Eschen-Lippold, L.; Scheel, D.; Lee, J. Teaching an old dog new tricks: Suppressing activation of specific
mitogen-activated kinases as a potential virulence function of the
bacterial AvrRpt2 effector protein Plant Signal Behav 11, e1257456, (2016) DOI: 10.1080/15592324.2016.1257456
AvrRpt2 is one of the first Pseudomonas
syringae effector proteins demonstrated to be delivered into host cells.
It suppresses plant immunity by modulating auxin signaling and cleavage
of the membrane-localized defense regulator RIN4. We recently uncovered
a novel potential virulence function of AvrRpt2, where it specifically
blocked activation of mitogen-activated protein kinases, MPK4 and MPK11,
but not of MPK3 and MPK6. Putative AvrRpt2 homologs from different
phytopathogens and plant-associated bacteria showed distinct activities
with respect to MPK4/11 activation suppression and RIN4 cleavage. Apart
from differences in sequence similarity, 3 of the analyzed homologs were
apparently “truncated.” To examine the role of the AvrRpt2 N-terminus,
we modeled the structures of these AvrRpt2 homologs and performed
deletion and domain swap experiments. Our results strengthen the finding
that RIN4 cleavage is irrelevant for the ability to suppress
defense-related MPK4/11 activation and indicate that full protease
activity or cleavage specificity is affected by the N-terminus.
Mönchgesang, S.; Strehmel, N.; Trutschel, D.; Westphal, L.; Neumann, S.; Scheel, D. Plant-to-plant variability in root metabolite profiles of 19 <i>Arabidopsis thaliana</i> accessions is substance-class-dependent Inter J Mol Sci 17, (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.
Chen, S.; Wirthmueller, L.; Stauber, J.; Lory, N.; Holtkotte, X.; Leson, L.; Schenkel, C.; Ahmad, M.; Hoecker, U. The functional divergence between SPA1 and SPA2 in
Arabidopsis photomorphogenesis maps primarily to the respective
N-terminal kinase-like domain BMC Plant Biol 16, 165, (2016) DOI: 10.1186/s12870-016-0854-9
BackgroundPlants have evolved complex mechanisms to adapt growth and
development to the light environment. The COP1/SPA complex is a key
repressor of photomorphogenesis in dark-grown Arabidopsis plants and
acts as an E3 ubiquitin ligase to ubiquitinate transcription factors
involved in the light response. In the light, COP1/SPA activity is
inhibited by photoreceptors, thereby allowing accumulation of these
transcription factors and a subsequent light response. Previous results
have shown that the four members of the SPA family exhibit partially
divergent functions. In particular, SPA1 and SPA2 strongly differ in
their responsiveness to light, while they have indistinguishable
activities in darkness. The much higher light-responsiveness of SPA2 is
partially explained by the much stronger light-induced degradation of
SPA2 when compared to SPA1. Here, we have conducted SPA1/SPA2 domain
swap experiments to identify the protein domain(s) responsible for the
functional divergence between SPA1 and SPA2.ResultsWe have
individually swapped the three domains between SPA1 and SPA2 - the
N-terminal kinase-like domain, the coiled-coil domain and the WD-repeat
domain - and expressed them in spa mutant Arabidopsis plants. The
phenotypes of transgenic seedlings show that the respective N-terminal
kinase-like domain is primarily responsible for the respective
light-responsiveness of SPA1 and SPA2. Furthermore, the most divergent
part of the N-terminal domain was sufficient to confer a SPA1- or
SPA2-like activity to the respective SPA protein. The stronger
light-induced degradation of SPA2 when compared to SPA1 was also
primarily conferred by the SPA2 N-terminal domain. At last, the
different affinities of SPA1 and SPA2 for cryptochrome 2 are defined by
the N-terminal domain of the respective SPA protein. In contrast, both
SPA1 and SPA2 similarly interacted with COP1 in light-grown seedlings.ConclusionsOur
results show that the distinct activities and protein stabilities of
SPA1 and SPA2 in light-grown seedlings are primarily encoded by their
N-terminal kinase-like domains. Similarly, the different affinities of
SPA1 and SPA2 for cry2 are explained by their respective N-terminal
domain. Hence, after a duplication event during evolution, the
N-terminal domains of SPA1 and SPA2 underwent subfunctionalization,
possibly to allow optimal adaptation of growth and development to a
changing light environment.
Genenncher, B.; Wirthmueller, L.; Roth, C.; Klenke, M.; Ma, L.; Sharon, A.; Wiermer, M. Nucleoporin-Regulated MAP Kinase Signaling in Immunity to a Necrotrophic Fungal Pathogen Plant Physiol 172, 1293-1305, (2016) DOI: 10.1104/pp.16.00832
Pathogen-responsive mitogen-activated protein
kinase (MAPK or MPK) cascades relay signals from activated immune
receptors across the nuclear envelope to intranuclear targets. However,
in plants, little is known about the spatial control of MAPK signaling.
