Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and powdery mildew susceptibility

Abstract

EXO70 proteins are essential constituents of the octameric exocyst complex implicated in vesicle tethering during exocytosis, while MLO proteins are plant-specific calcium channels of which some isoforms play a key role during fungal powdery mildew pathogenesis. We here detected by a variety of histochemical staining procedures an unexpected phenotypic overlap ofA. thaliana exo70H4andmlo2 mlo6 mlo12triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses of isolated trichomes corroborated deficiencies in the composition of trichome cell walls inexo70H4andmlo2 mlo6 mlo12mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO variants exhibited extensive co-localization of these proteins at the trichome plasma membrane and cell wall. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of themlotriple mutant and,vice versa, MLO6-GFP exhibited aberrant subcellular localization in trichomes of theexo70H4mutant. Transgenic expression of GFP-marked PMR4 callose synthase, a previously identified cargo of EXO70H4 dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in mlo triple mutant plants. In vivo protein-protein interaction assays uncovered isoform-preferential physical interaction between EXO70 and MLO proteins. Finally,exo70H4andmlomutants, 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.

Comprehensive comparative assessment of theArabidopsis thalianaMLO2-calmodulin interaction by variousin vitroandin vivoprotein-protein interaction assays

“…2021) cyclic nucleotide gated channels (CGNCs; Meng et al . 2020) and exocyst EXO70 subunits (Huebbers et al . 2022), no other plant proteins have been reported to date to associate in planta with MLO proteins.…”

“…While this approach was used in the context of the present work to probe the MLO2-CAM2 interaction, it could be deployed in future studies to identify novel interaction partners of MLO proteins. Apart from CAM (Kim et al 2002a;Kim et al 2002b;Kim et al 2014;Zhu et al 2021) cyclic nucleotide gated channels (CGNCs; Meng et al 2020) and exocyst EXO70 subunits (Huebbers et al 2022), no other plant proteins have been reported to date to associate in planta with MLO proteins.…”

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2–CALMODULIN2 interaction by various in vitro and in vivo protein–protein interaction assays

“…While this approach was used in the context of the present work to probe the MLO2–CAM2 interaction, it could be deployed in future studies to identify novel interaction partners of MLO proteins. Apart from CAM [ 22 , 23 , 26 , 24 ], cyclic nucleotide gated channels (CGNCs; [ 14 ]) and exocyst EXO70 subunits [ 46 ], no other plant proteins have been reported to date to associate in planta with MLO proteins. Being integral membrane proteins, the identification of protein interaction partners is notoriously difficult for MLO proteins.…”