Supplemental Figure 1. VLN5 retains conserved residues at both type 1 and type 2 Ca 2+ -binding

Transcription

1 Supplemental Figure 1. VLN5 retains conserved residues at both type 1 and type 2 Ca 2+ -binding sites in the G1 domain. Multiple sequence alignment was performed with DNAMAN Secondary structural elements of human villin were predicted by Predict Protein Server ( The six gelsolin-homology domains (G1 to G6) and the villin headpiece (VHP) domain are marked with lines above the sequence. Alpha-helices and β-strands are represented with revolving lines and broad arrows below the sequence, respectively. Amino acids with 100% conservation were marked with black blocks, whereas amino acids with greater than 50% identity were marked with gray blocks. The protein accession numbers are as follows: human villin (HV; NP_009058), lily 135-ABP (AAD54660), Arabidopsis thaliana VLN1 (NP_029567), VLN2 (NP_565958) and VLN5 (NP_200542). Type 1 and type 2 Ca 2+ ion coordinating residues are highlighted with green and yellow, respectively. Residues for site 1 and 1

2 site 2 calcium-regulation sites within the G1 domain are indicated with asterisks and closed circles, respectively. 2

3 Supplemental Figure 2. VLN5 is expressed preferentially in mature pollen and at higher levels than other Arabidopsis villins. The expression data for Arabidopsis villins was extracted from a currently available database ( Schmid et al., 2005). The expression data was normalized with the GCOS (Gene Chip Operating Software) method, Target intensity (TGT) value of 100, which was expressed as GCOS expression signal and was plotted against Arabidopsis tissues. Error bars represent SD (n = 3-5). (A) VLN1; (B) VLN2; (C) VLN3; (D) VLN4 and (E) VLN5. Arabidopsis tissues are as follows: (1) Dry seed; (2) Imbibed seed, 24 h; (3) 1st node; (4) Flower stage 12, stamens; (5) Cauline leaf; (6) Cotyledon; (7) Root; (8) Entire rosette after transition to flowering; (9) Flower stage 9; (10) Flower stage 10/11; (11) Flower stage 12; (12) 3

4 Flower stage 15; (13) Flower stage 12, carpels; (14) Flower stage 12, petals; (15) Flower stage 12, sepals; (16) Flower stage 15, carpels; (17) Flower stage 15, petals; (18) Flower stage 15, sepals; (19) Flower stage 15, stamen; (20) Flowers stage 15, pedicels; (21) Leaf 1 + 2; (22) Leaf 7, petiole; (23) Leaf 7, distal half; (24) Leaf 7, proximal half; (25) Hypocotyl; (26) Root; (27) Rosette leaf 2; (28) Rosette leaf 4; (29) Rosette leaf 6; (30) Rosette leaf 8; (31) Rosette leaf 10; (32) Rosette leaf 12; (33) Senescing leaf; (34) Shoot apex, inflorescence; (35) Shoot apex, transition; (36) Shoot apex, vegetative; (37) Stem, 2nd internode; (38) Mature pollen; (39) Seeds stage 3 w/ siliques; (40) Seeds stage 4 w/ siliques; (41) Seeds stage 5 w/ siliques; (42) Seeds stage 6 w/o siliques; (43) Seeds stage 7 w/o siliques; (44) Seeds stage 8 w/o siliques; (45) Seeds stage 9 w/o siliques; (46) Seeds stage 10 w/o siliques; (47) Vegetative rosette. (F) The expression signal of Arabidopsis villins in mature pollen was plotted. The results show that the expression of VLN5 is higher than that of other Arabidopsis villins in mature pollen. Error bars represent SD (n = 3-5). 4

5 Supplemental Figure 3. The level of VLN1 and VLN2 transcripts was not reduced in VLN5 RNAi flowers. Flowers from WT Col-0 and three VLN5 RNAi lines (Line 1 3) were subjected to RT treatment. Tubulin 2 was used as an internal loading control, whereas VLN1 and VLN2 primer pairs were used to detect whether VLN5 was silenced specifically. The number of PCR cycles was 25 for Tubulin 2 and 35 for VLN1 and VLN2, respectively. 5

6 Supplemental Figure 4. VLN5 loss-of-function does not affect pollen germination rate. Pollen grains from WT Col-0 and homozygous vln5 plants were germinated on germination medium. The germination rate was plotted versus time. White columns, gray columns and black columns represent WT Col-0, vln5-1 and vln5-2, respectively. Error bars represent ± SE, n =

7 Supplemental Figure 5. Pollen tube growth rate was reduced in VLN5 RNAi lines. WT Col-0, black bar; Line 1, gray bar; Line 2, white bar, Line 3, crosshatched bar. Error bars represent mean values ± SE, n 97. Pollen tube growth rate of VLN5 RNAi pollen tubes was significantly different from that of WT Col-0 pollen tubes as determined by ANOVA followed by Dunnett post hoc multiple comparisons, **P <

