Interplay of MLO and exocyst complex proteins in localized secretion in plant cells

• Applicant: Professor Dr. Ralph Panstruga
• Subject Area: Plant Cell and Developmental Biology ; Plant Genetics and Genomics ; Plant Physiology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 411779037

• Final Report Year: 2023

Final Report Abstract

In this joint DFG-GCAR co-funded project, carried out in collaboration with the group of Viktor Žárský (Charles University, Prague, Czech Republic), we aimed to test the hypothesis whether plant (Arabidopsis thaliana) exocyst EXO70 subunits and MLO integral membrane proteins co-function in localized secretion. The exocyst complex is an octameric protein complex that tethers vesicles prior to membrane-membrane fusion in exocytosis. Different from other eukaryotes, one of its subunits, EXO70, exists in multiple paralogs in plants. MLO proteins are plant-specific integral membrane proteins with seven membrane-spanning domains. They are best known for their role as susceptibility factors for the fungal powdery mildew disease. Like EXO70s, MLO proteins exist as medium-sized protein families. Starting from the initial observation that A. thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutants exhibit a similar phenotype of mislocalized callose deposition in the secondary cell walls of rosette leaf trichomes, we wondered whether EXO70 and MLO proteins may co-function in targeted exocytosis. We first established a robust protocol for the isolation of highquality trichomes for further analyses. We then expanded the phenotypic characterization of the mutants by a variety of histochemical staining procedures of detached trichomes, which further revealed possibly related cell wall alterations in exo70H4 and mlo2 mlo6 mlo12 triple mutant plants. Biochemical and Fourier transform infrared spectroscopic analyses of isolated trichomes corroborated deficiencies in the composition of trichome cell walls in exo70H4 and mlo2 mlo6 mlo12 mutants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoformpreferential physical interaction between EXO70 and MLO proteins, with the carboxyl-terminal region of MLO6 conferring the interaction with EXO70H4. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility, possibly by (co-)regulating focal secretion of cell wall-related cargo.

Publications

• An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome. Plant Methods, 18(1).
  Huebbers, Jan W.; Büttgen, Kim; Leissing, Franz; Mantz, Melissa; Pauly, Markus; Huesgen, Pitter F. & Panstruga, Ralph
  
• Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and susceptibility to powdery mildew. The Plant Cell, 36(4), 1007-1035.
  Huebbers, Jan W.; Caldarescu, George A.; Kubátová, Zdeňka; Sabol, Peter; Levecque, Sophie C. J.; Kuhn, Hannah; Kulich, Ivan; Reinstädler, Anja; Büttgen, Kim; Manga-Robles, Alba; Mélida, Hugo; Pauly, Markus; Panstruga, Ralph & Žárský, Viktor
  
• Proteomics dataset on detached and purified Arabidopsis thaliana rosette leaf trichomes. Data in Brief, 46, 108897.
  Huebbers, Jan W.; Mantz, Melissa; Panstruga, Ralph & Huesgen, Pitter F.