Browsing by Person "Schaller, Andreas"

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Charakterisierung der Matrixmetalloproteinasen (SlMMP1 & SlMMP2) aus Tomate (Solanum lycopersicum) und ihre Rolle in der pflanzlichen Entwicklung und Pathogeninteraktionen
(2010) Pasule, Christian; Schaller, Andreas
Matrixmetalloproteinases belong to the family of metzincins and are widely distributed in prokaryotic as well as eukaryotic organisms. Mammalian matrixmetalloproteinases function in the formation of peptide hormones, growth factors and receptor proteins and regulate important physiological processes. Little is known about the function of the related plant matrixmetalloproteinases, except for a few enzymes in plants. Their function in tomato plants is still unresolved. However, preliminary data suggest that matrixmetalloproteinases may play essential roles in the regulation of development and pathogen defense in tomato plants. The present study aimed at the elucidation of the function of the tomato matrixmetalloproteinases SlMMP1 and SlMMP2 by purification and biochemical characterization of the recombinant proteins, detailed expression analysis in tomato plants, as well as the phenotypical and molecular analysis of transgenic plants with reduced expression levels for SlMMP1/2 (SlMMP1/2-RNAi). SlMMP1/2 were expressed in E. coli and purified by affinity chromatography. Activity of recombinant MMPs was stimulated by Ca2+-ions. The highest activity was measured for SlMMP1 at pH 6,5 and for SlMMP2 at pH 7. The apparent Km-values for a fluorigenic peptide substrate were 19,5 µM for SlMMP1 and 19,9 µM for SlMMP2, with a catalytic efficiency (kcat/KM) of 0,010 and 0,024 s-1*µM-1 respectively. These data suggest redundant functions for SlMMP1 and SlMMP2. SlMMP1 was found to be expressed in all tested organs and developmental stages with highest expression levels in eight days old seedlings and stems from six weeks old plants. A constitutive expression in leaves from six weeks old plants was not observed for SlMMP2. In eight days old seedlings, SlMMP2 showed higher expression in roots and hypocotyls than in cotyledons. The tissue-specific expression of SlMMP1 and SlMMP2 suggests specific functions and substrates for the two enzymes in different tissues, inspite of their similar biochemical properties. SlMMP1/2 were localised to cell walls from hypocotyls and roots using immunohistochemistry. This confirms the assumed extracellular localisation of the enzymes. Transgenic plants with reduced SlMMP1/2 expression levels exhibit alterations in surface structure leading to necrotic lesions two weeks after germination. The lesions spread over the entire hypocotyl during later stages of development. Cellular organisation was altered in cortical tissues accompanied by cell death events in the epidermis and subepidermal cell layer. These effects spread over leaves during later stages of development. SlMMP1/2-RNAi-plants accumulate massive amounts of phenolic compounds, and exhibit impaired root development with a reduction in overall root length and reduced numbers of primary and secondary lateral roots. Many of the observed effects suggest an involvment of ethylene and a higher ethylene production was in fact confirmed in SlMMP1/2-RNAi-plants. The effects of the loss of SlMMP1/2 on gene expression were analyzd with microarrays in hypocotyls from SlMMP1/2-RNAi-plants. 522 genes were found to be differentially regulated in SlMMP1/2-RNAi-hypocotyls, with 332 up- and 190 downregulated as compared with widtype plants. Genes with roles in plant development, stress, reaction during abiotic and biotic stimuli, and genes with unknown roles in biological processes appeared to be overrepresented among the differentially regulated genes, however, statistically not significantly. Genes with functions in transport were found to be underrepresented in RNAi-plants. Based on the observation that infection with Sclerotinia sclerotiorum led to induction of SlMMP1/2 gene expression, a possible function for the enzymes in pathogen defense was analyzed. A direct involvment of the enzymes in plant defense against the fungus can be excluded, however, because no differences in fungal growth were detected between wildtype and SlMMP1/2-RNAi-plants. Furthermore, it was analyzed if there are differences in resistance against the biotrophic pathogen Xanthomonas campestris pv. vesicatoria (Xcv) between wildtype and SlMMP1/2-RNAi-plants. Bacterial growth was found to be reduced for virulent, avirulent and non-pathogenic bacteria, accompanied by strong necrosis in SlMMP1/2-RNAi-plants. These data suggest a function for SlMMP1/2 in the regulation of non-specific rections against biotic stresses. The results of the present study provide a basis for a more complete understanding of the function of SlMMP1/2 in development and pathogene defense of tomato plants.
