) was used. As secondary antibody on immunoblots we applied stabilized goat anti-mouse HRP-conjugated antibodies (product No. 1858413, Pierce). For immunofluorescence sheep-anti-mouse immunoglobulins G (catalogue No. 515-005-003, Dianova) were labeled with the fluorescent dye Atto532 or Atto647N (provided by K. H. Drexhage, Dept. of Chemistry, University of Siegen, Germany).

Supporting Online Material for Anatomy and Dynamics of a Supramolecular Membrane Protein Cluster

The Arabidopsis frill1 (frl1) mutant, that has serrated petals and sepals but no other large changes in plant morphology, was studied The frl1 had a mutation in STEROL METHYLTRANSFERASE 2 and an altered sterol composition It was found that the frl1 mutation causes ectopic endoreduplication in petal tips that do not normally endoreduplicate The rosette leaves of frl1 also showed an enhanced level of endoreduplication, but their morphology was hardly affected These facts suggest that the suppression of endoreduplication is important for petal morphogenesis and the normal sterol composition is required for this suppression

Ectopic endoreduplication caused by sterol alteration results in serrated petals in Arabidopsis

Cell polarity is a common response of single plant cells in response to bacterial or fungal attack, typically leading to a polar rearrangement of the cytoskeleton and directional movement of various organelles such as the nucleus and Golgi to pathogen contact sites at the cell surface. This process is accompanied by a re-localisation of a subset of proteins and directed vesicle trafficking that might contribute to focal deposition of de novo synthesized cell wall material (called papilla) between the preformed plant cell wall and the plasma membrane underneath pathogen contact sites. Secretory processes involving vesicles that release antimicrobial cargo and cell wall building blocks have been thought for a long time to contribute to plant defence. Recently, genetic evidence proved �soluble N-ethylmaleimide sensitive factor attachment protein receptor� (SNARE) proteins, which are known to drive intracellular vesicle fusions in various eukaryotes, to be required for resistance responses. A ternary SNARE complex containing plasma membrane-resident PEN1 syntaxin, SNAP33 and endomembrane resident VAMP721/722 is essential to restrict entry of several tested powdery mildew fungi into leaf epidermal sites. Focal accumulation of each of these three proteins at incipient fungal entry sites implies the existence of a secretory machinery that targets and discharges vesicle-resident antimicrobial cargo towards the fungal intruder into the extracellular space. However, the exact mechanism underlying focal SNARE protein accumulation at incipient entry sites remains elusive. In addition, it remains to be shown whether SNARE proteins must be concentrated at these sites to confer effective disease resistance. To reveal the specificity of focal PEN1 accumulation at the cell periphery diverse pathogenic interactions involving direct penetrating fungi were analysed using an Arabidopsis line expressing functional GFP-PEN1 driven by the 35S promoter in the pen1-1 mutant background. Neither non-adapted Magnaporthe grisea, Phakopsora pachyrhizi, or Colletotrichum destructivum nor adapted Hyaloperonospora parasitica or Colletotrichum higginsianum ascomycetes/oomycetes triggered detectable GFP-PEN1 accumulation. However, GFP-PEN1 accumulated at interaction sites with both non-adapted and adapted powdery mildews Erysiphe pisi, Blumeria graminis f sp. hordei, and Golovinomyces orontii. This suggests that only powdery mildews trigger PEN1 focal accumulation or that the other tested fungi/oomycetes suppress or avoid PEN1 accumulation at entry sites. It is conceivable that in pathogenic interactions where PEN1 accumulation was undetectable other syntaxins might become concentrated. Since PEN1 accumulation was detectable during compatible interactions with adapted and incompatible interactions with non-adapted powdery mildews, focal PEN1 concentration per se does not predict effective resistance responses. It is possible that the adapted powdery mildew fungi intercept the SNARE protein-dependent resistance response through inhibition of ternary SNARE complex formation or of other components in this secretory pathway. Alternatively, virulent powdery mildews might have the ability to detoxify PEN1-delivered antimicrobial cargo. Unexpectedly, GFP-PEN1 was also detected in the interior of haustorial encasements after fungal entry into epidermal cells in about 10% of G. orontii interaction sites containing a haustorial complex. Whilst haustoria are essential for pathogenesis and nutrient uptake, haustorial encasements are presumed to terminate fungal growth. Of eight additional tested transgenic lines, each expressing a different fluorochrome-tagged fusion protein known to accumulate at incipient powdery mildew entry sites, only YFP-SNAP33 accumulated at a higher incidence (~25%) at haustorial encasements. These findings support previous ideas that haustorial encasements might be an extension of cell wall-associated defense responses at the cell periphery. In a second experimental approach, I have developed a novel method for confocal high-throughput imaging at subcellular resolution. I have generated a chemically mutagenized M2 population of an Arabidopsis line expressing functional GFP-PEN1 driven by the 35S promoter in the pen1-1 mutant background. Following inoculation with B. g. hordei conidiospores, I aimed to identify mutant lines showing an aberrant PEN1 focal accumulation pattern at attempted fungal entry sites. Of several identified M2 candidates, one line, tentatively designated defect in focal accumulation 1 (dfa1), was validated in selfed M3 progeny. This line shows a reduced incidence of PEN1 accumulation beneath B. g. hordei appressoria. Further genetic and molecular characterization of this mutant line should provide deeper insights in mechanisms underlying the polar accumulation of PEN1 syntaxin upon powdery mildew attack.

Molecular characterization of pathogen-triggered cell polarity

In the last years, lateral compartmentalization has become a well-recognized topic in plant membrane research. Especially the membrane raft hypothesis receives particular attention, since it gives a conceivable explanation for the spatial and temporal organization of biological membranes. The aim of the work presented here was the investigation of the possible involvement of membrane rafts in biotic stress responses. In this study we elucidated in a quantitative proteomics approach immediate-early protein dynamics in plasma membrane-derived detergent-resistant membranes (DRMs) in response to bacterial flagellin. Proton ATPases and receptor-like kinases were the most prominently enriched protein classes. Strikingly, the flagellin receptor FLS2 was consistently relocalized to DMRs, indicative of its possible recruitment to membrane rafts prior to ligand-induced endocytosis. We performed reverse genetics and pharmacological interference to address the potential contribution of the identified proteins in flg22-triggered responses and thereby identified two novel players of elicitor-dependent oxidative burst control. In a second approach we aimed to elucidate the potential role of membrane rafts and/or sterols during the cellular compartmentalization process in the context of the Arabidopsis�powdery mildew interaction. We showed that the SNARE AtPEN1, which focally accumulates at fungal entry sites, partially associated with DRMs in a sterol-dependent manner. A complementary approach aiming at the genetic interference with membrane raft function revealed that a subset of sterol biosynthesis mutants displayed enhanced resistance towards the adapted powdery mildew pathogen, Golovinomyces orontii. This could be partially correlated with an aberrant focal accumulation of GFP-AtPEN1 underneath fungal attack sites, suggesting that the proper sterol composition, which might affect membrane raft integrity, could be important for the focal accumulation of GFP-AtPEN1 at attempted fungal entry sites. In sum our data indicate that membrane rafts might play a role in the compartmentalization of biological processes at the plasma membrane in response to biotic stimuli.

Nana Friederike Zappel

Papers

Membrane rafts in plant-pathogen interactions