Analysis 3

All digital images were analysed with the AnalySIS 3.2 (Olympus) software for image analysis. The thickness of the palisade and the spongy parenchymas were measured in five regions along the leaf lamina. The cell area and shape of upper and lower epidermis, palisade, and spongy parenchyma were quantified in 15 cells per each tissue per section. More specifically, cell shape was characterised as (a) aspect ratio (maximum width/height ratio of a bounding rectangle for the cell, defining how it is elongated), (b) sphericity (roundness of a particle with spherical particles having a maximum value of 1), and (c) convexity (the fraction of the cell's surface area and the area of its convex; a turgid cell has a maximum value of 1) [39 (link), 40 ]. The cell area occupied by phenolic compounds was measured in 3 regions (150 × 200 μm² each) of the mesophyll per section selected avoiding veins. Thus, the area occupied by phenolic compounds was calculated as the percentage of tissue/picture occupied by compounds appearing autofluorescent at the above-reported filter settings [41 ].

At harvest, mature leaves were sampled from five plants per treatment, fixed in a 38% formaldehyde, glacial acetic acid, 50% ethanol solution (5:5:90 by volume) and prepared for microscopy according to standard protocols (De Micco et al., 2011 (link); Carillo et al., 2020 (link)). In brief, subsamples of leaves were dehydrated and embedded in the acrylic resin JB4 (Polysciences, Hirschberg, Germany) and thin sectioned (5-μm thick) through a rotary microtome. The cross sections, stained with 0.5% Toluidine blue in water, were observed through a transmitted light microscope (BX60; Olympus, Hamburg, Germany). Digital images were captured (Camedia C4040; Olympus, Hamburg, Germany) and subjected to digital image analysis (AnalySIS 3.2; Olympus, Hamburg, Germany) to quantify the following traits: the thickness of leaf lamina, palisade, and spongy parenchyma, the quantity of intercellular spaces in the palisade parenchyma (in six regions per section). Another set of subsamples of leaves was devoted to peeling, and microphotographs of epidermal strips of the upper and the lower lamina surfaces were analyzed to quantify the following stomata traits: stomata frequency (number of stomata per mm² in three regions per peel) and stomata size (length and width of the guard cells in 10 cells per lower epidermis peel).

CHO-sCR3 cells were cultured in F12 medium supplemented with 10% FCS at 37°C and culture supernatants were harvested every week. sCR3 was purified from the filtered culture supernatant by using an immunoaffinity chromatography matrix prepared by a covalent linkage of MEM-174 mAb to Cyanogen bromide (CNBr)-activated Sepharose 4B beads (GE Healthcare, Piscataway, NJ). The bent and extended conformers of the sCR3 ectodomain were separated by gel filtration chromatography on Superdex 200 HR (GE Healthcare, Piscataway, NJ) in HBSS buffer complemented with 2 mM CaCl₂ and 2 mM MgCl₂. The freshly separated conformers of sCR3 were directly applied on glow-discharged carbon copper grids (Benada and Pokorny, 1990 (link)), stained with 0.75% uranyl formate, and visualized in a Philips CM 100 transmission electron microscope (FEI, Eidhoven, Netherlands) equipped with a MegaViewII slow scan camera controlled by AnalySis 3.2 software (Olympus Soft Imaging Solutions, Münster, Germany).

CD14-positive cells were treated with 22.5 pM CyaA toxin or CyaA-AC⁻ toxoid for 5 days as described above and fixed for 2 h with 2% glutaraldehyde–PBS. After three washes with ice-cold PBS, cells were postfixed with 0.5% osmium tetroxide–PBS and incubated overnight at 4°C. Fixed and washed samples were dehydrated with ethanol using a standard procedure. Cells were embedded in epoxy resin (EMBed-812 embedding kit; Electron Microscopy Sciences). Ultrathin sections were visually contrasted using lead citrate and uranyl acetate (54 (link)). Final samples were examined using an FEI Morgagni 268(D) electron microscope (FEI, Brno, Czech Republic) at 80 kV. Digital images were recorded with a MegaViewIII slow-scan camera and processed by AnalySis 3.2 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany) using standard software modules (shading correction, digital contrast enhancement).

RIPK4 in tissue samples was detected using immunohistochemistry as previously described [27 (link)]. Sections were evaluated under the BX41 microscope (Olympus Optical Co., Tokyo, Japan), the ColorView III camera (Soft Imaging System, Hanover, Germany), and analySIS 3.2 software (Soft Imaging System, Hanover, Germany). The sections were evaluated semi-quantitatively, as previously described [27 (link)]. Since we observed heterogenous, uniform, and granular staining, the assessment was performed separately for each of the staining patterns.