Funktionelle Charakterisierung der Subtilase At4g21630 aus Arabidopsis thaliana
(2012) Knappenberger, Mathias; Schaller, Andreas
The goal of this thesis was the molecular and functional characterization of the A. thaliana subtilase At4g21630.
Heterologe Expression und molekulare Charakterisierung des Dirigentproteins AtDIR6 aus A. thaliana
(2011) Pickel, Benjamin; Schaller, Andreas
Dirigent proteins are involved in the stereo- and regioselective control of plant secondary metabolism. Functionally described dirigent proteins from Forsythia intermedia and Thuja plicata couple coniferyl alcohol radicals to (+)-pinoresinol, a precursor of various lignans including the pharmaceutically relevant podophyllotoxin. The discovery of (?)-lariciresinol in A. thaliana roots and the accumulation of its precursor (?)-pinoresinol in a knock-out mutant lacking two pinoresinol reductases indicated the presence of a novel dirigent activity in A. thaliana which mediates the enantiocomplementary formation of (?)-pinoresinol. In this work AtDIR6 was identified as a candidate for this novel dirigent activity. The protein was cloned and heterologously expressed in a plant cell culture system. The recombinant protein was purified to appearent homogeneity by conventional chromatography methods. The purified protein was functionally active and directed the coupling of coniferyl alcohol radicals to (?)-pinoresinol in vitro. It was further shown that the stereoselectivity of AtDIR6 is opposed to that of known dirigent proteins in F. intermedia and T. plicata, and therefore, AtDIR6 is the first of the long-sought nantiocomplementary dirigent proteins. AtDIR6 was shown to possess a N-terminal signal peptide, which was cleaved during secretion between amino acids 29 and 30. Mature AtDIR6 accumulated extracellularly and remained non-covalently attached to the primary cell wall of suspension cultured cells. The native protein is glycosylated with two complex type and paucimannosidic Nglycans, respectively. It forms homodimers of app. 42 kDa and shows a high content of b-sheets. The functionally described dirigent proteins are small proteins that are characterised by the ability to bind coniferyl alcohol radicals and couple them enantiospecifically without possessing a catalytic activity of their own. Sequence identity between different dirigent proteins may be low. In these aspects dirigent proteins are similiar to lipocalins. A sequence alignment with dirigent proteins and lipocalins shows that the lipocalin-specific sequence motive, which is part of SCR II, is conserved among all functionally described dirigent proteins. Structural and mechanistic features of AtDIR6 suggest that dirigent proteins may belong to the calycin superfamily, which also includes lipocalins, and that their threedimensional structure may be that of a b-barrel.
