SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Phylogenetic tree of the BSK protein family.
The amino acid sequences of Arabidopsis BSK proteins belonging to RLCK-XII
subfamily were used for phylogenetic tree construction using the web service
Phylogeny.fr (www.phylogeny.fr; Dereeper et al., 2008, Nucleic Acids Res. 36: W465469). The phylogenetic tree was developed with the coordinated analysis of the amino
acid sequences by the programs MUSCLE for multiple sequence alignment, Gblocks for
automatic alignment curation, PhyML for tree building, and TreeDyn for tree drawing.
The CDG1 (constitutive differential growth 1) protein kinase, a member of the
Arabidopsis RLCK-VIIc subfamily, was used as an outgroup in this analysis. TAIR
accession numbers are shown in brackets. Scale above the tree represents branch length
measured by the number of amino acid replacements per position. Bootstrap values are
shown for each branch as percentage of 100 replicates.
Figure S2. Structure of BSK genes and position of T-DNA insertions in the mutants
characterized in this study.
Grey boxes represent exons, lines represent introns, yellow colored boxes represent 5'
untranslated regions, red colored boxes represent 3' untranslated regions, and arrowheads
represent 3' ends of the genes. The location of T-DNA insertions is indicated by inverted
triangles. Primers used for RT-PCR analysis to assess expression of the genes are
represented by solid arrows.
Figure S3. Expression of BSK transcripts in T-DNA insertion lines.
Semi-quantitative RT-PCR was used to determine the expression level of BSK1, BSK2,
BSK3, BSK4, BSK5, BSK6, BSK7 and BSK8 transcripts in 7-day-old wild-type Col-0 and
mutant seedlings. Expression of the GAPDH gene was used as an internal control for
cDNA template concentration. Primers used to amplify BSKs and GAPDH are listed in
Table S2.
Figure S4. Schematic representation of the crossing strategy used to generate bsk
multiple mutants.
Arrows above each mutant represent the parental lines. After each cross, seeds were
collected and progeny homozygous for all the mutant alleles were identified in the F2 and
F3 generations by PCR.
Figure S5. Developmental phenotype of bsk mutants grown in long-day conditions.
(a) Wild-type Col-0 and bsk mutants were grown under long-day conditions (16-h light/8h dark) at 25oC. Photographs were taken 32 days after sowing.
(b) Total leaf area of wild-type and mutant plants was measured using the ImageJ
program. Data are the means ± SD (n=3). The experiment was repeated three times with
similar results. An asterisk indicates a significant difference (P<0.05) between mutant
and wild-type plants.
Figure S6. Segregation of genotypes and developmental phenotypes in the F2 progeny
of the cross between the triple mutants bsk3,4,7 and bsk3,4,8.
(a) Wild-type Col-0 and 77 plants of the F2 progeny derived from the cross bsk3,4,7 X
bsk3,4,8 were grown under short-day conditions (8-h light/16-h dark) at 25oC. Plants
were genotyped and photographed 35 days after sowing. A representative plant for each
genotype is shown.
(b) Total leaf area of wild-type and mutant plants was measured using the ImageJ
program. Data are the means ± SD. An asterisk indicates a significant difference
(P<0.05) between mutant and wild-type plants. The number of plants analyzed for each
genotype, observed and expected segregation of the progeny are reported below the
graph.
Figure S7. GST is not phosphorylated by BIN2.
BIN2 and BIN2K69R fused to MBP, and GST were expressed in E. coli bacteria and
affinity-purified. Phosphorylation of GST by MBP-BIN2 and MBP-BIN2K69R was
assayed in vitro in the presence of [-32P]ATP, and proteins were fractionated by SDSPAGE. Gels were stained by Coomassie Blue (bottom panel), dried and exposed to
autoradiography overnight (top panel).
Figure S8. BIN2 phosphorylates BSK5, but not tomato MPK3K70R.
BIN2 and BIN2K69R were fused to MBP, while BSK5 and MPK3K70R were fused to GST,
expressed in E. coli bacteria and affinity-purified. Phosphorylation of GST-BSK5 and
GST-MPK3K70R by MBP-BIN2 and MBP-BIN2K69R was assayed in vitro in the presence
of [-32P]ATP, and proteins were fractionated by SDS-PAGE. Gels were stained by
Coomassie Blue (bottom panel), dried and exposed to autoradiography overnight (top
panel).
Figure S9. BSKs are not phosphorylated by tomato Pti1.
Tomato Pti1, Arabidopsis BSK1, BSK5, BSK6 and BSK8 were fused to GST, expressed
in E. coli bacteria and affinity-purified. Phosphorylation of GST-BSKs by GST-Pti1 was
assayed in vitro in the presence of [-32P]ATP, and proteins were fractionated by SDSPAGE. Gels were stained by Coomassie Blue (bottom panel), dried and exposed to
autoradiography overnight (top panel).
Figure S10. Expression of BSK genes in different tissues and development stages of
Arabidopsis.
Analysis of the expression pattern of BSKs in different tissues (a) and developmental
stages (b) of Arabidopsis was performed by using GenevestigatorV3
(https://www.genevestigator.com/gv/). The results are based on expression of the genes
in publicly available microarray experiments. The expression level is indicated by a
white-blue color scale shown in panel (a). The number of microarrays relevant to the
specific tissue or developmental stage is indicated.