Supporting Information Methods
Yeast two-hybrid (Y2H) assays
A cDNA library for Y2H was constructed from total RNA of ‘Ralls Janet’ pollen by using "Make
Your Own ‘Mate & Plate’ Library System User Manual" (Clontech). The cDNA library was
screened by using a ‘bait’ gene S1-RNase (accession no. GI:1018986), with the whole coding
region cloned into pGBKT7 vector. The screening was performed on SD/-Trp-Leu-His medium.
For interaction assays, S1, S2- (GI:643444), S5- (GI:642040), S7- (GI:7229072) and S9-RNases
(GI:642044) were inserted respectively into pGBKT7 vector. MdABCF (KF048865) and PhABCF
were inserted into pGADT7 vector. The non-S-RNase (Supplemental Table S3) and RNase I
(Beecher et al., 1998) were inserted into pGBKT7 vector as control. The interaction between
S-RNases and MdABCFs were tested by co-transforming them into AH109 strains. Interactions
were tested on SD/-Trp-Leu-Ade-His plate containing X-gal. A β-galactosidase activity assay of
the transformants was performed as Yeastmaker™ Yeast Transformation User Manual (Clontech).
Mating-based Split ubiquitin system assays
Protein interaction between S-RNase and MdABCF was examined in yeast using the DUAL
membrane Kit 3, which takes advantage of a split ubiquitin system, according to the
manufacturer’s protocol (Dualsystems Biotech). S1-, S2-, S5-, S7- and S9-RNases were cloned into
yeast prey vector fused to NubG at the N-terminus. MdABCF and PhABCF were inserted into bait
vector fused to Cub at the C-terminus. The two constructs and control vectors were cotransformed
into the NMY51 yeast strain, and the transformants were selected on SD-Trp-Leu media. Positive
clones were confirmed by PCR and cultured in SD-Trp-Leu liquid media to the early log phase for
growth assays. Ten microliters of transformed yeast, with an OD value of 0.5, were spotted on SD
selective plates (SD-Trp-Leu-His-Ade) containing 10 mM 3-aminnotriazole (3-AT) and grown at
30°C for 3 d. For more details see the user manual for DUAL membrane kit (1).
Recombinant protein expression and purification
The S1-, S2-RNase, non-S-RNase and RNase I genes were cloned into the pEASY-E1 vector
(Transgen) and expressed as a His-tagged fusion protein in E. coli strain BL21 Rosetta (DE3)
(Transgen). Cells were grown at 37°C in LB medium containing 50 µg/mL of ampicillin with
shaking at 250 rpm. When OD600 of the suspension reached 0.5, 0.2 mM IPTG was added to
induce protein expression by incubating at 23°C for 12-16h. The purification of this His-tag fusion
protein was performed as described in Novagen Ni-NTA His Bind.
The MdABCF, -TN2, -T, -NT, -N1 fragments were amplified by PCR. These fragments were
inserted respectively into the pGEX4T-1 vector (CWbiotech) to generate an in-frame fusion with
GST. The plasmid was introduced into E. coli strain BL21 Rosetta (DE3) and cells were grown
until OD600 reached approximately 0.5. Fusion protein expression was then induced by adding 0.5
mM IPTG and incubating at 18°C for 16 h. After centrifugation, the pellets were lysed in 1/10
culture volume of lysis buffer (50 mM sodium phosphate pH 7.2, 150 mM NaCl, 2 mM EDTA,
1% Triton X-100 and protease inhibitor cocktail) and cleared by centrifugation. The GST fusion
protein was purified by glutathione sepharose 4B beads (Amersham Pharmacia Biotech). All
purification steps were performed at 4°C. The recombinant proteins were then eluted with a buffer
containing 50 mM of Tris-HCl pH 7.9, 120 mM of NaCl and 10 mM of glutathione overnight at
4°C.
Pull-down and western blot assay
Purified His-tagged S1-RNase protein (10 nM) was absorbed onto Ni-NTA His Bind Resins. The
His-tagged non-S-RNase and RNase I protein (10 nM) were absorbed onto Ni-NTA His Bind
Resins as control. GST-tagged MdABCF, -TN2, -T, -NT proteins (10 nM of each) were purified.
For the negative control, GST-tagged protein was replaced with 10 nM of purified GST protein.
GST-protein and the His-protein were incubated at room temperature for 30 min, then unbound
proteins were washed away. Bound proteins were eluted with GST-tagged proteins using 20 μL of
phosphate buffer (pH 6.8) containing 50 mM KCl, 50 mM glutathione and 1mM DTT. Eluted
fractions were separated by SDS-PAGE and transferred onto Hybond C membranes (CWbiotech).
