New Phytologist Supporting Information Figs S1–S7 and Table S1
Article title: The putative Agrobacterium transcriptional activator-like virulence protein
VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell
nucleus
Authors: Yafei Wang, Wei Peng, Xu Zhou, Fei Huang, Lingyun Shao and Meizhong Luo
Article acceptance date: 28 April 2014
The following Supporting Information is available for this article:
Fig. S1 Western blot analysis of the expression of VirD5△13 in yeast cells
Fig. S2 Evolutionary relationships of VirD5 homologs and the NLSs on their amino acid
sequences.
Fig. S3 Subcellular localization assay for the Os12g0562400
Fig. S4 Coprecipitation assay for VirD5, AtVIP1 and VirE2 by glutathione sepharose
beads.
Fig. S5 Coprecipitation assay for VirD5, AtVIP1 and VBF by glutathione sepharose beads.
Fig. S6 Sequence analysis of virD operon and RT-PCR assays for VirD5, VirD2, and VirE2.
Fig. S7 A model for the putative functions of VirD5.
Table S1 List of oligonucleotides
Fig. S1 Western blot analysis of the expression of VirD5△13 in yeast cells. The total proteins
were extracted from yeast strain AH109 cells and analyzed by Western blotting using anti-Myc
antibody. VirD5△13, the total protein from yeast strain AH109 cells transformed with BDVirD5△13;
AH109,
the
total
protein
from
yeast
strain
AH109
Fig. S2 Evolutionary relationships of VirD5 homologs and the NLSs on their amino acid
sequences. (a) Evolutionary relationships of VirD5 homologs. The evolutionary history was
inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length =
1.49483880 is shown. The tree is drawn to scale, with branch lengths (next to the branches) in
the same units as those of the evolutionary distances used to infer the phylogenetic tree. The
evolutionary distances were computed using the Poisson correction method and are in the units
of the number of amino acid substitutions per site. Evolutionary analyses were conducted in
MEGA5. (b)The NLSs on the VirD5 homologous protein sequences
Fig. S3 Subcellular localization assay for the Os12g0562400. The YFP-tagged Os12g0562400
and CFP-tagged Ghd7 were coexpressed in the rice protoplast cells. Ghd7 was used as a nucleus
localization marker.
Fig. S4 Coprecipitation assay for VirD5, AtVIP1 and VirE2 by glutathione sepharose beads
Glutathione sepharose beads were used for coprecipitation. Lane 1, GST-AtVIP1+VirE2-CBD;
Lane 2, GST-AtVIP1+His-VirD5; Lane 3, GST-AtVIP1+VirE2-CBD+His-VirD5; Lane 4,
GST+VirE2-CBD+His-VirD5. Input, samples processed without precipitation.
Fig. S5 Coprecipitation assay for VirD5, AtVIP1 and VBF by glutathione sepharose beads.
Glutathione sepharose beads were used for coprecipitation. Lane 1, GST-AtVIP1+VBF-CBD;
Lane 2, GST-AtVIP1+His-VirD5; Lane 3, GST-AtVIP1+VBF-CBD+His-VirD5; Lane 4,
GST+VBF-CBD+His-VirD5. Input, samples processed without precipitation.
Fig. S6 Sequence analysis of virD operon and RT-PCR assays for VirD5, VirD2, and VirE2. (a)
Schematic representation of virD operon from Agropine-type Ti plasmid pTiBo542. Positions of
the primers used for RT-PCR, -10, -35 and RBS (ribosome binding site) regions are indicated.
(b) RT-PCR testing of VirD2 and VirD5. Total RNA was isolated from 2ml of Agrobacterium
strain EHA105 culture. After digestion with gDNA Eraser, the RNA samples were used to carry
out reverse transcription (+RT). The RNA samples without reverse transcription (-RT) were used
as controls to show that all of Ti plasmid DNA was digested out. The reverse transcription was
performed using gene-specific primer (D5-R07) of VirD5. The PCR reactions were carried out
on the cDNA using primers of VirD2 (D2-F and D2-R) or VirD5 (D5-F06 and D5-R06). (c)
Testing of VirD5 and VirE2 gene expression by RT-PCR. +AS: induced with acetosyringone
(AS), - AS: non-induced with acetosyringone (AS). Three independent RT-PCR experiments
were
performed
and
the
results
were
consistent.