8 Supplemental Figure 6. Root hair length of vln5 homozygous mutant plants is not significantly different from that of WT Col-0. (A) Micrographs of root hairs after germination for four days. (a) WT Col-0; (b) vln5-1; (c) vln5-2. Bar = 300 µm in (a) for (a-c). (B) The length of root hairs was plotted against each genotype. The average length of root hairs is ± 30.7 (n = 144), ± 18.9 (n = 155) and ± 18.9 (n = 179) for WT Col-0, vln5-1 and vln5-2, respectively. Error bars represent ± SE. There is no significant difference of the length of root hairs between vln5 mutants and WT Col-0 (P = 0.62 for vln5-1 and P = 0.56 for vln5-2). 8

9 9

10 Supplemental Figure 7. Actin distribution in WT Col-0 and VLN5 loss-of-function pollen tubes. (A) Actin distribution in WT Col-0 pollen tubes. (a-f) showing different actin staining patterns in WT Col-0 pollen tubes. (B) Actin distribution in vln5-1 pollen tubes. (a-f) showing different actin staining patterns in vln5-1 pollen tubes. (C) Actin distribution in vln5-2 pollen tubes. (a-f) showing different actin staining patterns in vln5-2 pollen tubes. (D) Actin distribution in VLN5 RNAi line 1 pollen tubes. (a-f) showing different actin staining patterns in VLN5 RNAi line 1 pollen tubes. (E) Actin distribution in VLN5 RNAi line 2 pollen tubes. (a-f) showing different actin staining patterns in VLN5 RNAi line 2 pollen tubes. Bar = 10 µm. 10

11 Supplemental Figure 8. VLN5 loss-of-function does not alter the level of actin polymer in pollen tubes. Quantification of actin polymer level in WT Col-0 and vln5 mutant pollen tubes. The relative amount of F-actin was determined by measuring the fluorescence pixel intensity of phalloidin staining. WT Col-0, black bar; vln5-1, white bar; vln5-2, gray bar. Error bars represent ± SE (n > 39), (P = 0.13 for vln5-1 and P = 0.87 for vln5-2 by a student s t-test). 11

12 Supplemental Figure 9. Pollen tube growth of VLN5 RNAi plants is hypersensitive to LatB treatment. To determine the effect of LatB on pollen tube growth rates of VLN5 RNAi plants, 3 nm LatB was added to the germination medium. The growth rate of pollen tubes from WT Col-0, and VLN5 RNAi plants in standard germination medium was normalized to 100%. WT Col-0 grew significantly better than did VLN5 RNAi pollen in the presence of LatB. WT Col-0, black bar; Line 1, gray bar; Line 2, white bar, Line 3, crosshatched bar. Error bars represent mean ± SE (n 65), **P < 0.01 (Student s t-test). 12

13 Supplemental Figure 10. VLN5 loss-of-function renders the growth of pollen tubes resistant to cytochalasin D (CD). To determine the effect of CD on pollen tube growth rates, 200 nm and 500 nm CD were added to the germination medium. The growth rate of pollen tubes from WT Col-0 and vln5 homozygous mutant plants in standard germination medium was normalized to 100%. vln5 pollen tubes grew significantly better than did WT Col-0 plant in the presence of CD. WT Col-0, black bar; vln5-1, gray bar; vln5-2, crosshatched bar. Error bars represent mean ± SE, n 121, *P < 0.05 and **P < 0.01 (student s t-test). Experiments were repeated three times for each treatment. 13

14 Supplemental Figure 11. The amount of VLN5 sedimented was decreased in the presence of Ca 2+ /calmodulin (CaM). A high-speed cosedimentation assay was employed to determine whether the binding of VLN5 to actin filaments was regulated by Ca 2+ /CaM. Three micromolar polymerized actin was incubated with 500 nm VLN5 with or without 50 µm CaM in the presence of 1 mm free Ca 2+. The mixtures were centrifuged at 200,000g for 1 h to separate bound versus unbound VLN5. (A) SDS-PAGE separation showing that the amount of VLN5 bound to actin filaments was decreased in the presence of Ca 2+ /CaM. Lanes 1 and 2 represent samples of supernatant and pellet for actin alone; lanes 3 and 4 represent samples of supernatant and pellet for actin plus 500 nm VLN5; lanes 5 and 6 represent samples of supernatant and pellet for actin plus 500 nm VLN5 in the presence of 50 µm CaM, respectively. (B) Plot of the percentage of VLN5 in the pellet. Error bars represent mean ± SE (n = 3), *P < 0.05 (student s t-test). 14

