Browsing by Subject "Subtilase"

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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.
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.