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1 Supplementary Figures and Tables SI Fig. 1. Schematic diagrams of the Gateway-system based T. pseudonana transformation vectors. A-C, for expression and in vivo localization of egfp-tagged SIT genes under the transcriptional control of respective native promoters. Upper, pmhl_243; middle, pmhl_290; lower, pmhl_293. These vectors were cotransformed with a plasmid vector expressing Nat1 conferring resistance to the antibiotic nourseothricin.
2 Relative fluorescence unit Relative fluorescence unit A B 1.60E E E E E E E E E E E E E E E E+00 Si replete (0.25%) Si starvation (97%) Si replete (0.11%) Si starvation (90%) SI Fig. 2. Flow cytometry analysis of net egfp expression. The relative whole cell fluorescence was measured, using a imaging flow cytometer, after incubating Thalassiosira pseudonana cells in siliconreplete and silicon-deplete media for 24 hr. SIT1-eGFP (A) and SIT2-eGFP (B) were transcribed under the transcriptional control of the respective native promoters to analyze expression level in response to Si starvation. 488nM laser with 100 mw power output was used to excite egfp. Number in parentheses indicate the percentage of fluorescent cells in the gated population.
3 SI Fig. 3. Multiple sequence alignments and neighbor joining tree of SITs from centric and pennate diatoms. T. pseudonana SIT1 and SIT2 share 92% amino identities, and only 56% to SIT3.
4 SI Fig. 4. Schematic diagrams of the intron-spliced hairpin RNA (RNAi) and antisense RNA expression used for downregulation of silicon transporters in the diatom T. pseudonana. For efficient selection of transgenic clones, nourseothricin acetyl transferase gene (NAT) conferring resistance against herbicide nourseothricin was placed at the 3 end of the constitutive promoter FCP (fucoxanthin chlorophyll a/c binding protein ).
5 SI TABLE 1 Primers used in vector constructions and RT-PCR Primers Sequence Gateway Vector Construction Primers Description GW-13 GGGGACAACTTTGTATACAAAAGTTGTACATTCAACGGAATCTGATTATCAAC SIT1 as-attb5 GW-14 GGGGACCACTTTGTACAAGAAAGCTGGGTAACCCTTCCATCTCTCCAGGAC SIT1 as-attb2 GW-19 GGGGACAACTTTTCTATACAAAGTTGTAATGTCTACCGCTGAAATCCAATC SIT1 +ve RNAi-attB4r GW-20 GGGGACAACTTTATTATACAAAGTTGTCTGTAAGGTCAAAGAGAGCAACG SIT1 +ve RNAi-attB3r GW-21 GGGGACAACTTTGTATAATAAAGTTGTAACAGACGATAAGAGAGACCCATG SIT1 -ve RNAi-attB3 GW-22 GGGGACCACTTTGTACAAGAAAGCTGGGTAATGTCTACCGCTGAAATCCAATC SIT1 -ve RNAi-attB2 GW-25 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACCACTCTTGACGACACGGC NAT-attB1 GW-26 GGGGACAACTTTTGTATACAAAGTTGTTCAGGGGCAGGGCATGCTCATG NAT-attB5r GW-35 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACCACTCTTGACGACACGGC NAT-attB1 GW-36 GGGGACAACTTTGTATAGAAAAGTTGGGTGTCAGGGGCAGGGCATGCTCATG NAT-attB4 GW-56 GGGGACAACTTTTCTATACAAAGTTGTAATGGTGAGCAAGGGCGAGGAG egfp-f GW-57 GGGGACAACTTTATTATACAAAGTTGTTTACTTGTACAGCTCGTCCATGC egfp-r GW-104 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATTACAATTACAGTGTACAGATAATG SIT1 prom-f GW-157 GGGGACAACTTTGTATAGAAAAGTTGGGTGGGCATCCTCGGCAAGAGCATC SIT1 gene-r GW-106 GGGGACAACTTTGTATAATAAAGTTGTAACTTCTGGCAATCTGTTGTAAATAG SIT1-term-F GW-107 GGGGACCACTTTGTACAAGAAAGCTGGGTAAGCAGAAACGAAATCATGCGGAG SIT1-term-R GW-182 GGGGACAAGTTTGTACAAAAAAGCAGGCTTATCACCAGTTTCATAACAACACAACC SIT2 prom-f GW-183 GGGGACAACTTTGTATAGAAAAGTTGGGTGAGCCTGCGCGTCAACAGCCTC SIT2 gene-r GW-184 GGGGACAACTTTGTATAATAAAGTTGTAGCAGTGTGTTTGTGTCATCGTG SIT2 term F GW-185 GGGGACCACTTTGTACAAGAAAGCTGGGTAATGATCTCGGGTGGAGGAGTC SIT2 term R GW-186 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACTTCATCTGACAAACGCCGC SIT3 prom-f GW-187 GGGGACAACTTTGTATAGAAAAGTTGGGTGCCACTTCGCCAATCCACTTTCA SIT3 gene-r GW-188 GGGGACAACTTTGTATAATAAAGTTGTAGAAGCGTGATGATAATGCTCTGTC SIT3 term-f GW-189 GGGGACCACTTTGTACAAGAAAGCTGGGTAGTGGCTGTGTCAAAAAGCCCGC SIT3 term-r
6 SI TABLE 1 Primers used in vector constructions and RT-PCR (cont d) RT-PCR Primers Primers Sequence Description RT-19 CTCTACATGAGCATGCCCTG RNAi RT-20 GAAGGATGTTGAAGGGAGTGAG RT-25 AGGTCACCAACGTCAACG Antisense RNA RT-26 TCGTCAGTTGCTCTCTATGTTG RT-30 CATGGGTCTCTCTTATCGTCTG RT qpcr RT-31 TCTTGTAAGTCAATGGGTGGC RT-32 TGTAAAGATCAGGGTCAACGG
7 TABLE S2 Gateway vectors used to construct egfp-tagged SITs Fragments PCR primers Donor vectors used Entry vector created by BP reaction SIT1 promoter and coding region GW-104/GW-157 pdonr-attp1-attp4 pmhl_241 egfp GW-56/GW-57 pdonr-attp4r-attp3r pmhl_139 SIT1 terminator GW-106/GW-107 pdonr-attp3-attp2 pmhl_207 pdestination vector used pmhl_71 pexpresssion vector created by LR reaction pmhl_243 SIT2 promoter and coding region GW-182/GW-183 pdonr-attp1-attp4 pmhl_288 egfp GW-56/GW-57 pdonr-attp4r-attp3r pmhl_139 SIT2 terminator GW-184/GW-185 pdonr-attp3-attp2 pmhl_289 pmhl_71 pmhl_290 SIT3 promoter and coding region GW-186/GW-187 pdonr-attp1-attp4 pmhl_291 egfp GW-56/GW-57 pdonr-attp4r-attp3r pmhl_139 SIT3 terminator GW-188/GW-189 pdonr-attp3-attp2 pmhl_292 pmhl_71 pmhl_293
8 SI TABLE 3 Signal peptide prediction for SIT1-3 Hectar 1 Predotar 2 ptarget 3 WoLF PSORT 4 SIT1 Signal peptide (0.5995) - Plasma membrane (87.6 %) plas: 10 SIT2 Signal peptide (0.5999) - Plasma membrane (81.4 %) plas: 10 SIT3 Type II signal anchor (0.4954) Endoplasmic reticulum (0.95) Peroxisomes (81.4 %) plas:
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Nature Biotechnology: doi: /nbt.4166
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