To determine callose deposition induced by chitosan, DIMBOA, DIMBOA-Glc, HDMBOA-Glc, and 4MO-I3M, callose abundance after chemical infiltration was analyzed from 15 randomly collected leaf segments from five different 10-day-old wheat seedlings and six 5-week-old Arabidopsis accession Columbia-0 (Col-0) plants per treatment. Each leaf was infiltrated with 500 μl of 0.2% (w/v) low viscous chitosan (Sigma, USA), which has a molecular weight of approximately 190 kDa. Chitosan was dissolved to 1% in 1% acetic acid and diluted to 0.2% by distilled water. DIMBOA, DIMBOA-Glc, and HDMBOA-Glc were dissolved to 80 μg/ml by 1.96% methanol and 0.04% acetic acid. 4MO-I3M (Phytoplan, Germany) was dissolved to 80 μg/ml by distilled water. Twenty-four hours after infiltration, leaf segments around the infiltration spots (approximately 4 cm2) were collected and destained by 96% ethanol to remove chlorophyll. Leaf segments with cell death symptoms were excluded. Destained leaf segments were stained with 70 mM phosphate buffer (pH 9.0) containing 0.01% aniline blue (Sigma, USA). Callose deposition was observed by epifluorescence microscopy (Zeiss, Germany), and callose spots were counted by eye. To determine callose deposition induced by aphid feeding, five aphid adults were placed in cages (2-cm diameter) on the leaves of 14-day-old wheat plants. After 7 days, all aphids were removed. Aphid-infested leaf segments were collected from 10 different plants per line. Leaf segment destaining and callose staining were performed as described above. As callose deposition following aphid infestation showed variable shapes and intensity, we first determined the total area of callose per plant (μm2) using Digimizer (MedCalc Software, Belgium). We then assessed the intensity of the staining on a relative scale from 1 (weak intensity) to 5 (strong intensity). Then, to obtain an integrated measure of callose induction, we multiplied intensity with callose area for each plant individually.

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