Supplemental Data. Ko et al. (2014). Plant Cell /tpc Supplemental Figure 1. Evidence of T-DNA Insertion in ms142 Mutant.
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1 Supplemental Figure 1. Evidence of T-DNA Insertion in ms142 Mutant. (A) DNA gel blotting to hptii probe confirmed single T-DNA insertion (marked with arrow) in ms142 mutant. (B) T-DNA tagged construction map of TRIM. (C) Gene structure and T-DNA insertion site in the mutant at 3 rd intron (+1257bp from ATG). (D) Genotyping heterozygous T4 mutant progeny using primer designed for the mutant allele (R+RB, amplicon = 340 bp) and wild-type allele (F+R, amplicon = 458 bp). Three primers sets were incorporated in one PCR reaction for genotyping. Genotype: WT, wild-type like; He, heterozygous; Ho, homozygous. Grain fertility: F, fertile, S, sterile.
2 Supplemental Figure 2. Meiotic Analysis and Expression of Meiotic Regulatory Genes in the Wild-type and ms142. (A) Acetocarmine staining of microspore mother cells and meiocytes of WT and ms142. Arrow, degenerated meiocytes. Bar = 10µm. (B) RT-PCR analysis gene expression pattern of meiotic regulatory genes in various anther developmental stages of WT and ms142. SC, sporogenous cell; MMC, microspore mother cell; Mei, meiosis; YM, young microspore; VP, vacuolated pollen; PM, pollen mitosis; MP, mature pollen.
3 Supplemental Figure 3. Differential interference contrast (DIC) images of anther cross section relevant to TUNEL section. Scale bar = 50 µm.
4 A ATG bhlh TAA NLS NLS D P35S:eGFP:Tnos P35S egfp Tnos C P35S:NLS:mRFP:Tnos P35S vird2-nls mrfp Tnos P35S:bHLH142:Tnos P35S bhlh142 egfp Tnos E bhlh142 Supplemental Figure 4. Scheme of bhlh142 Gene, Multiple Alignment, and Subcellular Localization of rice bhlh142 Fused with GFP. (A) Scheme of the bhlh142 gene and T-DNA insertion position. Gray boxes represent exons and intervening lines represent introns. The ATG start codon and TGA stop codon are indicated. bhlh, basic helix-loop-helix domain (amino acids 182 to 228); NLS, two nuclear localization signals (amino acids 159 to 165 and 235 to 240, respectively). (B) Alignment of bhlh domains. The bhlh142 protein was aligned with the bhlh domains of the analogous proteins from other species. Asterisks indicate the conserved basic amino acid Arg that is important for binding DNA. Pound signs indicate conserved Leu residues important for forming the α-helix. (C) Predicted 3D protein structure of rice bhlh142 as constructed by MD simulation. The bhlh domains are marked in green color. (D) Schemes of fusion constructs. P35S, cauliflower mosaic virus 35S promoter; Tnos, nopaline synthase gene terminator. The NLS domain of VirD2 fused with mrfp was used as the nuclear localized control. (E) In vivo targeting of fusion protein. DIC (bright field, left column) and overlays (Merge, right column) are shown. Scale bar = 20 µm.
5 Supplemental Figure 5. Phylogenetic Analysis of bhlh142-related Proteins. Aral, Arabidopsis lyrata; Aegt, Aegilops tauschii; At, Arabidopsis thaliana; Brad, Brachypodium distachyon; Os, Oryza Sativa; Sei, Setaria italica; Sb, Sorghum bicolor; Selm, Selaginella moellendorffii ; Triu, Triticum urartu; Zm, Zea mays.
6 A B C Relative expression level Relative expression level Relative expression level bhlh142 MMC Mei YM VP Anther developmental stage bhlh142 TNG ms142 TNG udt1 MMC Mei YM VP Anther developmental stage bhlh142 Hitomebore eat1 MMC Mei YM VP D E Relative expression level Relative expression level CP1 Hitomebore eat1 MMC Mei YM VP Anther developmental stage C6 Hitomebore eat1 MMC Mei YM VP Anther developmental stage Anther developmental stage Supplemental Figure 6. Real-time PCR Analysis of Relative Gene Expression of bhlh142 mrna in the Anthers of ms142 (A), udt1 (B), eat1 (C), and the Expression of CP1 and C6 in eat1 Anther. WT of ms142 and udt1 is TNG67, WT of eat1 (Tos17 mutant HA0530 ) is Hitomebore. MMC, meiocyte mother cell; Mei, meiosis; VP, vacuolated pollen. Error bars indicate SD (n = 3).
