Fig. 3: Fine scale ankyrin deletion analysis.
a–f vpy-4 was transformed with full-length VPYpro:VPY-cpVenus or ankyrin deletion constructs, colonized with D. epigeae, and harvested 4 weeks post planting. a Representative epifluorescence images of vpy-4 WGA-AlexaFluor 488 stained roots complemented with VPYpro:VPY-cpVenus, or not complemented, as seen for vpy-4 transformed with VPYpro: cpVenus. Scale bars are 100 μm. b Number of arbuscules per infection unit in vpy-4 transformed with ankyrin deletion constructs. Different letters indicate statistically significant differences using one-way ANOVA, p < 0.05, n = 9 infection units, sampled from three biological replicates with three infection units per biological replicate. c Arbuscule length of vpy transformed with VPYpro:VPY-cpVenus (n = 406 arbuscules), or VPYpro:VPYANKΔ2-cpVenus (n = 388 arbuscules), sampled from three biological replicates with three infection units per replicate. d Number of arbuscules per infection unit in vpy-4 transformed with ankyrin deletion constructs. Different letters indicate statistically significant differences using one-way ANOVA, p < 0.05, n = 9, sampled from three biological replicates with three infection units per biological replicate. e Arbuscule length of vpy-4 transformed with VPYpro:VPY-cpVenus (n = 344 arbuscules), or VPYpro:VPYANKΔ4-cpVenus (n = 459 arbuscules). *** indicates a significance of p < 0.001 using a two-tailed Student’s t test sampled from three biological replicates with three infection units per replicate. In (b)–(e), lines in boxplots represent the median value, box limits represent the upper and lower quartiles, whiskers represent 1.5 times the interquartile range. f WT roots were transformed with vectors containing VPYpro:VPYANKΔ1-cpVenus, VPYpro:VPYANKΔ2-cpVenus, VPYpro:VPYANKΔ4-cpVenus, VPYpro:VPYANKΔ5-cpVenus, VPYpro:VPYANKΔ7-cpVenus, or VPYpro:VPYANKΔ8-cpVenus and PM and PAM marker BCP1pro:PT1-mCherry colonized with R. irregularis to determine the subcellular distribution of the truncated fusion proteins in arbuscule-containing cells. Samples are representative images of at least two independent experiments, with three root systems and ten root pieces imaged per experiment, n = 37 (VPYpro:VPYANKΔ1-cpVenus), n = 24 (VPYpro:VPYANKΔ2-cpVenus), n = 33 (VPYpro:VPYANKΔ4-cpVenus), n = 25 (VPYpro:VPYANKΔ5-cpVenus), n = 38 (VPYpro:VPYANKΔ7-cpVenus), or n = 14 (VPYpro:VPYANKΔ8-cpVenus) arbuscule-containing cells. Fluorescence images are projections of 10 optical sections on the z-axis taken at 0.5 µm intervals. Scale bars, 10 µm. N, nucleus (arrowhead), P, punctus (arrow), VS, vacuolar sphere (open arrowhead).
