Figure 3.

Functionality of NTS sequences and their phosphovariants from the HopQ1 and AtMPK6. (A) Amino acid sequence of the phosphopeptide comprising putative nuclear translocation signal (NTS) derived from HopQ1. NTS motif is framed and the phospho-Ser is highlighted in red. (B) Comparison of NTS surrounding from metazoan MAP kinases with the corresponding sequence identified in plant MAP kinases activated in immunity. NTS motif is highlighted in red. Hs Homo sapiens, Nt N. benthamiana, At A. thaliana. (C) The quantification of the ratio of the fluorescence intensity of nuclear and cytoplasmic fraction of mDendra2 in N. benthamiana epidermal leaf cells at 72 h after infiltration with A. tumefaciens strains carrying constructs indicated: 1) a control protein consisting of GUS and mDendra2, 2) wild-type NTS peptide derived from HopQ1 and 3) AtMPK6 (NTS-Q1-TPS, NTS-MPK6-TPS,respectively) 4) phosphomimic (EPE) or 5) phosphonull (APA) variants (NTS-Q1-EPE, NTS-Q1-APA, NTS-MPK6-EPE, NTS-MPK6-APA, respectively) fused to GUS- mDendra2. mDendra2 fluorescence intensity was measured for five areas within the nucleus and cytoplasm, and the average was calculated for each set of measurements. Then the ratio of average nuclear (N) to cytoplasmic (C) fluorescence was calculated for each tested cell. Error bars correspond to ± SEM. Letters indicate homogenous groups detected by one-way ANOVA and Tukey HSD post hoc test (p < 0.05).