Fig. 4
ARK-mRFP localizes to the peri-arbuscular membrane. Multiphoton laser scanning microscopy (a−d) shows subcellular localization of a mRFP (magenta) signal labels PAM around arbuscule fine branches (A) at 20 days post-inoculation (dpi). Auto fluorescence from plant cell walls and autofluorescent bodies are false colored in green and white, respectively. NA nonarbusculated cell. Scale bar 10 μm, z-stack 10 μm. b ARK1-mRFP localizes to PAM surrounding collapsing arbuscular (CA) fine hyphae. Scale bar 30 μm, z-stack 20 μm. c Mock-inoculated (MI) ARK1-mRFP-expressing roots and d nontransformed colonized WT lack RFP signal; c, d Scale bar 15 μm, z-stack 10 μm. Representative images of three roots from at least three plants are provided. e Quantification of R. irregularis root colonization levels of the ark1-1 mutant containing the empty vector (EV) control or pARK::ARK-mRFP. T total colonization, EH extra-radical hyphae, H hyphopodia, IH intra-radical hyphae, A arbuscules, S spores, V vesicles. For statistical analysis Kruskal−Wallis tests with the Holm adjustment method were performed using the agricolae package and selecting the option for “treatment groups formation” with P value set at ≤0.05. Bars show median levels while the letters above each bar indicate colonization values that were not significantly different in the post hoc pairwise comparisons. f−h Transmission electron micrograph (TEM) shows immuno-gold labeling (IGL) of ARK1-mRFP to the PAM (f, g) using an RFP antibody and secondary antibody conjugated to 10-nm immuno-gold particles (black arrows) and not to ER (white arrow) or host tonoplast (white arrowhead). Black arrowheads show nonspecific labeling to the fungal cell wall (FCW). Scale bar 500 nm (f) and 100 nm. Representative images from arbuscule-containing cells (n > 4) of independently sampled roots derived from two different experiments (g). h PT11-eGFP control roots cross-reacted with RFP antibodies, showing nonspecific labeling (black arrowheads) of the FCW. Scale bar 100 nm. HC host cytosol, FH fungal hypha, FT fungal tonoplast, FPM fungal plasma membrane
