Supplemental Information. Boundary Formation through a Direct. Threshold-Based Readout. of Mobile Small RNA Gradients

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1 Developmental Cell, Volume 43 Supplemental Information Boundary Formation through a Direct Threshold-Based Readout of Mobile Small RNA Gradients Damianos S. Skopelitis, Anna H. Benkovics, Aman Y. Husbands, and Marja C.P. Timmermans

2 Supplemental Figure 1. Altering the source and level of mirarf perturbs leaf flatness; Related to Figure 1. (A-I) Tissue specificity of promoters driving artificial mirna expression. (A-C) Representative seedling images of (A) pas2:gus, (B) pmir166a:gus, and (C) pfil:gus reporter lines. (D-I) Transverse sections through young leaf primordia of (D and G) pas2:gus, (E and H) pmir166a:gus and (F and I) pfil:gus reporters in (D-F) wild-type and (G-I) rdr6. The AS2 and MIR166A promoters are active in the adaxial and abaxial leaf epidermis (insets), respectively, while FIL is expressed throughout the abaxial domain. Note that expression patterns in rdr6 are unchanged in comparison to wild-type and that the MIR166A and FIL promoters show activity in the vasculature of mature leaves (arrowheads), but not in the vasculature of young leaf primordia (D-I). Scale bars, 50 µm. (J) Leaf-curling phenotypes seen in T1 seedlings of rdr6 mutants carrying the pas2:mirarf, pmir166a:mirarf, or pfil:mirarf transgenes can be grouped into four classes: (K) severe upwardcurling (dark blue), (L) mild upward-curling (bright blue), (M) flat (light blue) and (N) rdr6-like

3 (lavender). Yellow arrowheads, downward-curling leaves; white arrowheads, upward-curling leaves. (O) Levels of tasiarf (wild-type and rdr6) or mirarf (rdr6 carrying the pas2:mirarf, pmir166a:mirarf, or pfil:mirarf transgenes) as determined by small RNA qrt-pcr. Note the frequency of upward-curled seedlings in (J) positively correlates with the levels of tasiarf/mirarf in (O). tasiarf is not detected (nd) in rdr6 mutants. Expression levels (means ± SE) normalized to wildtype tasiarf expression were calculated based on at least three independent biological replicates.

4 Supplemental Figure 2. Signal intensity in colorometric alkaline phosphatase reactions linearly correlates with probe amount; Related to Figures 1 and 3. (A and B) In situ hybridization reveals endogenous tasiarf forms an abaxially-dissipating concentration gradient in (A) wild-type, and is not detected in (B) rdr6 leaf primordia. (C) Quantification of the colorometric alkaline phosphatase reaction signal shows this to be linearly correlated to probe amount over a wide range of levels provided that the reaction is not saturated. Signal intensities (mean ± SE in arbitrary units) were quantified based on four independent experiments. Note, 30 fmol approximates the estimated total amount of mirnas found in a typical mammalian cell (Bissels et al., 2009).

5 Supplemental Figure 3. Functional complementation and characterization of pphb:phb-yfp and pphb:phb*-yfp lines; Related to Figure 2. (A to C) In contrast to (A) wild-type, (B) phb rev double mutants show defects in meristem maintenance and organ initiation; these defects are completely rescued by (C) the pphb:phb-yfp transgene. (D) Representative PCR genotyping of phb rev, complemented phb rev pphb:phb-yfp, or wild-type seedlings confirms complementation requires the pphb:phb-yfp transgene. Primer sets: 1, phb-6 allele; 2, PHB wild-type; 3, rev-9 allele; 4, REV wild-type; 5, YFP. (E) PHB*-YFP (middle strand) harbors a point mutation that reduces complementarity to wild-type mir166 (top strand) and blocks mir166-directed transcript cleavage. The complementary mutation in mir166* (bottom strand) restores cleavage of PHB*-YFP transcripts. Mutated nucleotides are noted in red. (F and G) Transverse sections show expression of (F) pas2:gus throughout the epidermis of adaxialized pphb:phb*-yfp leaf primordia, whereas (G) pmir166a:gus expression remains polar and restricted to the bottom leaf surface. Scale bars, 50 µm.

6 Supplemental Figure 4. mirgfp efficiently silences GFP expression; Related to Figure 3. (A and B) GFP fluorescence in (A) p35s:3xnls-gfp seedlings (no mirgfp) is completely eliminated in (B) such seedlings ubiquitously expressing mirgfp (p35s:mirgfp). (C and D) 5 RLM-RACE analysis yields (C) a single GFP cleavage product that (D) initiates at the expected 10 th nucleotide in the mirgfp target site (black arrowhead). (E) Small RNA blot showing mirgfp is 21 nt in size and accumulates specifically in p35s:mirgfp lines. U6 hybridization confirms near even loading of RNA samples. (F) Read counts for mirgfp and GFP-derived secondary sirnas in reads per million (rpm) normalized to the total number of mapped nt small RNA reads in libraries constructed from p35s:3xnls-gfp (no mirgfp) and p35s:3xnls-gfp p35s:mirgfp lines. The absence of GFP-derived secondary sirnas confirms the lack of transitivity in these lines.

