietz, S.; Herz, K.; Gorzolka, K.; Jandt, U.; Bruelheide, H.; Scheel, D.; Root exudate composition of grass and forb species in natural grasslands Sci. Rep. 10, 10691, (2020) DOI: 10.1038/s41598-019-54309-5

Plants exude a diverse cocktail of metabolites into the soil as response to exogenous and endogenous factors. So far, root exudates have mainly been studied under artificial conditions due to methodological difficulties. In this study, each five perennial grass and forb species were investigated for polar and semi-polar metabolites in exudates under field conditions. Metabolite collection and untargeted profiling approaches combined with a novel classification method allowed the designation of 182 metabolites. The composition of exuded polar metabolites depended mainly on the local environment, especially soil conditions, whereas the pattern of semi-polar metabolites was primarily affected by the species identity. The profiles of both polar and semi-polar metabolites differed between growth forms, with grass species being generally more similar to each other and more responsive to the abiotic environment than forb species. This study demonstrated the feasibility of investigating exudates under field conditions and to identify the driving factors of exudate composition.
Printed publications

Jiang, X.; Hoehenwarter, W.; Scheel, D.; Lee, J.; Phosphorylation of the CAMTA3 transcription factor triggers its destabilization and nuclear export bioRxiv (2019) DOI: 10.1101/825323

The calmodulin-binding transcription activator 3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced genes in plants. Post-transcriptional or -translational mechanisms have been proposed to control CAMTA3’s role in the crosstalk between immune and chilling responses. Here, we show that treatment with the bacterial flg22 elicitor, but not cold stress, induces a phospho-mobility shift of CAMTA3 proteins. Correspondingly, CAMTA3 is directly phosphorylated by two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, which triggers CAMTA3 nuclear export and destabilization. SR1IP1, a substrate E3 ubiquitin ligase adaptor required for pathogen-induced CAMTA3 degradation, is shown here to be likely plasma-membrane-localized and therefore cannot physically interact with the nuclear CAMTA3. Despite the flg22-inducible re-localization of CAMTA3 to the cytoplasm, we failed to detect CAMTA3-SR1IP1 complexes. Hence, the role of SR1IP1 for CAMTA3 degradation needs to be re-evaluated. Surprisingly, flg22 elicitation can still induce nuclear export and phospho-mobility shift of a phospho-null CAMTA3 that cannot be phosphorylated by MAPKs, suggesting the participation of additional flg22-responsive kinase(s). A constitutively-active calcium-dependent protein kinase, CPK5, can stimulate a phospho-mobility shift in CAMTA3 similar to that induced by flg22. Although CPK5 can interact with CAMTA3, it did not directly phosphorylate CAMTA3, suggesting the requirement of a still unidentified downstream kinase or additional components. Overall, at least two flg22-responsive kinase pathways target CAMTA3 to induce degradation that presumably serves to remove CAMTA3 from target promoters and de-repress expression of defence genes.
Publications

Nietzschmann, L.; Gorzolka, K.; Smolka, U.; Matern, A.; Eschen-Lippold, L.; Scheel, D.; Rosahl, S.; Early Pep-13-induced immune responses are SERK3A/B-dependent in potato Sci. Rep. 9, 18380, (2019) DOI: 10.1038/s41598-019-54944-y

Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.
Publications

Matern, A.; Böttcher, C.; Eschen-Lippold, L.; Westermann, B.; Smolka, U.; Döll, S.; Trempel, F.; Aryal, B.; Scheel, D.; Geisler, M.; Rosahl, S.; A substrate of the ABC transporter PEN3 stimulates bacterial flagellin (flg22)-induced callose deposition in Arabidopsis thaliana J. Biol. Chem. 294, 6857-6870, (2019) DOI: 10.1074/jbc.RA119.007676

Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro. Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens.
Publications

Westphal, L.; Strehmel, N.; Eschen-Lippold, L.; Bauer, N.; Westermann, B.; Rosahl, S.; Scheel, D.; Lee, J.; pH effects on plant calcium fluxes: lessons from acidification-mediated calcium elevation induced by the γ-glutamyl-leucine dipeptide identified from Phytophthora infestans Sci. Rep. 9, 4733, (2019) DOI: 10.1038/s41598-019-41276-0

Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.
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.
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