Here, we report that the Arabidopsis (Arabidopsis thaliana) nuclear pore
complex protein Nup88/MOS7 is essential for immunity to the
necrotrophic fungus Botrytis cinerea. The mos7-1 mutation, causing a
four-amino acid deletion, compromises B. cinerea-induced activation of
the key immunoregulatory MAPKs MPK3/MPK6 and reduces MPK3 protein levels
posttranscriptionally. Furthermore, MOS7 contributes to retaining a
sufficient MPK3 abundance in the nucleus, which is required for full
immunity to B. cinerea. Finally, we present a structural model of MOS7
and show that the mos7-1 mutation compromises interactions with
Nup98a/b, two phenylalanine-glycine repeat nucleoporins implicated in
maintaining the selective nuclear pore complex permeability barrier.
Together, our analysis uncovered MOS7 and Nup98 as novel components of
plant immunity toward a necrotrophic pathogen and provides mechanistic
insights into how these nucleoporins coordinate nucleocytoplasmic
transport to mount a robust immune response.
Mönchgesang, S.; Strehmel, N.; Schmidt, S.; Westphal, L.; Taruttis, F.; Müller, E.; Herklotz, S.; Neumann, S.; Scheel, D. Natural variation of roots exudates in Arabidopsis thaliana - linking metabolomic and genomic data. Sci Rep 6, 29033 , (2016) DOI: 10.1038/srep29033
Many metabolomics studies focus on aboveground parts of the plant, while metabolism within roots and the chemical composition of the rhizosphere, as influenced by exudation, are not deeply investigated. In this study, we analysed exudate metabolic patterns of Arabidopsis thaliana and their variation in genetically diverse accessions. For this project, we used the 19 parental accessions of the Arabidopsis MAGIC collection. Plants were grown in a hydroponic system, their exudates were harvested before bolting and subjected to UPLC/ESI-QTOF-MS analysis. Metabolite profiles were analysed together with the genome sequence information. Our study uncovered distinct metabolite profiles for root exudates of the 19 accessions. Hierarchical clustering revealed similarities in the exudate metabolite profiles, which were partly reflected by the genetic distances. An association of metabolite absence with nonsense mutations was detected for the biosynthetic pathways of an indolic glucosinolate hydrolysis product, a hydroxycinnamic acid amine and a flavonoid triglycoside. Consequently, a direct link between metabolic phenotype and genotype was detected without using segregating populations. Moreover, genomics can help to identify biosynthetic enzymes in metabolomics experiments. Our study elucidates the chemical composition of the rhizosphere and its natural variation in A. thaliana, which is important for the attraction and shaping of microbial communities.
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.
Ziegler, J.; Schmidt, S.; Chutia, R.; Müller, J.; Böttcher, C.; Strehmel, N.; Scheel, D.; Abel, S. Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation. J. Exp. Bot. 67, 1421-1432, (2016) DOI: 10.1093/jxb/erv539
Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation.
Dobritzsch, M.; Lübken, T.; Eschen-Lippold, L.; Gorzolka, K.; Blum, E.; Matern, A.; Marillonnet, S.; Böttcher, C.; Dräger, B.; Rosahl, S. MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides. Plant Cell 28, 583-596, (2016) DOI: 10.1105/tpc.15.00706
The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface.
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
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
plant defence genes, and correlates with enhanced
pathogen infection in AvrRpt2-expressing transgenic plants. Diverse
homologs can be found in some phytopathogens,
plant-associated and soil bacteria. Employing these putative bacterial
homologs and inactive AvrRpt2 variants, we can
uncouple the inhibition of MPK4/MPK11 activation from the cleavage of
and related members from the so-called NOI family,
as well as from auxin signalling. Thus, this selective suppression of
MAPKs is independent of the previously known
AvrRpt2 targets and represents potentially a novel virulence function of
Brömme, T.; Schmitz, C.; Moszner, N.; Burtscher, P.; Strehmel, N.; Strehmel, B. Photochemical Oxidation of NIR Photosensitizers in the Presence of Radical Initiators and Their Prospective Use in Dental Applications ChemistrySelect 1, 524–532, (2016) DOI: 10.1002/slct.201600048
Photochemical oxidation of near infrared (NIR) photosensitizers in the presence of diaryl iodonium salts bearing either bis(trifluoromethylsulfonyl)imide or hexafluorophosphate was investigated by exposure with NIR LEDs emitting either at 790 nm, 830 nm, 850 nm or 870 nm. Four different cyanines with barbituryl group at the meso position exhibit similar absorption in the NIR. These photosensitizers initiate in combination with diaryliodonium salts radical photopolymerization of dental composites with the focus to cure large thicknesses. Furthermore, the mixture comprising the cyanine and the iodonium salt was used to generate brown color in dental composites on demand. This required to understand the mechanism of dye decomposition in more detail applying exposure kinetics and a coupling of Ultra Performance Liquid Chromatography (UPLC) with mass spectrometry (MS) to analyze the photoproducts formed. Data showed cleavage of the polymethine chain at typical positions in case of the oxidized species. These were formed as result of electron transfer between the excited state of the photosensitizer and the iodonium salt. UPLC-MS experiments additionally indicated a certain sensitivity of the system upon adding of acids and radicals generated by thermal treatment of azobisisobutyronitrile (AIBN). Thus, treatment of the photoinitiator composition led almost to the same products no matter the system was either exposed with NIR light or treated with acids or radicals generated by thermal decomposition of AIBN. These findings helped to understand the large curing depth of 14 mm upon NIR exposure at 850 nm and the brown color formed.