Fig. 3: A stretch of 53 amino acids at the N′ end of the protein mediates uptake of BcCrh1 by plant cells.
Following analysis of a series of deletion constructs, a minimal region suspected of being necessary for protein uptake was defined. a To test the delivery of heterologous proteins using the putative translocation signal, we generated two expression constructs with enzymatic inactive version of the apoplastic NIP BcXYG1 (MBcXyg1) (Zhu et al.5). Blue—BcCrh1 SP, Red—MBcXyg1, Green—the suspected delivery sequence. Middle left— images of N. benthamiana leaves three days after Agroinfiltration. Leaves were bleached with ethanol (right images) and the necrotic area was calculated. Center lines of the boxplots show the medians, box limits indicate the 25th and 75th percentiles; whiskers cover the full range of values; all data points are plotted as black dots. Data are from 10 sample points from three independent biological replications. Different letters indicate statistical differences at P ≤ 0.001 (P = 6.05e−10) according to unpaired two-tailed Student’s t-test. b Images of tomato leaves 48 h after infiltration with 11 μM of purified BcCrh1-derived peptides, BcCrh175–144 and BcCrh121–144. c Tomato cell cultures (MsK8) were incubated for 18 h with 5.5 μM solution of GFP-tagged peptides (GFP-BcCrh175–144, GFP-BcCrh121–144, and GFP-BcCrh121–74), washed three times and visualized by a confocal microscope. Bars = 50 μm. The experiments were repeated three times with similar results.