7 Supplemental Figure 5. Target readout is affected by mirgfp levels at the source; Related to Figures 3 and 4. (A-C) Sequential optical sections of a pas2:mirgfp seedling leaf show cells in the presumptive spongy parenchyma and abaxial epidermal layers all retain fluorescence. Nuclei not visible in one section (e.g. arrowheads) show fluorescence in adjacent focal planes. Fluorescence is never detected in presumptive palisade parenchyma and adaxial epidermal cells, regardless of the focal plane. (D) Fluorescence signal intensity (mean ± SE in arbitrary units) quantified from multiple nuclei per cell layer (see Materials and Methods) shows GFP fluorescence is completely silenced in the two adaxialmost cell layers in pas2:mirgfp and the two abaxial-most cell layers in pmir166a:mirgfp primordia. GFP fluorescence intensity in the remaining cell layers is reduced and less variable, showing a lower coefficient of variation (CV: standard deviation divided by mean) than the p35s:3xnls-gfp (no mirgfp) control. (E-G) Sequential optical sections through a 24 hours estradiol-induced pas2>>mirgfp seedling leaf show a lack of GFP fluorescence in adaxial epidermal cells, regardless of the focal plane. Cells in the

8 remaining layers all retain fluorescence with nuclei not visible in one section (e.g. arrowheads) showing fluorescence in adjacent focal planes. (H-J) Independent leaf sections of estradiol-induced pas2>>mirgfp seedlings 72 hours post-induction demonstrating GFP fluorescence on the abaxial side has a stochastic distribution. Ep, epidermis; Pp, presumptive palisade parenchyma; Sp, presumptive spongy parenchyma.

9 Supplemental Figure 6. Characterization of the estradiol-inducible mirgfp system; Related to Figure 4. (A) Schematic of the epidermal-specific, estradiol-inducible patml1>>3xnls-gfp construct. (B-E) GFP fluorescence progressively increases in patml1>>gfp seedlings induced with 20 µm estradiol for (B) 0, (C) 6, (D) 12, and (E) 24 hours. (F-H) Transverse sections of estradiol-induced patml1>>3xnls-gfp seedlings (F) 0, (G) 12, and (H) 24 hours post-induction show GFP fluorescence, and hence OlexA accumulation, is cell-autonomous. (I) Schematic of the adaxial epidermal-specific, estradiol-inducible pas2>>mirgfp construct. (J-M) GFP fluorescence progressively decreases in pas2>>mirgfp seedlings upon (J) 0, (K) 24, (L) 48, and (M) 72 hours of induction with 20 µm estradiol. Seedlings after 48h of induction resemble (N) stably-silenced pas2:mirgfp seedlings. (O) Small RNA qrt-pcr shows a time-dependent increase in mirgfp levels upon induction with 20 µm estradiol that negatively correlates with GFP fluorescence (K to L). mirgfp levels (means ± SE) normalized to U6 were calculated based on at least three independent biological replicates, and plotted

10 relative to mean mirgfp levels 24 hpi. n.d., not detected; no mirgfp, input RNA from p35s:3xnls- GFP seedlings; no RT, input RNA from 72 hpi pas2>>mirgfp seedlings.

11 Supplemental Figure 7. Aligning the mir166 and tasiarf gradients compromises the adaxialabaxial boundary and robust flat leaf architecture; Related to Figure 5. (A and B) Images of 28-dayold plants showing that unlike (A) wild-type, (B) pmir166a:mirarf leaves are not flat, presenting substantial downward-curling (yellow arrowheads) and upward-curling (blue arrowhead) phenotypes. (C and D) Dissected (C) wild-type and (D) pmir166a:mirarf mature leaves reveal the latter develop ectopic outgrowths on their abaxial surface (white arrowheads). (E) Transverse sections of pmir166a:mirarf primordia show misexpression of the pas2:gus adaxial epidermal reporter in random cells of the abaxial epidermis (black arrowheads). (F) Leaves of pphb:phb*-yfp

12 pas2:mir166* seedlings display a range of phenotypes including downward-curling (yellow arrowheads), upward-curling (blue arrowheads), and radial leaf morphologies (black arrowhead). (G) Transverse sections of primordia from a single pphb:phb*-yfp pas2:mir166* seedling show a variable misexpression of the pas2:gus reporter demonstrating adaxial-abaxial polarity and flat leaf production are no longer robustly achieved.