Fig. 4: BcCrh1 triggers plant immunity responses and enhances plant resistance to Botrytis infection.

a ROS accumulation. N. benthamiana leaves were Agroinfiltrated with free GFP, and native or enzymatic inactive (MBcCrh1) BcCrh1. After 48 h samples were stained with DAB, they were decolorized with ethanol and staining intensity was quantified with ImageJ. b Calose deposition. N. benthamiana leaves were infiltrated with 11 μM of purified proteins. Photomicrographs indicating callose deposition induced by BcCrh1 and MBcCrh1. After 24 h leaves were stained with aniline blue, bleached with ethanol, and images were captured with a fluorescent microscope (Bar = 50 μm). Callose levels were estimated by quantification of the number of spots per square millimeter using ImageJ. All data (n = 15 in (a), n = 12 in (b)) from three independent biological replicates are plotted as black dots. c Defense gene expression. Tomato leaves were infiltrated with purified proteins and relative expression levels of selected defense genes were determined by qRT-PCR 24 after 24 and 48 h. Values represent mean ±;SD (n = 9) from three independent biological replicates and three technical replicates. d Infection assay. N. benthamiana leaves were infiltrated with 11 μM of GFP (mock) or purified proteins, 48 h later the leaves were inoculated with B. cinerea mycelia plugs, the plants were incubated for an additional 72 h in a moist chamber and then symptoms were recorded. Data are from three independent experiments, each with four replications. e Infection assay of A. thaliana transgenic plants. Leaves of transgenic plants that express GFP (35S:GFP) or BcCrh1 (two independent plants, 35S:BcCrh-1# and 35S:BcCrh-2#) were inoculated with droplets of B. cinerea spore suspension, the plants were incubated in a moist chamber and symptoms were recorded 48 hpi. Images show all the inoculated leaves from a single plant. Graph shows data of three independent biological replications (n = 44, 36, 36). Whiskers of the boxplots in (a), (b), (d), and (e) show the minimum and maximum values; center lines of boxplots display the median values; box limits indicate the 25th and 75th percentiles. f Defense gene expression of transgenic A. thaliana plants. Relative expression levels of defense-related marker genes were measured by qRT-PCR. Values are means ± SD (n = 9) from three independent biological replicates and three technical replicates. Asterisks indicate significant difference between control (35S:GFP) and BcCrh1 transgenic lines according to one-way ANOVA, P ≤ 0.001. In all other graphs, different letters indicate statistical differences at P ≤ 0.01 according to one-way ANOVA.