Supporting Information
Table S1. Disease scores for plants shown in Figure S1b inoculated with virulent DC3000.
Table S2. PCR primers used in this study.
Figure S1. Evaluation of transgenic Arabidopsis lines expressing HA-SRFR1 tagged with
NES, NLS, nes or nls sequences.
(a) Disease scoring scale used to evaluate multiple lines per construct. The lower half of
leaves was infiltrated with DC3000 or DC3000(avrRps4) at a density of 1  106 cfu/ml and
scored 5 days after inoculation.
(b) Two examples of disease assays on independent homozygous transgenic srfr1-1 lines
in the T3 generation infiltrated with DC3000(avrRps4). Col-0 (resistant), RLD (susceptible)
and srfr1-1 (intermediate resistance) were used as non-transgenic controls. Transgenic
lines used for in planta bacterial growth assays shown in Figure 1b are indicated in bold.
Black circles represent disease scores from individual leaves, horizontal bars indicate
means, and letters indicate statistically significant differences determined by WilcoxonMann-Whitney tests of unpaired samples (P<0.05). Plant lines were also tested with
virulent DC3000 and were scored as susceptible as RLD (see Table S1). Results shown
here are representative of at least two (nes and nls lines), three (NES 17.2, NES 28.3, NLS
4.1, NLS 5.5) and six (NES 18.1, NLS 2.7) assays.
(c) Protein blots of lines shown in (b). Total protein probed with anti-HA (top) and antiGAPDH antibody as a loading control (bottom).
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Figure S2. Protein blot of soluble fractions from HA-SRFR1 expressing plants shown in
Figure 1.
Immunoblot analyses were performed as described in legend to Figure 1a. A sample of nlsHA-SRFR1 from the microsomal fraction was loaded as a positive control. Compared to
the soluble fraction, the nuclear and microsomal fractions shown in Figure 1 are 20-fold
and 4-fold concentrated, respectively. Molecular mass values of protein standards (in kD)
are shown on the left of the panels. The assay was repeated three times with similar results.
Figure S3. Yeast two-hybrid analyses of SRFR1 and TCP8 fragments.
(a) Diagrams of the SRFR1 and TCP8 fragments inserted into yeast two-hybrid vectors.
(b) Interactions between full-length or truncated SRFR1 and full-length TCP8. The
Gal4BD-SRFR1 and Gal4AD-SGT1b interaction is included as a control (Li et al., 2010).
Both the N-terminal and C-terminal fragment of SRFR1 binds to TCP8, although the latter
interaction appears weaker.
(c) ONPG assay of protein interactions in yeast using the indicated BD-SRFR1 fragments
and TCP8, TCP14, and TCP15. The negative control (-) is BD-SRFR1 and pEXP-AD502.
The positive control (+) is BD-HY5 and AD-STO.
(d) Interactions between full-length SRFR1 and TCP8 fragments.
(e) TCP8 fragments are stable when transiently expressed in N. benthamiana. Yeast twohybrid constructs were recombined into pBA-Myc and expressed using Agrobacteriummediated transient expression.
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Figure S4. Subcellular localization of TCP proteins.
(a) TCP proteins are predominantly localized to nuclei. GFP-TCP3, GFP-TCP8, GFPTCP14, and GFP-TCP15 fusion constructs were transiently expressed in N. benthamiana
leaves using Agrobacterium. GFP (bottom row) was included as a control. RFP was coexpressed to label the cytoplasm and nucleus of transiently transformed cells. Scale bars:
20 µm in each panel.
(b) Magnification of Figure S2a to show GFP-TCP localization in nuclei. Scale bars: 10
µm in each panel.
(c) Protein blot of total protein isolated from transiently transformed N. benthamiana.
Molecular weights of protein standards (in kD) are shown on the left. Coomassie-stained
blot is shown below to indicate relative amount of protein on gel.
Figure S5. BiFC analysis of SRFR1-TCP interactions with reciprocal tags.
Transient co-expression of cCFP-SRFR1 with the indicated nVenus-TCPs, together with
RFP as a cellular marker for cytoplasm and nucleus. Scale bars: 10 µm in each panel.
Figure S6. TCP homo- and heterodimerizations analyzed by BiFC.
