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1 DOI: /ncb2294 Figure S1 Localization and function of cell wall polysaccharides in root hair cells. (a) Spinning-disk confocal sections of seven day-old A. thaliana seedlings stained with 0.1% S4B were collected with a 10X objective (scale bars = 50 mm) using either brightfield (left panel) or appropriate filter sets to image S4B fluorescence (right panel). Young root hairs in a zone in which active tip-growth occurs display highly enriched S4B staining predominantly in the root hair apex (arrowheads), while mature root hairs display much more uniform staining along the entire root hair shaft (brackets). Quantification of the time- and dose-dependent effects of cellulase (b), xyloglucanase (c), and pectate lyase (d) treatment on growing root hairs. Characterization of the effect of xyloglucanase treatment on cellulose or cellulose-like polysaccharides (e), or xyloglucan (f) in growing root hairs. Medial confocal sections of root hairs from zones of active tip-growth were collected using a 40X (left; scale bar = 10 mm) or 10X lens (right; scale bar = 50 mm) and brightfield (upper panels) or appropriate filter sets to visualize S4B (e; lower panels) or anti-ccrc-m1 immunofluorescence (f; lower panels, 40X lens) either prior to (left panels) or after xyloglucanase treatment (+XGase; right panels) (scale bars = 10 mm). 1
2 Figure S2 (a). Brightfield image of root hairs from seven day-old wt(col-0) seedlings, (b). Root hairless kjk-2 seedling, (c). kjk-2 seedling transformed with 35S::EYFP-CSLD3. (d-e) Medial plane optical sections of kjk-2 seedlings rescued with (d) 35S::EYFP-CSLD3 or (e) the endogenous promoter (endop::eyfp-csld3) collected using spinning-disk confocal microscopy and appropriate filter sets. (f) Root hair lengths in wt(col-0), and kjk-2 lines rescued with EYFP-CSLD3 driven by either endogenous CSLD3 promoter (endop) or 35S EYFP-CSLD3::A6CD chimera driven by 35S promoter. Average length of root hairs (n = 30) from three independent seedlings were measured, error bars = +/- SD (scale bars = 100 mm for A-C; 10 mm for D-E). 2
3 Figure S3 Polarized EYFP-CSLD3 localizes into discrete regions of fluorescence that are motile in the tips of root hair cells. (a) Three successive Z-plane optical sections of a growing root hair cell stably expressing EYFP- CSLD3 were collected using spinning-disk confocal microscopy. EYFP- CSLD3 fluorescence was collected for 50 milliseconds with a 100X oil objective (1.46 NA) and optical sections are separated by 1 mm distances. EYFP-CSLD3 is enriched in a tip-restricted plasma membrane domain in the apex of the hair cell, and does not appear to be uniform but localizes in discrete regions of high fluorescence (highlighted by arrowheads; scale bar = 2 mm). (b) EYFP-CSLD3 dynamics within the apical plasma membrane domain. Successive frames of a time-lapse movie were collected for 50 milliseconds with a 100X oil objective (1.46 NA) at five second intervals. Zones where discrete regions of higher EYFP-CLSD3 fluorescence were detected are indicated by white or yellow brackets. While some of these appeared motionless relative to the growing tip (white asterisk) multiple fluorescent regions appeared and disappeared from the confocal plane, or could be observed to move position relative to the growing root hair apex (arrow heads; scale bar = 2 mm). 3
4 Figure S4 Fluorescence recovery after photobleaching growing root hairs. Medial confocal sections were collected from growing root hairs from seven day-old seedlings expressing EYFP-CSLD3. After establishing root hairs were growing, photobleaching was performed within small (a) or large (c) ROI (red circles). Quantification of fluorescence recovery of EYFP-CSLD3 within the apical plasma membrane domain in two independent root hairs (black squares and grey triangles) was measured at 2-second intervals within the small (b) or large (d) photobleached ROI. 4
5 Figure S5 Fluorescence recovery after photobleaching in latrunculin B treated root hairs. Medial confocal sections were collected from growing root hairs from seven day-old seedlings expressing EYFP-CSLD3. After establishing root hairs were growing, they were treated with 200 nm latrunculin B. Upon loss of cytoplasmically localized EYFP-CSLD3 vesicles within the root hair apex, photobleaching was performed within small (a) or large (c) ROI (red circles). Quantification of fluorescence recovery of EYFP- CSLD3 within the apical plasma membrane domain in two independent root hairs (black squares and grey triangles) was measured within the small (b) or large (d) photobleached ROI (scale bar = 1 mm). 5
6 Primers Primer Sequence (5-3 ) A: CSLD3-upper ATGGCGTCTAATAATCATTTCATGAACAGTAG B: CSLD3-lower TCATGGGAAAGTGAAAGATCCTCCGATTT C: kjk-4-lower TCATTTGATCTCGAGGAGAGCGAGTA D: CSLD3-Nterm-upper ATGGCGTCTAATAATCATTTCATGAACAGT E: CSLD3-Nterm-lower GGAAACCACTTGGGAAACTGATCAAGTAGC F: CSLD3-Cterm-upper GAGAGGTTTGCGTATGTGAACGTTGGAATCTAC G: CSLD3-Cterm-lower TCATGGGAAAGTGAAAGATCCTCCGATT H: CESA6-CD-upper ATCAGTTCCCTAAATGGTACCC I: CESA6-CD-lower GAGTTAATGTAGGACAACCGCT Table S1 Primers used for PCR amplification and assembly of CSLD3, kjk-4, and CSLD3::A6CD constructs. 6
7 Supplementary Movie Legends Movie 1: DCB treatment results in rapid root hair rupture. Movie 2A: Cellulase treatment results in root hair rupture, but can be competitively inhibited by 0.8 mg/ml CM-cellulose. Movie 2B: Xyloglucanase treatment inhibits root hair growth. Movie 2C: Pectate lyase treatment inhibits root hair growth. Movie 3: EYFP-CESA6 is restricted to internal compartments in growing root hair cells. Movie 4: A functional EYFP-CSLD3 fusion localizes to membranes in the tips of growing root hair cells. Movie 5: Delivery of EYFP-CSLD3 to tip-enriched plasma membranes requires an intact actin cytoskeleton. Movie 6: EYFP-CSLD3 localizes as discrete particles in tip-enriched plasma membranes in growing root hair cells. Movie 7: EYFP-CSLD3 dynamics within the apical plasma membranes in growing root hair cells. Movie 8A: FRAP analysis of EYFP-CSLD3 in growing root hair cells; small ROI. Movie 8B: FRAP analysis of EYFP-CSLD3 in growing root hair cells; large ROI. Movie 8C: FRAP analysis of EYFP-CSLD3 in the apical plasma membrane domain of latrunculin B treated root hair cells; small ROI. Movie 8D: FRAP analysis of EYFP-CSLD3 in the apical plasma membrane domain of latrunculin B treated root hair cells; large ROI. Movie 9: DCB effect on EYFP-CSLD3 dynamics in the tips of root hair cells. Movie 10: Isoxaben has no effect on EYFP-CSLD3 localization in growing root hair cells. Movie 11: CGA treatment inhibits root hair growth and induces cell rupture and bulging. Movie 12: Subcellular distribution of the EYFP-CSLD3::A6CD chimera in a growing root hair cell from a rescued kjk-2 seedling. 7
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