Mobile signals in plant parasitism
(2024) Greifenhagen, Anne; Schaller, Andreas
Close to two percent of all flowering plants evolved parasitism, with some parasitic species, like Striga spp. from the Orobanchaceae family, posing a prevailing threat to crop yield. Parasitic weed management is challenging and requires a deeper understanding of the complex parasite-host relationship (Section 1.1). Parasitic plants infect and parasitize host plants through a multicellular feeding organ, the haustorium. This organ may either develop from the root tip as a single terminal haustorium or emerge multiple times along the growing roots, called lateral haustoria. In both cases, protohaustoria develop into mature haustoria that enable the withdrawal of water and nutrients. Parasitism depends on parasite and host endogenous signaling but also on communication between both partners (Section 1.2). This is similar to the development of other plant organs like lateral roots and symbiotic nodules, whose number is adjusted by an autoregulation of nodulation (AON) system. The induction of parasitic organs by pathogenic nematodes, but in particular also by parasitic plants, involves the manipulation, neofunctionalization, and interspecific exchange of mobile signals (Section 1.3). However, most of these molecular cues remain elusive in the parasitic plant-host plant interaction. This work aimed to address the biogenesis and function of parasite-derived endogenous and interspecific mobile signals involved in early till late stages of parasitism in the model system Phtheirospermum japonicum infecting Arabidopsis thaliana (Section 1.4). Transcriptome and genome studies on parasitic plants paved the way to unravel signaling cues contributing to parasitism. The evolution of parasitism correlates with the expansion of certain gene families followed by their parasitism-related neofunctionalization as seen for the KARRIKIN-INSENSITIVE 2 ‘divergent’-type (KAI2d) gene family in parasitic plants of the Orobanchaceae. Likewise, subtilisin-like serine protease (subtilase, SBT) genes in P. japonicum and Striga underwent an expansion, and some show haustorium tip-specific expression. The proteolytic activity of PjSBTs is required for proper haustorium formation and development. Despite their importance, no substrates of PjSBTs have been identified. In this work, PjSBT1.2.3 was found to be co-expressed with CLAVATA3(CLV)/EMBRYO-SURROUNDING REGION-related 3 (PjCLE3) during infection in the same domain of the haustorium. PjSBT1.2.3 cleaved PjCLE3 in vitro, thereby releasing the bioactive mature PjCLE3 peptide (Section 2.1, Fig.2.1.1). Sensing host-derived haustorium-inducing factors (HIFs) initiates haustorium organogenesis. In the absence of a host, the synthetic mature PjCLE3 induced protohaustorium formation similar to a host-derived benzoquinone HIF. Combined treatment of both HIFs potentiated their activity (Section 2.1, Fig.2.1.2). Pj cle3 knock-out hairy roots (HRs) formed fewer haustoria, particularly due to the absence of secondary protohaustoria (Section 2.1, Fig.2.1.3). These data demonstrate the existence of an autoregulation of haustoria formation (AOH) system as part of which the PjSBT1.2.3-PjCLE3 module, in analogy to AON, regulates the number of P. japonicum lateral haustoria. During the early stages of parasitism, the parasitism-related PjSBT1.2.3-PjCLE3 module promotes protohaustorium formation by sensitizing the parasite root for host-derived HIFs (Section 3.1). A homologous SBT-CLE module also exists in Striga, even though the parasite forms a terminal haustorium. Striga CLE2s are identical to host CLEs and this mimicry might improve nutrient allocations from the host during later stages of parasitism (Section 2.1, Fig.S2.1.2; Section 3.2). Similarly, parasite-derived cytokinin (CK) translocates through the haustorium inducing host hypertrophy, a swelling of host tissue above the infection site, thereby potentially benefiting parasite nutrient acquisition. In agrobacteria and plants, isopentenyltransferases (IPTs) synthesize CK precursors. Similar to SBTs and KAI2ds from parasitic plants, P. japonicum and S. hermonthica IPT1 genes exist as multiple copies, with one copy, PjIPT1a showing specific expression at the tip of haustoria (Section 2.2, Fig.2.2.1, Fig.2.2.2). Bioinformatic tools predicted a chloroplast transit peptide (CTP) for PjIPT1s, but PjIPT1-GFP fusions localized to cytoplasm and nucleus suggesting the CTP to be non-functional (Section 2.2, Fig.2.2.2, Fig.S2.2.2). To test substrate specificity and activity of both PjIPT1 copies, isoprenylation-activity was probed in vitro. PjIPT1b used both AMP and ATP as substrates, whereas PjIPT1a displayed a higher affinity for AMP, indicating that PjIPT1b may be the canonical, whereas PjIPT1a is the parasitism-related IPT (Section 2.2, Fig.2.2.4). This is further supported by the observation that CRISPR/Cas9-mediated mutation of PjIPT1a abolishes CK responses in the infected host (Section 3.3, Section 2.2, Fig.2.2.3). SBT-CLE, IPT-CK together with KAI2ds all have in common that their parasitism-related function may evolutionally result from gene duplication combined with neofunctionalization. Targeting duplicated and neofunctionalized genes may prove to be a promising strategy to combat parasitic weeds. (Sections 3.4, 3.5).
