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Supporting Information Figs S1–S10 and Table S1
Fig. S1 XopDXcc8004 does not affect bacterial growth in Xcc-infected radish leaves.
Four-week-old radish plants were inoculated with a 105 CFU ml-1 inoculum of Xcc8004
and Xcc8004ΔxopD by clipping. Bacterial growth of strains Xcc8004 and Xcc8004ΔxopD
were assessed at 0, 2, 4, 6, 8, and 10 dpi. Data points represent mean log10 CFU cm-2 of
three independent experiments. Error bars indicate SD. Different letters above the bars
indicate values that are significantly different (P<0.05) from each other as determined by
one-way ANOVA (SPSS version 17.0).
Fig. S2 XopDXcc8004 does not affect the pathogenicity of Xcc.
Four-week-old wild-type Arabidopsis (Col-0) plants were inoculated with a 105 CFU ml-1
inoculum of Xcc8004 and Xcc8004ΔxopD by stabbing. Bacterial growth was performed at
0 and 3 dpi. Data points represent mean log10 CFU cm-2 of three independent experiments.
Error bars indicate SD. Different letters above the bars indicate values that are significantly
different (P<0.05) from each other as determined by one-way ANOVA (SPSS version 17.0).
Photographs were taken at 3 dpi.
Fig. S3 XopDXcc8004 does not promote the growth of PstDC3000 in Arabidopsis leaves.
Four-week-old Arabidopsis (Col-0) plants including wild-type (WT) and transgenic lines
were sprayed with 50 mM estradiol for 24 h before inoculation with a 104 CFU ml-1 inoculum
of PstDC3000. Data points represent mean log10 CFU cm-2 of three independent experiments.
Error bars indicate SD. Different letters above the bars indicate values that are significantly
different (P<0.05) from each other as determined by one-way ANOVA (SPSS version 17.0).
Fig. S4 XopDXcc8004 delays disease symptoms in PstDC3000-infected transgenic
Arabidopsis line (RGAp::GFP-RGA).
Four-week-old Arabidopsis leaves were sprayed with H2O, 50 µM GA3 for 2 h, 50 mM
estradiol for 24 h, or 50 mM estradiol for 24 h and 50 µM GA3 for 2 h before inoculation with
a 104 CFU ml-1 inoculum of PstDC3000. Numbers at the bottom of each panel indicate the
number of leaves surveyed (denominator) and the number of leaves showing the phenotypes
(numerator). Photographs were taken at 2 dpi. Total proteins of leaves were extracted and
used for anti-FLAG and anti-GFP immnoblot assays.
Fig. S5 Schematic diagram of three truncated RGA proteins.
Three partial RGA encoding sequences were identified from potential yeast transformants
screened by XopDXcc8004. Three partial RGA sequences encode residues 19-261, 3-153 and
40-220 amino acid sequence in RGA protein. Three deduced amino acid sequence contain the
amino acid 44-48 DELLA and amino acid 89-93 VHYNP motifs.
Fig. S6 XopDXcc8004 and XopDXcc80041-210 interact with RGA.
Yeast two-hybrid assays were performed to detect the interaction of XopDXcc8004,
XopDXcc80041-210, and XopDXcc8004221-442 with RGA. Total proteins were extracted from yeast
cells and were used for anti-myc and anti-HA immunoblot assays.
Fig. S7 XopDXcc8004 interacts with DELLA proteins GAI, RGL1, RGL2 and RGL3.
Yeast two-hybrid assays were performed to detect the interaction of XopDXcc8004 with GAI,
RGL1, RGL2, and RGL3. Total proteins were extracted from yeast cells and were used for
anti-myc and anti-HA immunoblot assays.
Fig. S8 GA reduces the growth of PstDC3000 in Arabidopsis leaves.
Four-week-old Arabidopsis (Col-0) plants including wild-type (WT) and transgenic lines 2
and 4 were sprayed 50 mM estradiol for 24 h and/or were then sprayed 50 µM GA3 for 2 h
before inoculating a 104 CFU ml-1 inoculum of PstDC3000. Data points represent mean log10
CFU cm-2 of three independent experiments. Error bars indicate SD. Different letters above
the bars indicate values that are significantly different (P<0.05) from each other as determined
by one-way ANOVA (SPSS version 17.0).
Fig. S9 DELLA motif is required for RGA to interact with XopDXcc8004 and its truncated
protein XopDXcc80041-210.
Yeast two-hybrid assays were performed to detect the interaction of XopDXcc8004,
XopDXcc80041-210, and XopDXcc8004221-442 with the RGA mutant ΔDELLA (lacks the DELLA and
VHYNP motifs). Total proteins were extracted from yeast cells and were used for anti-myc
and anti-HA immunoblot assays.
Fig. S10 XopDXcc8004 represses the bacterial flagellin flg22-induced H2O2 production.
Four-week-old Arabidopsis (Col-0) plants including wild-type (WT) and transgenic line 2
were sprayed with 50 mM estradiol for 24 h and/or with 50 µM GA3 for 2 h. The means of
eight replicates of relative luminescence are shown. Error bars indicate SD. The experiment
was repeated three times with similar results.
