Legends to the Supplementary Figures
Figure S1. Activity range of the 5xUAS tag. (a) Diagram showing the position of the 5xUAS
insertion (blue arrowheads) relative to the SHR genomic fragment. (b) Confocal images of
representative transgenic plants with a normal ground tissue pattern (left), with supernumerary
ground tissue layers (middle), and a root fasciation phenotype (right). (c) A graph showing the
proportion of independent transgenic lines exhibiting various numbers of ground tissue layers and
root fasciation. For each UAS-tagged SHR transgene, 47-63 independent transgenic lines were
observed by CLSM, and classified according to the number of ground tissue layers and root
fasciation, Scale bar: 50 m.
Figure S2. Expression patterns of GFP in the GAL4 enhancer trap lines used as host plants. (a)
Q2610 and (b) J0571. A magnified view of the root tip region is shown alongside of the seedling
image of each line.
Figure S3. Co-segregation and recapitulation analyses of urp mutants. The phenotype of each plant
is indicated above the gel images; w, wild type; m, mutant; m* mutant with an intermediate
phenotype, where the mutant phenotype segregated as a semi-dominant trait. The (+) and (-)
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symbols below the gel image indicate the presence and absence of the 5xUAS insertion, respectively.
In the case where PCR analysis was performed to distinguish homozygous from hemizygous
insertions, labels H and h, respectively, are used in place of (+) to distinguish these genotypes. The
confocal images on the right show recapitulation of the urp phenotype by the introduction of each
UAS-URP genomic fragment into the Q2610 host. Gray boxes and arrows, protein-coding regions;
thick bars with five black triangles, T-DNAs with the 5xUAS tag at their left border; black arrows,
positions of the primers used in the genotyping experiment. Primer sequences can be found in Table
S1. Mutant analyses were carried out as follows: (a) In urp1-D, a 5xUAS insertion was identified
upstream of a hypothetical open reading frame. This insertion was detected in all 84 T2 seedlings
exhibiting the urp1-D phenotype, while it was not detected in all 36 T2 seedlings with a wild-type
appearance. Introduction of the UAS-URP1 genomic fragment into Q2610 plants recapitulated the
same phenotype as the urp1-D mutant. (b) In urp2-D, a 5xUAS insertion was identified upstream of
PLT3. This insertion was not detected in all seven T2 seedlings with a wild-type appearance, while it
was detected in all 22 T2 seedlings with the mutant phenotype. The 5xUAS insertion had either a
homozygous or hemizygous status. (c) urp3-D contained two T-DNA insertion loci, one (locus A)
was upstream of OBP2, and the other (locus B) was between At2g31450 and At2g31460. In both loci,
the T-DNA was arranged in a back-to-back configuration. Co-segregation analysis revealed that the
phenotypic strength was correlated with the insertion status at locus A; seedlings with a strong
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phenotype (almost no root growth; strong patterning defects) were homozygous, while those with a
weaker phenotype (retarded root growth; weak patterning defects) were hemizygous. The 5xUAS
insertion status at locus B did not correlate with the seedling phenotype. (d) urp5-D contained three
T-DNA insertion loci, one (locus A) was upstream of RKD1, and the other two (locus B and C) were
intragenic regions of A3g07660 (locus B) and At5g65750 (locus C). Co-segregation analysis
revealed that the mutant phenotype was correlated with the insertion at locus A, but not with those at
locus B and C. For urp4-D, we did not perform a co-segregation analysis and moved directly on to
the recapitulation experiment, since a homologous gene (RKD1) had already been identified as being
responsible for the cell proliferation phenotype of urp5-D, a defect shared by urp4-D. (e) In urp7-D,
a 5xUAS insertion was identified upstream of SMB. This insertion was absent in all four T2
seedlings with a wild-type appearance, while it was detected in all 11 T2 seedlings exhibiting the
mutant phenotype.
Figure S4. RNA gel blot analysis of urp mutants. Lower panels show the blots stained with
methylene blue to visualize equal RNA loading. Lane S, RNA from shoots; lane R, RNA from roots.
Figure S5. Recapitulation of urp1-D root phenotype by UAS-URP1 transgene. (a) Schematic
representation of the UAS-URP1 construct and its frame-shift version. (b) Introduction of
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UAS-URP1, but not its frame-shift version, to Q2610 plants recapitulated the urp1-D root phenotype.
Representative T1 seedlings 10 days post-germination are shown. Scale bar, 1 cm.
Figure S6. GUS-staining of the promoter-nls:YFP:GUS reporter lines. (a-f) GUS staining of
cotyledons and the shoot meristem region of lines expressing nls:YFP:GUS fused to the promoter
regions of the indicated genes. Arrows indicate weak GUS staining. (g-l) GUS staining in the root
meristem region. Scale bar: 0.5 mm (a-f); 100 m (g-l).
Figure S7. Use of the Q2610 enhancer sequence as a post-embryonic and root-specific promoter. (a)
Schematic representation of the enhancer trap locus in Q2610 (yellow box). (b) Use of the
insertion-flanking genomic sequence as a promoter to drive the root-specific expression of GVG and
GFP-tagged RKD1/2 proteins.
Figure S8. RKD gene structures and T-DNA insertion positions. Arrows indicate the points of the
T-DNA insertions. Thick colored segments, protein-coding regions; thin gray segments, other
genomic regions.
Table S1. Primers used in this study.
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