Fig. S7 A model for the putative functions of VirD5
Table S1 List of oligonucleotides
Gene name and Gene ID
VirD5
Gene ID 6382152
Primer
name
Sequence
Restriction site
D5-F06
TGGTTTACTGCTTCTGGGTCA
D5-R06
GCGATACACTTGCTGCTCACG
D5-R07
TTTCCGTCTTACGCCGACTGATT
D-F09
gtcgacATGACAGGAAAGTCGAAAGTT
Sal1
D-R9
ccatggcGCGTTTAAACGCTTTGTCTTG
Nco1
D-F17
ACGCgtcgacATGACAGGAAAGTCGAAAGTT
Sal1
D-R17
CATGccatggcATCTGTCCGTTGACCAATGGG
Nco1
D-F18
ACGCgtcgacTCCTCCGATTCTTTTGTGTAT
Sal1
D-R18：
CATGccatggcGCGTTTAAACGCTTTGTCTTG
Nco1
D-F12
CGCggatccgtATGACAGGAAAGTCGAAAGTT
BamH1
D-R08
CGCggatccTCAGCGTTTAAACGCTTTGTC-3'
BamH1
D-R12
ggatccATCCGGCGGAACAAGGACAGG
BamH1
D-R13
ggatccCGCACCGGTTTCGGTCGCGAG
BamH1
D-R14
ggatccCGTCAAGCTTGTGTACGACGG
BamH1
D-R15
ggatccTTCATAAGCAGTTCGCTCAGG
BamH1
D-R16
ggatccGCCCAAGGGAACACCGTTGTA
BamH1
D-R19
CGCggatccTGTATACTCGCCGGTAATCCC
BamH1
D-R20
CGCggatccTAAATCTCGGAGCCAGGTCAC
BamH1
D-F19
CGCggatccgtGAAGAGGACACCCCCTCAACC
BamH1
D-F20
CGCggatccgtCGCTGGAGTAAGAGGGAACGC
BamH1
D-F21
5CGCggatccgtGATAGATCAGACGTCCCTTTA
BamH1
D-F22
CGCggatccgtTATGAGTCGGAGCATATTTTC
BamH1
D-F10
ggatccATGACAGGAAAGTCGAAAGTT
BamH1
D-R10
ggtaccGCGTTTAAACGCTTTGTCTTG
Kpn1
D-F03
ggatccATGACAGGAAAGTCGAAAGTT
BamH1
D-R03
gtcgacTCAGCGTTTAAACGCTTTGTC
Sal1
D-F25
ccatggctATGACAGGAAAGTCGAAAGTT
Nco1
D-R25
gcggccgccGCGTTTAAACGCTTTGTCTTG
Not1
D-F26
ccatggtcATGACAGGAAAGTCGAAAGTT
Nco1
D-R26
ggatcccGCGTTTAAACGCTTTGTCTTG
BamH1
D-F27
CGGggtaccATGACAGGAAAGTCGAAAGTT
Kpn1
D-R27
GCtctagaGCGTTTAAACGCTTTGTCTTG
Xba1
VirD2
D2-F
GAATGGGCAGCCGAGATGTTT
Gene ID:6382149
D2-R
CGGGCGAGAAGTATCGGAATT
VirE2-F01
GCAGCGAGCACGGAAATCAAG
VirE2-R01
TATCGGGCAGCAGCGAACCAG
14640-F1
actagtATGGATCCGTCTAGCAATGAGA
Spe1
14640-R1
ggtaccAAAGCTGTTGACGCTTTGGCTA
Kpn1
VirE2
14640-F2
CATGccatggctATGGATCCGTCTAGCAATG
Nco1
Gene ID:6382154
14640-R2
CCGctcgagAAAGCTGTTGACGCTTTGG
Xho1
14640-F3
CATGccatggtcATGGATCCGTCTAGCAATG
Nco1
14640-R3
CGGggtaccgAAAGCTGTTGACGCTTTGGC
Kpn1
14640-F4
CGGggtaccATGGATCCGTCTAGCAATGAG
Kpn1
14640-R4
CATGccatggcAAAGCTGTTGACGCTTTGGC
