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. 2022 Sep 5;13:5228. doi: 10.1038/s41467-022-32124-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a–e vpy-4 was transformed with VPY_pro:VPY-cpVenus or ankyrin deletion constructs, colonized with Diversispora epigeae, and harvested 4 weeks post planting. Roots were stained with wheat germ agglutinin conjugated to AlexaFluor 488 (WGA-AlexaFluor 488) to visualize the fungus. a Representative epifluorescence images of vpy-4 WGA-AlexaFluor 488 stained roots complemented with VPY_pro:VPY-cpVenus, or not complemented, as seen for vpy-4 transformed with VPY_pro:VPY_ANKΔ3-5-cpVenus. Scale bars, 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-4 transformed with VPY_pro:VPY-cpVenus (n = 396 arbuscules), VPY_pro:VPY_ANKΔ9-cpVenus (n = 496 arbuscules), or VPY_pro:VPY_ANKΔ7-9-cpVenus (n = 536 arbuscules). Different letters signify statistically significant differences from one-way ANOVA, sampled from three biological replicates with three infection units per replicate. In (b)–(d), lines in boxplots represent the median value, box limits represent the upper and lower quartiles, whiskers represent 1.5 times the interquartile range. d Overlapping histogram of arbuscule length between VPY_pro:VPY_ANKΔ9-cpVenus (purple bars) and VPY_pro:VPY-cpVenus (striped bars). Arbuscule length is plotted on the x-axis, and probability densities are plotted on the y-axis to normalize sample size. e Overlapping histogram of arbuscule length between VPY_pro:VPY_ANKΔ7-9-cpVenus (pink bars) and VPY_pro:VPY-cpVenus (striped bars). Arbuscule length is plotted on the x-axis, and probability densities are plotted on the y-axis to normalize sample size. f Subcellular localization of VPY ankyrin deletions. WT roots expressing vectors containing VPY_pro:VPY_ANKΔ9-cpVenus, VPY_pro:VPY_ANKΔ7-9-cpVenus, VPY_pro:VPY_ANKΔ3-5-cpVenus, or VPY_pro:VPY_ANKΔ4-9-cpVenus and PM and PAM marker BCP1_pro:PT1-mCherry were colonized with R. irregularis to determine the subcellular distribution of the truncated fusion proteins in arbuscule-containing cells. Overlay images display both cpVenus and mCherry channels. Samples are representative images of at least two independent experiments, with three root systems and ten root pieces imaged per experiment, n = 47 (VPY_pro:VPY_ANKΔ9-cpVenus), n = 19 (VPY_pro:VPY_ANKΔ7-9-cpVenus), n = 44 (VPY_pro:VPY_ANKΔ4-9-cpVenus), or n = 22 (VPY_pro:VPY_ANKΔ3-5-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 (arrow); CA, cytoplasmic aggregate (arrowhead).