Biology subjects

Moschou, P.

Publications and source records attributed to Moschou, P..

4 recordsLinked to original sources

bioRxiv2020

Protein Detection and Localization in Plant Cells Using Spot-Tagging

Fluorescent labelling of proteins without compromising their activity is crucial for determining their spatiotemporal localization while retaining their functionality. Spot-tag is a 12-amino acid peptide recognized by a single-domain nanobody. Here we introduce the spot-tag as a labelling strategy for proteins in fixed and living plant cells, using as an example the microtubule motor centromeric protein E-related Kinesin 7.3. Spot-tagging of ectopically introduced Kinesin 7.3 does not interfere with microtubules and spot staining results in a close-grained fluorophore labelling revealing a localization pattern that resembles "beads-on-a-string". We anticipate that our protocol will apply to many more demanding protein cellular targets, offsetting activity perturbations and low photon quantum yields imposed by other protein-tagging approaches.

plant biology↗

bioRxiv2019

Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems

Proximity-dependent biotin labelling (PDL) uses a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest. This enables covalent biotin labelling of proteins and allows subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of PDL in plants. Here we present the results of a systematic study applying a variety of PDL approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both non-biotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.

plant biology↗

bioRxiv2018

The Rhizoctonia solani LysM and RlpA-like effector proteins contribute to virulence suppressing chitin-triggered immunity and hypersensitive response

Rhizoctonia (in Greek \"root-killer\") species and particularly R. solani attacks a broad range of plant species and crops. It belongs to Basidiomycota and is a soil-borne pathogen causing mainly damping-off diseases of seedlings and root rot, although it can infect plants in any stage. Despite the severity of this disease, many aspects in R. solani infection biology still remain to be elucidated. Here we investigated the role of two effector candidates, predicted from the genome of a R. solani AG2-2IIIB strain that uses sugar beet as a host. Gene expression analysis showed that genes encoding for a LysM effector and a rare lipoprotein-A-like protein (RsRlpA) were induced upon early infection stages. When heterologous expressed in Cercospora beticola the two genes contributed to virulence. The RsLysM effector showed chitin- binding affinity and suppression of chitin-triggered immunity but could not protect hyphae from hydrolysis. The RsRlpA effector suppressed hypersensitive response in Nicotiana benthamiana leaves. Overall, this study provides us with valuable information on R. solani infection biology, implying that this organism relies on mechanisms similar to hemibiotrophic pathogens in order to establish a successful infection.

plant biology↗

bioRxiv2017

The Proteolytic Landscape Of An Arabidopsis Separase-Deficient Mutant Reveals Novel Substrates Associated With Plant Development

Digestive proteolysis executed by the proteasome plays an important role in plant development. Yet, the role of limited proteolysis in this process is still obscured due to the absence of studies. Previously, we showed that limited proteolysis by the caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) modulates development in plants through the cleavage of unknown substrates. Here we used a modified version of the positional proteomics method COmbined FRActional DIagonal Chromatography (COFRADIC) to survey the proteolytic landscape of wild-type and separase mutant RADIALLY SWOLLEN 4 (rsw4) root tip cells, as an attempt to identify targets of separase. We have discovered that proteins involved in the establishment of pH homeostasis and sensing, and lipid signalling in wild-type cells, suggesting novel potential roles for separase. We also observed significant accumulation of the protease PRX34 in rsw4 which negatively impacts growth. Furthermore, we observed an increased acetylation of N-termini of rsw4 proteins which usually comprise degrons identified by the ubiquitin-proteasome system, suggesting that separase intersects with additional proteolytic networks. Our results hint to potential pathways by which separase could regulate development suggesting also novel proteolytic functions.

plant biology↗