Nco1
V1-F01
ggatccATGGAAGGAGGAGGAAGAGGA
BamH1
V1-R01
ggtaccGCCTCTCTTGGTGAAATCCAT
Kpn1
V1-F02
CGCggatccATGGAAGGAGGAGGAAGAGGA
BamH1
V1-R02
CCGctcgagTCAGCCTCTCTTGGTGAAATC
Xho1
V1-F03
CCGgaattcATGGAAGGAGGAGGAAGAGG
EcoR1
V1-R03
CGCggatcccTCAGCCTCTCTTGGTGAAAT
BamH1
V1-F06
ccatggtcATGGAAGGAGGAGGAAGAGGA
Nco1
VIP1
V1-R06
ggatcccGCCTCTCTTGGTGAAATCCAT
BamH1
Gene ID: 840957
V1-NR
CCGctcgagTTCGATATTAAACGACGCCG
Xho1
V1-CF
CGCggatccTCGATTTTAGCTTCTGTGAGT
BamH1
V1-R05
CCGctcgagGCCTCTCTTGGTGAAATCCAT
Xho1
V1-CF01
CATGccatggagTCGATTTTAGCTTCTGTGAGT
Nco1
V1-F08
CGGggtaccATGGAAGGAGGAGGAAGAG
Kpn1
V1-R08
CCGgaattcGCCTCTCTTGGTGAAATCC
EcoR1
6250-F
gaattcATGATGATGTTACCAGAAGCA
EcoR1
6250-R
gagctcTGTTTTAGGCCTCACTTCAAT
Sac1
6250-F1
CGCggatccATGATGATGTTACCAGAAGCA
BamH1
6250-R1
CGGggtaccTGTTTTAGGCCTCACTTCAAT
Kpn1
VBF
6250-F2
ccatggtcATGATGATGTTACCAGAAGCA
Nco1
Gene ID: 842078
6250-R2
ggatcccTGTTTTAGGCCTCACTTCAAT
BamH1
6250-R3
CCGctcgagTTATGTTTTAGGCCTCACTTC
Xho1
6250-F4
CATGccatggctATGATGATGTTACCAGAAGCA
Nco1
6250-R4
CCGctcgagTGTTTTAGGCCTCACTTCAAT
Xho1
IMPA-1 importin alpha-1
06720-F
ggatccATGTCACTGAGACCCAACGCT
BamH1
Gene ID: 819857
06720-R
ggtaccGCTGAAGTTGAATCCTCCGGA
Kpn1
IMPA-4 importin alpha-4
Gene ID: 837448
09270-F
ggatccATGTCGCTGAGGCCGAGCAC
BamH1
09270-R:
ggtaccGGCAAATTTGAATCCACCAAC
Kpn1
Primers for RDSA
RDSA_1
TAGTTGAGTCTCACAAACGAACAC(N20)CATTCCAAAATCCATGGCTGATA
RDSA_1fo
TAGTTGAGTCTCACAAACGAACAC
RDSA_1re
TATCAGCCATGGATTTTGGAATG
RDSA_2
AATGGATCCAAGCTTAAGC(N18)CGTTGAATTCCCATGGACA
RDSA_2fo
AATGGATCCAAGCTTAAGC
RDSA_2re
TGTCCATGGGAATTCAACG
Primers for Y1H assay
D5RE-S
agctACCAGCGACCGCGCTCAGGC
Hind3
D5RE-AS
tcgaGCCTGAGCGCGGTCGCTGGT
Xho1
D5REm-S
agctACCAGCGAatGCGCTCAGGC
Hind3
D5REm-AS
tcgaGCCTGAGCGCatTCGCTGGT
Xho1
pAbAi-S
GTTCCTTATATGTAGCTTTCGACAT
D5REm1-S
agctACCAGCGAatatGCTCAGGC
Hind3
D5REm1-AS
tcgaGCCTGAGCatatTCGCTGGT
Xho1
Primers for EMSA assay
D5RE2-F
ACCAGCGACCGCGCTCAGGC
D5RE2-R
GCCTGAGCGCGGTCGCTGGT
mD5RE2-F
ACCAGCGAatGCGCTCAGGC
mDRE2-R
GCCTGAGCGCATTCGCTGGT