Analysis of extracellular vesicles released by the phytopathogen Pseudomonas syringae pv. tomato DC3000 in planta

Krassini, Laura (2026): Analysis of extracellular vesicles released by the phytopathogen Pseudomonas syringae pv. tomato DC3000 in planta. Dissertation, LMU München: Faculty of Biology

Abstract

Pseudomonas syringae pathovar tomato DC3000 (Pto DC3000) is a phytopathogen colonising the leaf apoplast of the important crop tomato and the model plant Arabidopsis thaliana (At), resulting in bacterial speck disease. Recently, extracellular vesicles (EVs) of Pto DC3000 became of interest as they were shown to carry various proteins important for the plant infection process. In this dissertation, Outer membrane porin F (OprF) and Ampicillin C (AmpC) were established as protein markers for the detection of in vitro and in planta released Pto-EVs by immunoblotting. Having established a detection method, the proteomic cargo of plant EVs and of Pto-EVs isolated from Pto-infected Arabidopsis apoplast was determined by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Comparing the proteinaceous cargo of apoplastic At-EVs from mock and from infected plants revealed mild changes associated with plant immunity. The protein composition in Pto-EVs from Arabidopsis apoplast exhibited greater divergence from previously reported protein compositions in Pto-EVs released in culture conditions, with indications for potential roles in iron uptake by Pto-EVs. Next, protein candidates for generating Pto-EV biomarkers were selected based on the proteomic dataset. These Pto DC3000 biomarker lines were created by introducing mCherry fusion proteins encoded on a plasmid. After confirming that the transformation did not induce any growth- or vesiculation-related phenotypes, the EV-biomarker line Pto DC3000 mCherry(mCh)-tagged Pto-EV-MARKER 1 (PEM1) was established for subsequent analyses. Interactions between mCherry-tagged Pto-EVs from Pto DC3000 mCh-PEM1 with Pto DC3000 cells, with Arabidopsis mesophyll protoplasts and roots, and with the necrotrophic fungus Botrytis cinerea were investigated by microscopy. While there were no interactions between mCherry-tagged Pto-EVs with Pto DC3000 cells and Arabidopsis mesophyll protoplast and root cells visible, Botrytis cinerea hyphae were stained positively for mCherry after incubation with EVs derived from Pto DC3000 mCh-PEM1. Incubation of Arabidopsis roots with FM4-64-stained Pto-EVs from Pto DC3000 wild type (WT) and Pto DC3000 mCh-PEM1 revealed a fluorescent signal of FM4-64 at the plant plasma membrane. Since the incubation with FM4-64-stained mCh-PEM1 Pto-EVs revealed both signals, FM4-64 and mCherry, at the plant cell membranes, it is likely that the staining led to side effects, which allowed the visualisation of Pto-EVs at the plant plasma membrane.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 570 Life sciences
• Faculties: Faculty of Biology
• Language: English
• Date of oral examination: 23. March 2026
• 1. Referee: Robatzek, Silke
• Signature of the printed copy: 0001/UMC 31908