Prolines within the PRR are essential to MT-association of XopL_Xe.

(A-B) Western blot analysis of MT co-sedimentation assay utilizing (A) 5 μg of affinity-purified XopLXe (88.1 kDa) protein tagged with 6xHis-SUMO-strep or (B) the tag-alone control. Supernatant fractions are marked ‘S’ and pellet fractions ‘P’. Samples with MTs are marked ‘+’ and without by ‘-’. Recombinant protein was detected using a strep-specific antibody (*). The intensity of the XopL signal (abundance) is shown below corresponding lane. Ponceau stained membranes show tubulin at 55 kDa in the pellet fraction with MTs (m derivatives from confocal images of agroinfected GFP-TUA6 lower leaf epidermis (2 dpi, OD600 of 0.8). MT binding is expressed as ‘Relative MT fluorescence’ (see Materials and Methods). Treatments that are not significantly different (p>0.05) are labeled with the same letter (Kruskal-Wallis One Way Analysis of Variance on Ranks, pairwise Dunn’s post-hoc). Background colors are included for ease of comparison between key controls XopLm (blue) and mCherry (red). (D) Quantification of confocal images to evaluate MT density following the expression of XopLXe derivatives in agroinfected GFP-TUA6 lower leaf epidermis (2 dpi, OD600 of 0.8). Treatments that were significantly different than XopLXe are marked with black asterisks (* = p0.001; Kruskal-Wallis One Way ANOVA on Ranks, Dunn’s post-hoc against XopL). ex2 proline to alanine exchanges that were significantly different than ex2 are marked with red asterisks (*** = p>0.001; Kruskal-Wallis One Way ANOVA on Ranks, Dunn’s post-hoc against ex2). Red box in the background is included for ease of comparison with the mCherry control (red). (C and D) Boxes represents first to third quartiles and the median is represented by a horizontal line. Whiskers represent the distribution of remaining data points. (E and F) Western blot analysis of protein extracts isolated from the experiments in (C) and (D), respectively. Signals of XopL derivatives and the mCherry control were detected using an mCherry-specific antibody (*). ‘NI’ and ‘Ch’ refer to not inoculated and mCherry control, respectively. Protein mass is expressed in kDa. (F) Amido black staining is shown as a loading control.