7 Supplemental Figure 7. Analysis of Protein Interaction Between bhlh142, TDR1, and EAT1 by (A) yeast two-hybrid assay and (B) BiFC in rice leaf protoplasts expressing the indicated constructs. Scale bars = 20 µm.
8 Supplemental Figure 8. RNAi Knockdown (KD) of bhlh142 Inhibited Pollen Development. (A) Construct of RNAi vector. (B) RT-PCR showed downregulation of bhlh142 in the anthers of four RNAi knockdown lines. (C) Comparison of the wild-type and KD line #3 after bolting. (D) Comparison of the wild-type and KD line #3 spikelets at one day before anthesis. (E) Comparison of the wild-type and KD line #3 anther. (F) Comparison of the wild-type and KD line #3 pollen grains stained by I2/KI solution. Scale bars = 20 cm in (C), 0.5 mm (D), 1 mm (E) and 20 µm (F).
9 Supplemental Table 1. Agronomic Traits and Grain Yields of ms142 Mutant and its BCF2 Population. Results are presented as mean values ± SD.
10 Supplemental Table 2. Genetic Determination of Male Fertility in ms142 Mutant in Different Cropping Seasons. Chi-square test for the ratio of 3 fertile: 1 sterile, P = 0.05, X 2 = a Total number of plants that had been transplanted into the field. Average temperature during heading stage in the 1 st cropping season of 2012, minimum temperature was 24.6 ± 1.2 o C and maximum 29.9 ± 3.3 o C; in the 2 nd cropping season, minimum 19.9 ± 3.5 o C and maximum 26.1 ± 3.7 o C.
11 Supplemental Table 3. Homologs of rice bhlh142 in Other Cereal Crops. Crop Accession No. Amino acid similarity Maize ZmLOC % Millet XP_ % Wheat EMS % Sorghum XP % Brachypodium XP_ %
12 Supplemental Table 4. Primers used in this work. Primer Name RAP Accession No. Sequence (5 3 ) Screening of s80 Mutant T-DNA inserted S80qPCR-F3 Os01g GGAGCACGTACATCCAGCGG S80-GT-R3 ACTCATCCACCACTTCAATCAGCC RB-13 AACTCATGGCGATCTCTTACC Hyg-F GATGTAGGAGGGCGTGGATA Hyg-R CGTCT GCTGC TCCAT ACAAG Quantitative/ RT- PCR S80qPCR-F3 Os01g GGAGCACGTACATCCAGCGG S80-GT-R3 ACTCATCCACCACTTCAATCAGCC OsMS2-qRT-F Os03g TGGAGCAGTTCGCCAGCTACG OsMS2-qRT-R CTTCTCCTCCTCCGACATCTCCC OsTDR-F Os02g CGCTCGCTCGTCCCAAACAT OsTDR-R CGGTCATTGCTGGGTCCTTGT Os C6-F Os11g TCCTCCTCGTCCTGCTCGTC Os C6-R GGTTCACGATGTGGCACAGG 18SrRNA-F2 TTAGG CCACG GAAGT TTGAG G 18SrRNA-R2 ACACT TCACC GGACC ATTCA A Udt1-F Os07g GATCTTCTGGACCAAGAGGGCAG Udt1-R GTCAGGAGTGTCTCAGATGCTTGG OsCP1-F Os09g GGACCACCTGCTGCTGCAACT OsCP1-R GAACACTTCGTGCCATCGCC bhlh141-f Os04g TGGTGGAACAGAAGAGGCATGG bhlh141-r GCATGAAGCAGAGAGTTGGCCTT CYP703A3-F Os08g GGCGTCATCCTGGTGCTCAT CYP703A3-R CGACGCCTAGCTCAAACTTGC CYP704A2-F Os03g GGTTGAGATCGGGACGCTGT CYP704A2-R ATCACCTTCTCTTGCTTGCCG OsUBQ5-F Os01g GCGGAAGTAAGGAAGGAGGAGG OsUBQ5-R GGCATCACAATCTTCACAGAGGTG MSP1-F Os01g GAGAACTTCGAGCCGAGGGTCT MSP1-R CCAGCCGACGAGGTTTCCAC
13 AP37-F Os04g AGGCGGGCAGCGTCTCCAT AP37-R CCATAAGCCAGCCACGATGATGA AP25-F Os03g CCGCAGAACGTCAACTCCGT AP25-R GCACAAGCCGTTCTCGTGC Mel1-F Os03g CGAGGTCTGGTCTCTGCTCAACC Mel1-R GAAGCACATGGCTGAACTGACCT SDS-F Os03g GCTGCCGAGCATGGAGGAGT SDS-R CTCCCTGTTCCTCGCCTTGG PAIR1 Os03g GGACCCAGATTAACCAGACACCA GGTGCCACCCTGTAGTAGCTCG PAIR3 Os10g TCTTCACGAGAAGTTCAAGGAGGAG CGCCGACTGTCTTCTCAGCAT GAMyb-F Os01g CATCCTGGTCCATTCCTCAATGAC GAMyb-R TTCAGGATGAGGTGAAGTGTCCC In-situ hybridization analysis S80qPCR-F2 CATGTTCAACACCAAGATTCATTCG S80FLcds-R2 TGCAAACCATGACATACCAAAGATC Subcellular localization analysis S80cDNA-XbaI-F TATCTAGAGTGGTAGAGTGCGAGGAAG