Supplementary materials
Figure S1. The maximum-likelihood tree of OsCASP1 homologs with their protein sequences. The scale bar indicates the simple matching distance. The alignment and phylogenetic tree were constructed with MEGAX (http://megasoftware.net/). The numbers for interior branches indicate the bootstrap values for 500 replications.
Figure S2 . (A) Alignment of CASPs in Arabidopsis and rice. IC: Intracellular loop; TM: Transmembrane domain; EC: extracellular loop. (B) (C) Transmembrane domains of OsCASP1 was predicted with TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (B) and Protter (http://wlab.ethz.ch/protter/#) (C)
Figure S3. Models of the constructs for complementation test, tissue-specific expression, and subcellular localization of OsCASP1.CASP1-N includes a transmembrane region predicted by TMHMM with a lower probability. TM: Transmembrane region.
Figure S4. The phenotypes of the WT and els1 mutant and characterization of els1.(A) Internode length and plant height; IN = internode. (B) The tiller number of the WT and els1 mutant at the heading stage. (C) and (D) The tiller phenotype of the seedling at 33-d-old. The emergence of tiller in the els1 mutant was delayed. (E) The els1 locus was fine-mapped on chromosome 4. The numbers of recombinants are shown in brackets.
Figure S5. The genotype and phenotype of CRISPR/cas9 mutants.(A) Schematic map of OsCASP1 gene and sequence alignment of the sgRNA target region showing altered bases in different mutant lines. The target region of the sgRNA was indicate with arrow, and the heterozygous transgenic plants with different mutant sites were marked with rectangles. (B) The typical phenotypes of CRISPR/cas9 transgenic plants.
Figure S6. Subcellular localizations of OsCASP1 protein. (A)OsCASP1pro:OsCASP1-GFP in rice root. (B) and (C)OsCASP1pro:OsCASP1-GFP in onion and Arabidopsisprotoplast. (D) and (E) 35S::DsRed-OsCASP1 in onion andArabidopsis protoplast. Circle indicates the nuclear position. (F) 35S::DsRed-CASP1-N in rice protoplasts. CASP1-N includes a transmembrane region predicted by TMHMM with a lower probability. ER: Endoplasmic reticulum.
Figure S7. Subcellular localizations of OsCASP1 protein in transgenic plant roots carrying 35S::DsRed-OsCASP1. (A) OsCASP1 in the cell membrane in small lateral roots. (B) and (C) OsCASP1 is concentrated in the stele of lateral roots. (D) and (E) OsCASP1 in main roots. Arrows indicate the nuclei.
Figure S8. The localization of OsCASP1pro:OsCASP1-GUSexpression after salt treatment. (A) Whole roots treated with NaCl. (B) Crown root with lateral roots. (C) Lateral root. (D) Transection of the crown root. (E) Longitudinal section of the crown root. ep, epidermis; ex, exodermis; sc, sclerenchyma; co, cortex; en, endodermis; pe, pericycle. Scale bar: (A) 1cm; (B), (C), (D) and (E) 0.1cm.
Figure S9. The representative cross-sections at different zones in primary roots stained with berberine-aniline blue and phloroglucinol. (A) Staining with berberine-aniline blue. Autofluorescence of cell walls is detected as blue. Arrow head indicates CS at endodermis. (B) Staining with phloroglucinol. Arrow head indicates endodermis. ep, epidermis; ex, exodermis; sc, sclerenchyma.
Figure S10. The representative cross-sections stained with periodic acid (A) and permeability of CS was evaluated using periodic acid (B). Purple color indicates that the periodic acid penetrated into root tissues. (C) The roots stained with periodic acid.