Transient pair-wise co-expression in N. benthamiana of TCP3, TCP8, TCP14 and TCP15
as either nVenus or cCFP fusions in the indicated combinations, together with RFP as a
cellular marker for cytoplasm and nucleus. Scale bars: 5 µm in each panel.
Figure S7. TCP8 over-expressing plants do not show changes in bacterial resistance.
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(a) Western blot of total extracts of Col-0 (Col) and T2 generation of six independent MycTCP8 OX lines probed with HRP-conjugated Myc antibody (A-14). The lane marked "M"
contained molecular mass markers.
(b) In planta bacterial growth was measured in Col-0 and the T3 generation of two
independent Myc-TCP8 over-expression (OX) lines (line 2 and line 9) on day 0 and day 3
after inoculation with DC3000 (top) and DC3000(avrRps4) (bottom). Plants were
inoculated with a bacterial suspension at a density of 5  104 cfu/ml. Values represent
averages of cfu/cm2 leaf tissue from triplicate samples, and error bars denote standard
deviation. This experiment was repeated once with similar results.
Figure S8. Molecular characterization of the tcp8-1 tcp14-5 tcp15-3 triple T-DNA
insertion line.
(a) Schematic representation of the gene structures of TCP8 (top), TCP14 (middle) and
TCP15 (bottom) with exons in orange, T-DNA insertion sites as determined by sequencing
of flanking DNA, and location of primers used for RT-PCR.
(b) Ethidium bromide-stained gels showing PCR products obtained with TCP genespecific primers on opposite sides of the respective T-DNA insertions using RNA isolated
from Col-0 (left) and tcp8-1 tcp14-5 tcp15-3 (right) plants.
(c) Gel showing PCR products obtained with TCP gene-specific primers downstream of
the respective T-DNA insertion sites. Numbers indicate the positions of DNA size
standards. ACTIN2 primers were included as a control for the amount of RNA used for
RT-PCR.
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Figure S9. Bacterial growth assays in single and double T-DNA insertion lines of TCP8,
TCP14 and TCP15.
In planta bacterial growth was measured in Col-0, tcp8-1, tcp14-5, tcp15-3, tcp8-1 tcp14-5
(t8 t14), tcp8-1 tcp15-3 (t8 t15),, and tcp14-5 tcp15-3 (t14 t15) on the indicated days after
inoculation with DC3000 (top) and DC3000(avrRps4) (bottom). Plants were inoculated
with a bacterial suspension at a density of 5  104 cfu/ml. Values represent averages of
cfu/cm2 leaf tissue from triplicate samples, and error bars denote standard deviation. This
experiment was repeated twice with similar results.
Figure S10. Reduced effector-triggered immunity to DC3000 expressing avrRps4,
avrRpm1, avrRpt2, and hopA1, but no increased susceptibility to the Type III secretiondeficient strain DC3000(hrcC-), in tcp8-1 tcp14-5 tcp15-3 mutant lines.
(a) In planta bacterial growth in Col-0 (C0) or tcp8-1 tcp14-5 tcp15-3 (3-t) plants on day 0
(green bars) and day 3 (orange bars) of DC3000, DC3000(avrRps4), DC3000(hopA1),
DC3000(avrRpt2), or DC3000(avrRpm1) inoculated at a bacterial density of 5  104
cfu/ml.
(b) Experiments as in (a) with indicated plant lines inoculated with DC3000(hrcC-).
Values in (a) and (b) represent averages from triplicate samples, and error bars denote
standard deviation. Asterisks indicate statistically significant differences determined by a
two-tailed Student’s t-test (P<0.05). These experiments were repeated three times (a) or
twice (b), with similar results.
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Figure S11. Growth analysis of the tcp8-1 tcp14-5 tcp15-3 srfr1-4 quadruple mutant line.
(a) Two independent replicates of plants grown under short-day conditions (8 h light/16 h
dark, 24C). Shoots were harvested after 24 days (top) or 25 days (bottom).
(b) Three independent replicates of plants grown under long-day conditions (16 h light/8 h
dark, 22C). Shoots were harvested after 26 days for all replicates.
In (a) and (b), error bars denote standard deviation, and letters indicate statistically
significant differences determined by a two-tailed Student’s t-test (P<0.05), with sample
sizes given in parentheses.
Figure S12. Replicate PR2 protein blots of plant lines shown in Figure 6.
Total protein was isolated from two separate batches of plants grown 3 months apart.
GAPDH is shown as a loading control.
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