KpnI-S80-nonstop-R GAGGTACCAGGTACTCATCCACCACTTCAA BiFC construction BiFC-TDR-BamHI-F AGGGATCCCACCACATGGGAAGAGGAGACC BiFC-TDR-SalI-R ATGTCGACTCAAACGCGAGGTAATGCAGG BiFC-UDT-BamHI-F GTGGATCCATGCCGCGGCGCGCGAGGGCGA BiFC-UDT-Xho-R TGCTCGAGATGCTTGGAACCTCCACAATGCTG G BiFC-bHLH141-Pst-F AGCTGCAGTTTGCCAAAATGATTGTTGGG BiFC-bHLH141-Sal-R GCGTCGACTTGAATATGTCGAGGGCCTGG S80-Y2H-AD-BamH-F GTGGATCCCGAGGAAGATGTATCACC S80-Y2H-AD-Xho-R AGCTCGAGCTAGTTAGTACTCATCCACCAC Yeast Two Hybrid S80-Y2H-AD-BamH-F GTGGATCCCGAGGAAGATGTATCACC S80-Y2H-AD-Xho-R AGCTCGAGCTAGTTAGTACTCATCCACCAC S80-Y2H-BD-Pst-R CGCTGCAGTTAGTACTCATCCACCACTT 141-Y2H-BD-Eco-F AGGAATTCTTTGCCAAAATGATTGTTGGG
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Figure S1. Figure S2 RT-PCR. qpcr RT-PCR. Northern. IVSwt ΔIVS IVS IVS IVS. NTC mock IVSwt ΔIVS IVS IVS. mock IVSwt ΔIVS
Figure S1 40 cycles IVS IVS IVS NTC wt Δ Δ mut pri-mirna163 * Fig. S1 Transcripts generated from the MIR163 gene variants in which splice sites have been mutated are not spliced. products were separated
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Supplementary Information
Supplementary Information Supplementary Fig. 1. Seed dormancy and germination responses of RVE1 and PIF1. (a) Diagram of RVE1 and the T-DNA insertion of the rve1-2 mutant (SAIL_326_A01). Black boxes represent
Supplemental Figure 1 A
Supplemental Figure 1 A Supplemental Data. Han et al. (2016). Plant Cell 10.1105/tpc.15.00997 BamHI -1616 bp SINE repeats SalI ATG SacI +1653 bp HindIII LB HYG p35s pfwa LUC Tnos RB pfwa-bamhi-f: CGGGATCCCGCCTTTCTCTTCCTCATCTGC
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Supplemental Figure legends Figure S1. (A) (B) (C) (D) Figure S2. Figure S3. (A-E) Figure S4. Figure S5. (A, C, E, G, I) (B, D, F, H, Figure S6.
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Supplemental Data. Benstein et al. (2013). Plant Cell /tpc
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Supplementary Data 1.
Supplementary Data 1. Evaluation of the effects of number of F2 progeny to be bulked (n) and average sequencing coverage (depth) of the genome (G) on the levels of false positive SNPs (SNP index = 1).
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Supplementary information, Figure S1
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S156AT168AY175A (AAA) were purified as GST-fusion proteins and incubated with GSTfused
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Supplemental Materials Supplemental Figure S1 (a) Phenotype of the wild type and grik1-2 grik2-1 plants after 8 days in darkness.
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Supplemental Data. Zhou et al. (2016). Plant Cell /tpc
Supplemental Figure 1. Confirmation of mutant mapping results. (A) Complementation assay with stably transformed genomic fragments (ComN-N) (2 kb upstream of TSS and 1.5 kb downstream of TES) and CaMV
Supplementary Methods
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Supplemental Figure 1
Supplemental Figure 1 A gta2-1 gta2-2 1kb AT4G08350 B Col-0 gta2-1 LP+RP+LBb1.3 C Col-0 gta2-2 LP+RP+LB1 D Col-0 gta2-2 gta2-1 GTA2 TUBLIN E Col0 gta2-1 gta2-2 Supplemental Figure 1. Phenotypic analysis
Supplemental Table 1. List of PCR primers
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Supplementary information
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