Unraveling the interplay of MLO and exocyst complex proteins in localized secretion in plant cells

  • Untersuchung des Zusammenspiels von MLO und EXO70 Proteinen in der lokalisierten Sekretion in Pflanzenzellen

Hübbers, Jan Wilhelm; Panstruga, Ralph (Thesis advisor); Buyel, Johannes Felix (Thesis advisor)

Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


Mildew resistance Locus O (MLO) genes encode plant-specific integral membrane proteins that are subdivided into seven clades. Clade IV and clade V MLO proteins gained economic relevance as loss-of-function mutant alleles of their coding genes confer a durable resistance to the fungal powdery mildew disease. Though MLO proteins were recently identified as putative calcium channels, it remains enigmatic how this calcium channel-activity blends into mlo-mediated powdery mildew resistance and other MLO-associated cellular processes. In the model plant Arabidopsis thaliana, loss-of-function of certain MLO proteins is linked to defects in root thigmomorphogenesis, pollen tube guidance to the ovule and growth of the pollen tube apex, suggesting a role of MLO proteins in localized secretion. Localized secretion is the redirection of secretory processes to specific domains in the cellular cortex for a targeted exocytosis of cellular compounds. The notion that MLO proteins play a pivotal role during vesicle tethering was spurred by almost identical alterations in callose deposition in trichomes on leaves of the powdery mildew resistant mlo2 mlo6 mlo12 triple mutant and trichomes deprived of the exocyst complex subunit EXO70H4. The EXO70 subunits of the conserved octameric exocyst complex are widely regarded as important actuators of the yet elusive mechanism of localized secretion in plant cells. Based on the similar phenotypes in exo70H4 and mlo2 mlo6 mlo12 mutant trichomes, I addressed the question whether MLO proteins and EXO70 proteins interplay in the secretory pathway to mediate localized secretion in plant cells. Initially, I developed an efficient method for the isolation of A. thaliana rosette leaf trichomes to facilitate a comprehensive analysis of the cell walls of wild type and mutant trichomes. I demonstrated that the trichomes provided by this procedure are suitable for various downstream analyses, including a proteomic profiling of trichomes. Afterwards, I subjected isolated wild type as well as exo70H4 and mlo2 mlo6 mlo12 mutant trichomes to various cell wall analyses. Histochemistry confirmed near identical defects in the targeted deposition of cell wall components in exo70H4 and mlo2 mlo6 mlo12 mutant trichomes, while a biochemical quantification of monosaccharides and Fourier-transform infrared spectroscopy indicated globally altered cell wall characteristics in these mutant trichomes. Luciferase complementation revealed that MLO2, MLO6 and MLO12 preferentially interact with distinct EXO70 subunits. I confirmed the interaction for the MLO6-EXO70H4 tandem by yeast two-hybrid, which further uncovered that the interaction of EXO70H4 and MLO6 is mediated by the carboxy-terminus of the MLO6 protein. In this context, I exploited published sequence data of 341 MLO proteins from different plant species and identified a conserved motif in the carboxy-terminus of clade IV and clade V MLO proteins that probably mediates the binding of target proteins. Eventually, infection assays with the adapted powdery mildew fungus Erysiphe cruciferarum revealed a synergistically enhanced resistance of combined mlo exo70H4 double mutants to powdery mildew infection. My data uncover a shared function of MLO proteins and EXO70H4 in the secretory pathway and raise the question whether MLO-EXO70 membrane modules are a common theme in localized secretion in plant cells.