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

Huebbers, J. W.; Caldarescu, G. A.; Kubatova, Z.; Sabol, P.; Levecque, S. C. J.; Kuhn, H.; Kulich, I.; Reinstaedler, A.; Buettgen, K.; Manga-Robles, A.; Melida, H.; Pauly, M.; Panstruga, R.; Zarsky, V.

doi:10.1101/2022.12.30.521597

bioRxiv · 10.1101/2022.12.30.521597

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

Huebbers, J. W.·Caldarescu, G. A.·Kubatova, Z.·Sabol, P.·Levecque, S. C. J.·Kuhn, H.·Kulich, I.·Reinstaedler, A.·Buettgen, K.·Manga-Robles, A.·Melida, H.·Pauly, M.·Panstruga, R.·Zarsky, V.

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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 of A. thaliana exo70H4 and mlo2 mlo6 mlo12 triple 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 in exo70H4 and mlo2 mlo6 mlo12 mutants. 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 the mlo triple mutant and, vice versa, MLO6- GFP exhibited aberrant subcellular localization in trichomes of the exo70H4 mutant. 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, exo70H4 and mlo 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.

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Huebbers, J. W., Caldarescu, G. A., Kubatova, Z., Sabol, P., Levecque, S. C. J., Kuhn, H., Kulich, I., Reinstaedler, A., Buettgen, K., Manga-Robles, A., Melida, H., Pauly, M., Panstruga, R., Zarsky, V.. 2022-12-30. Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and powdery mildew susceptibility. https://doi.org/10.1101/2022.12.30.521597

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