15 Supplemental Figure 12. Ca 2+ /CaM inhibits the bundling activity of VLN5. A low-speed cosedimentation assay was employed to determine whether the bundling activity of VLN5 was regulated by Ca 2+ /CaM. Three micromolar polymerized actin was incubated with 500 nm VLN5 with or without 50 µm CaM in the presence of 1 mm free Ca 2+. The mixtures were centrifuged at 13,600g for 30 min. (A) SDS-PAGE separation showing that the amount of sedimented actin filaments was decreased in the presence of CaM. Lanes 1 and 2 represent samples of supernatant (S) and pellet (P) for actin alone; lanes 3 and 4 represent samples of supernatant and pellet for actin plus 500 nm VLN5; lanes 5 and 6 represent samples of supernatant and pellet for actin plus 500 nm VLN5 in the presence of 50 µm CaM, respectively. (B) Plot of the percentage of VLN5 and actin in the pellet. Error bars represent mean ± SD (n = 3), (*P < 0.05 by a student s t-test). (C-E) Micrographs of actin filaments stained with rhodamine-phalloidin. (C) Individual actin filaments in the absence of villin. The image was captured at a 500-ms exposure time. (D) Actin filament bundles formed in the presence of 0.5 µm VLN5. The image was captured at 150-ms exposure time. (E) Actin bundles formed in the presence of 0.5 µm VLN5 with the addition of CaM. The image 15

16 was captured at a 150-ms exposure time. Bar in (D) = 10 µm. 16

17 Supplemental Figure 13. Direct visualization of VLN severing activity by time-lapse TIRFM. The detail method is described in the method section and Figure 11A legend. (A) Time-lapse images of actin filaments severing in the presence of 0.5 nm human villin at 1 µm free Ca 2+. See Supplemental Movie 4 for the entire time series. (B) Time-lapse images of actin filaments severing in the presence of 5 nm VLN5 at 100 nm free Ca 2+. See Supplemental Movie 5 for the entire time series. (C) Time-lapse images of actin filaments severing in the presence of 5 nm VLN5 at 10 µm free Ca 2+. See Supplemental Movie 6 for the entire time series. (D) Time-lapse images of actin filaments severing in the presence of 5 nm VLN5 at 100 µm free Ca 2+. See Supplemental Movie 7 for the entire time series. (E) Time-lapse images of actin filaments severing in the presence of 5 nm VLN5 at 1 mm free Ca 2+. See Supplemental Movie 8 for the entire time series. Scale bar = 20 µm. 17

18 Supplemental Table 1. List of primers used in genotyping, plasmid construction and RT-PCR Name Primer sequence(5-3 ) Description LP5-1 CCAAGAATCAGAGGTTCCACC For RP5-1 AAAATTCAGGTCTGGCGAATC genotyping of LP5-2 AAAGATCCTTCTCGAAGCAGC VLN5 T-DNA RP5-2 GAGGATCACTCTCTCCATCCC LP2-1 CCAAGAATCAGAGGTTCCACC insertion lines RP2-1 AAAATTCAGGTCTGGCGAATC LP2-2 AAAGATCCTTCTCGAAGCAGC RP2-2 GAGGATCACTCTCTCCATCCC LBGABI ATATTGACCATCATACTCATTGC LBSAIL GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC v5pf GGGGACAAGTTTGTACAAAAAAGCAGGCTACTCGTTAGTCCGTTTTGTT For VLN5 v5pr GGGGACCACTTTGTACAAGAAAGCTGGGTCTCTGGTTTTTGCAAATCTTT promoter GUS fusion construction v5if TCTAGACCATGGCTAAATATAAGAAACCAATC For VLN5 v5ir GGATCCATTTAAATCCTCTGAGTCGGTTTTAAGG RNAi plat52f GAATTCTGTCGACATACTCGACTCAG construction plat52r CTCGAGTTTAAATTGGAATTTTTTTT v5f1 GCCCATGGCGTTTTCCATGAGAGATTTA For VLN5 v5r1 CGGCGGCCGCTTAGAAGAGATTGACAGACAT full-length v5f2 CTCGGTAAAGATTCCAGCCA CDS cloning v5r2 CAATGTATGGCTTCGGTTCG v5f3 ACAAGTTGACCCAAAGAAGA and VLN5 v5r3 TTAGAAGAGATTGACAGACA insertion lines v1tf TCTTACTCTTGGTCTGAAAT RT-PCR v1tr TTAGAAAAGATGAAGAGATA analysis v2tf CATCGTTGTTATTTGGCACT v2tr CTAGAACAAGTCGAACTTCT eif4af GGGTATCTATGCTTACGGTTTCG eif4ar CAGAGAACACTCCAACCTGAATC v5f1 GCCCATGGCGTTTTCCATGAGAGATTTA For RT-PCR V5bR CTTAACCAGGCCTTGAACGTTAACTCCTTG (VLN5 tissue Tubulin2F GGTATCCAGGTCGGAAATGC distribution) Tubulin2R TCCCGTAGTCAACAGAAAGT qvln5f GTTTCGGGTTCAAGGTTCTG For Real-time qvln5r GAGGAAGTAAGATTGCCACACC PCR (vln5 qeif4af TGACCAGAGGCTGAATGAAGT RNAi lines) qeif4ar CGTAAGCATAGATACCCCTAAGAA 18

19 Supplemental References: Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel, D., and Lohmann, J.U. (2005). A gene expression map of Arabidopsis thaliana development. Nat Genet 37,