Table S1 List of primers used in this study
Primer name: sequence (5’-3’)
Description
For the mutant
Xcc8004ΔxopD
strain
(HindIII)xopD-1: CCCAAGCTTAGAGTTTGAACGGCG CTAC
(PstI)xopD-2: AACTGCAGTTCCACGTTTCTTCTGGC
(PstI)xopD-3: AACTGCAGTTCTAGCAATTTGACAATC
(EcoRI)xopD-4: CGGAATTCCTTTCATCGCGGCAACCT
For the complemented
strain
Xcc8004ΔxopD
(XopDXcc8004)
or
(XopDXccB100)
8004HM-F(BamHI): AAGCTTGGATCCATGGAATCCCAAGAC
8004HM-R(KpnI):GAATTCGGTACCCTATCAGTGATGATGATGATGATGGAA
CTTTTTCC
B100HM-F(BamHI): AAGCTTGGATCCATGGACAGATTATT
B100HM-R(KpnI):GAATTCGGTACCTTATCAGTGATGATGATGATGATGCTG
GAACTTCCAC
For the yeast two-hybrid
screening
(NdeI)XopD-F: CCCATATGATGGAATCCCAAGACC
(PstI)XopD-R: AACTGCAGCTAGAACTTTTTCCAC
(PstI)XopD-N-R: AACTGCAGCTAGTCCGTCACTTGAGG
(NdeI)XopD-C-F: CCCATATGATGACTTCGTGGCTGCTG
(NdeI)RGA1-F: CCCATATGATGAAGAGAGATCATCACC
(SacI)RGA1-R: CGAGCTCTCAGTACGCCGCCGTCGA
(EcoRI)GAI-F: CGGAATTCATGAAGAGAGATCATCA
(SacI)GAI-R: CGAGCTCCTAATTGGTGGAGAGTT
(NdeI)RGL1-F: CCCATATGATGAAGAGAGAGCACAAC
(SacI)RGL1-R: CGAGCTCTTATTCCACACGATTGAT
(NdeI)RGL2-F: CCCATATGATGAAGAGAGGATACGGA
(BamHI)RGL2-R: CGGGATCCTCAGGCGAGTTTCCAC
(NdeI)RGL3-F: CCCATATGATGAAACGAAGCCATCAAG
(SacI)RGL3-R: CGAGCTCCTACCGCCGCAACTCC
For RGAK33A,K65A
RGAΔDELLA
RGADELLA-F: GGTAACATGGACGACGGTGGAGGTAACATG
RGADELLA-R: GTTACCTCCACCGTCGTCCATGTTACCTCCACC
RGAVHYNP-F: GCGACGGATACTGATAATATGCTCTCTGAG
RGAVHYNP-R: CATATTATCAGTATCCGTCGCGAGATGAG
RGA-K33A-F: ATGATGATGGTGAGAAAAGAAGAAGACGGT
RGA-K33A-R: GTCTTCTTCTTTTCTCACCATCATCATCTT
RGA-K65A-F: GAGGTTGCTTTGAGACTCGAACAATTAGAG
RGA-K65A-R: TAATTGTTCGAGTCTCAAAGCAACCTCCGC
and
For
the
XopDXcc8004
transgenic plants
(XhoI)XopD-F: CCGCTCGAGATGGAATCCCAAGACC
(Csp45I)XopD-R: CCTTCGAAGAACTTTTTCCACCAC
For
the
protoplast
transformation assay
(KpnI)RGA1-F: GGGGTACCATGAAGAGAGATCATCACCAATT
(SalI)RGA1-R: GCGTCGACGTACGCCGCCGTCGAGAGTTTC
(XhoI)RGA-F: CCGCTCGAGATGAAGAGAGATCATCACCAATT
(Csp45I)RGA-R: CCTTCGAAGTACGCCGCCGTCGAGAGTTTC
(KpnI)XopD-F: GGGGTACCATGGAATCCCAAGACC
(SalI)XopD-R: GCGTCGACGAACTTTTTCCACCAC
(KpnI)XopD-C-F: GGGGTACCATGACTTCGTGGCTGCTG
(SalI)XopD-N-R: GCGTCGACGTCCGTCACTTGAGG
(XhoI)GID1-F: CCG CTCGAGATGGCTGCGAGCGATG
(Csp45I)GID1-R: CCTTCGAAACATTCCGCGTTTAC
For the transient protein
expression
(XhoI)GFP-F: CCGCTCGAGATGGTGAGCAAGGGCG
(PstI)GFP-R: AACTGCAGTTACTTGTACAGCTCGTCCA
For the BiFC assay
XopD-F-XbaI: GCTCTAGAATGGAATCCCAAGAC
XopD-R-XmaI: TCCCCCCGGGGAACTTTTTCCACC
RGA-F-XbaI: GCTCTAGAATGAAGAGAGATCAT
RGA-R-XmaI: TCCCCCCGGGGTACGCCGCCGTCG
For
the
quantitative
RT-PCR assay
qRGA-F: AGAAGCAATCCAGCAGA
qRGA-R: GTGTACTCTCTTCTTACCTTC