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

Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis 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 corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. 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.

Efficient Isolation and Purification of High-Quality Arabidopsis thaliana Trichomes.

Trichomes are fine outgrowths on the surface of aerial plant organs which play a role in protecting plants against water loss, UV radiation, and herbivore feeding. Throughout the years, trichomes have become a popular paradigm in biological research. For example, trichomes on rosette leaves of the reference plant Arabidopsis thaliana have been used as a model to investigate cell development, cell differentiation, and, more recently, cell wall biogenesis. State of the art -omics studies on specific cell types or tissues often require physical separation, enrichment, and purification. This, of course, also applies to leaf trichomes, and various methods have thus been proposed to separate trichomes and leaf tissue. Though most of these methods are indeed suitable for trichome isolation, they suffer in part from tedious operating procedures, low yield, poor sample purity, and reduced trichome integrity. We have thus revised a previously reported method for trichome isolation, and report here an efficient and scalable procedure for the isolation and gradient centrifugation-based purification of high-quality A. thaliana trichomes. We describe the preparation of plant material and trichome release, which is based on prolonged gentle agitation of plant seedlings in the presence of a cation-chelating agent that weakens trichome-leaf interactions. We also outline the steps for the subsequent recovery and purification of the isolated crude trichome fraction, which is based on the use of discontinuous sucrose gradient centrifugation. In addition to A. thaliana, we have found that this procedure can be applied to release and enrich glandular and non-glandular trichomes from various species, including Solanum lycopersicum and Nicotiana benthamiana. The resulting purified leaf trichomes can be subjected to different types of bioassays, including histochemistry, biochemical quantification of cell wall monosaccharides, and transcriptomics, as well as proteomic profiling. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of plant material for release and enrichment of A. thaliana trichomes Basic Protocol 2: Purification of A. thaliana trichomes by density gradient centrifugation.

An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome.

BACKGROUND: Rosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses. RESULTS: To take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species. CONCLUSIONS: We provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.