The formation of an apoplastic diffusion barrier in Arabidopsis seeds is regulated by peptide hormone signaling
(2022) Royek, Stefanie; Schaller, Andreas
Diffusion barrier formation is a critical factor in plant development. The most well described diffusion barriers in Arabidopsis are the Casparian strip and the cuticle. They function in the formation of organ boundaries, prevent water and molecule loss, and protect the plant against environmental stresses. The Casparian strip surrounds the root vascular tissue, whereas the cuticle covers aerial plant organs and is formed de novo during seed development. Embryonic cuticle formation is regulated by a peptide hormone signaling pathway, involving the leucine rich repeat receptor like kinases GASSHO1 (GSO1), GASSHO2 (GSO2) (Tsuwamoto et al. 2008) and the subtilisin-like serine protease ABNORMAL LEAF SHAPE 1 (ALE1). Whereas the latter pathway components have been identified in 2001 and 2008, the peptide hormone mediating the signaling has remained elusive. One aim of this work was to identify the missing pathway element. It was hypothesized that the peptide hormone is released from a larger precursor by ALE1 protease activity to trigger cuticle formation via interaction with the GSO receptors. To uncover the unknown element, the signaling pathway for Casparian strip formation, prooved to be a useful lead. Remarkably, Casparian strip and embryonic cuticle formation employ the same receptor (GSO1), and for Casparian strip formation the GSO1 ligands are known to be members of the CASPARIAN STRIP INTEGRITY FACTOR (CIF) protein family (Doblas et al. 2017, Nakayama et al. 2017). Based on its similarity to the mature CIF peptides and on its phenotypic appearance, it was speculated that a seed expressed protein, called TWISTED SEED1 (TWS1), could serve as the sought ALE1 substrate. As it can be challenging to link proteases to their physiological substrates, this work describes methods how to identify protease specific cleavage sites. One of them was applied to test if TWS1 serves as ALE1 substrate. GFP-tagged TWS1 was transiently coexpressed with ALE1 in Nicotiana benthamiana via agroinfiltration. An ALE1-specific TWS1 cleavage product was detected in the protein extract of coinfiltrated leaves. It was identified by pull down via GFP immunoprecipitation, subsequent separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) analysis. Another method, described in this work, is the identification of protease cleavage sites by in-gel reductive dimethylation: cleavage product-containing gel bands are treated with formaldehyde and cyanoborohydride, prior to in-gel tryptic digest, to achieve a dimethylation of N-terminal free amino groups. The chemically modified N-termini can rapidly be identified and assigned to previous cleavage by the protease of interest. With the method described above, it was found that TWS1 is c-terminally cleaved by ALE1. The two amino acids directly flanking the cleavage site were found to be important for ALE1 cleavage site selection, as their substitution caused a loss of ALE1- dependent cleavage. Our cooperation partners demonstrated an interaction of mature TWS1 with the GSO receptors. The binding affinity of mature TWS1 was reduced by a 3 amino acid C-terminal extension, demonstrating the biological relevance of ALE1-mediated TWS1 processing. Like the CIFs, TWS1 contains a DY tyrosine sulfation motive at its N-terminal processing site. The role of tyrosine sulfation in precursor processing is largely unexplored and was addressed in this work by comparing in-vitro cleavage of different sulfated versus nonsulfated TWS1 precursors. SBT1.8 was found to cleave TWS1 at the N-terminal processing site, and cleavage site selection was influenced by the sulfation state of TWS1 P2´ tyrosine. A homology based 3D model of SBT1.8 was created, which suggested that SBT1.8 interacts with the negatively charged sulfate via a positively charged arginine residue (R302). The role of R302 in substrate binding and recognition was confirmed by in-vitro cleavage assays with mutated SBT1.8 versions, in which R302 was replaced. N-terminal TWS1 cleavage was no longer observed when R302 was substituted. Likewise, no N-terminal cleavage was observed for two other seed expressed Arabidopsis subtilases (SBT1.1 and SBT5.4) that feature an arginine at the corresponding position, indicating that the sole presence of R302 is not sufficient for N terminal cleavage site recognition.