Electrophoresis and western blotting were performed according to standard methods. GST
antibody (1:3,000) was used as primary antibody and horse radish peroxidase-conjugated (HRP)
anti-rat as secondary antibody (CWbiotech). eECL Western Blot Kit (CWbiotech) was employed
to detect the signals according to the manufacturer’s instructions.
Particle bombardment-mediated transient expression in pollen
The
MdABCF
was
cloned
into
pCAMBIA1300
holding
CFP.
The
ER-marker
(Lat52:SYP73:mCherry, ER maker) and MdABCF-CFP were co-transformed to the pollen by
particle bombardment. One µg of recombinant plasmid was mixed with 4 µL of spermidine (0.1
M), 10 µL of CaCl2 (2.5 M), 6 µL of standard concentration gold (BioRad). The parameters of
bombardment were as follows: 1300 p.s.i. rupture disc, 29 mm Hg vacuum, 1 cm gap distance and
9 cm particle flight distance. The transformed pollen were subsequently cultured in dark for 1 h.
We then added FITC-S-RNase into GM. After culturing for 30 min, an Olympus BX61 FV1000
confocal microscope was used to observe the co-localization. A 100×objective was used for
imaging. All images were recorded using a stepper motor to make Z-series.
To determine the role of microfilament in transporting S-RNase and its relationship with MdABCF,
MdABCF was cloned into pCAMBIA1300 holding CFP, and MdActin was cloned into
pCAMBIA1300 holding mCherry. Both of these two fusion genes were under the Lat52 promoter.
Then the two vectors were co-transformed to the pollen by particle bombardment as described
above.
RT-PCR
Reverse transcription PCR was used for MdABCF gene expression analysis. Total RNA was
extracted from anther and style at different development stages of ‘Ralls Janet’ apple (S1S2-pollen),
as well as from sepal, leaf, ovary and filament at the mature stage by using Plantol efficient plant
total RNA kit (Lab kit). Gene-specific primers are listed. Apple actin gene (AB638619.1) was
used as internal control. Samples were cycled at 94°C for 5min, 30 cycles of: 94°C for 30s, 55°C
for 30 s, 72°C 90 s.
Real-Time Quantitative PCR Analysis
One microgram of total RNA was used for cDNA synthesis with the primeScript Master Mix Kit
(TaKaRa). Quantitative reverse transcription-PCR was performed with the SYBR Premix ExTaq II
Kit (TaKaRa), and amplification was real-time monitored on an Option Continuous Fluorescence
Detection System (Bio-Rad). Actin (AB190176) was used for normalization. Primer (Lat52-F2
and FragR) information is given in Table S4.
Plant transformation
As shown in Fig. S9, for overexpression assay, the coding region of MdABCF and PhABCF were
respectively cloned into pFGC5941 vector used Lat52 promoter (pollen specific): Lat52:MdABCF
and Lat52:PhABCF. For silencing assay, a interfering fragment targeting PhABCF was designed
and cloned into pFGC5941 vector to generate pFGC5941-Lat52-pro:RNAi recombinant plasmids,
under the Lat52 promoters. The primers used for these constructions are listed in Supplemental
Table S4. These constructs were transformed into Agrobacterium tumefaciens EHA105 which
were then used to infect the P. inflata using leaf disc method (2).
Southern blot analysis
Genomic DNA from transgenic P. inflate plants (single copy) was digested with Bam HI and
transferred onto NC membrane and hybridized with relative probe. The probe for overexpression
was a DNA fragment amplified from Lat52 promoters as shown in Supplemental Table 2. The
probe for silenced lines was a DNA fragment from the promoter and the target gene as Table S2.
Southern-blots were performed as described previously (3).
LatA, Oryzalin or Taxol Treatment of pollen
To test whether MdABC proteins transport S-RNase in cooperation with F-actin, we performed in
vitro pollen germination experiments. Pollen were hydrated in GM for 15 min and treated with
as-ODN as described in section “Antisense oligonucleotide treatment”, and then the pollen were
divided into nine independent groups, with different groups treated with each of the following
items: LatA+S1-RNase (50 µg/mL), LatA+S1-RNase (100 µg/mL), LatA+S1-RNase (200 µg/mL);
Oryzalin+S1-RNase (50 µg/mL), Oryzalin+S1-RNase (100 µg/mL), Oryzalin+S1-RNase (200
µg/mL); Taxol+S1-RNase (50 µg/mL), Taxol+S1-RNase (100 µg/mL) or Taxol+S1-RNase (200
µg/mL). Pollen treated with 50 µg/mL of S1-RNase were used as control. Pollen were grown at
25oC for 3 h and 50 were picked randomly for measurement of tube length, which was repeated
three times (n=3). Imaging of pollen was carried out using light microscopy (Olympus BX61).
Preparation of style crude protein extract
The method of preparing style crude protein extract were as Dought et al. (1998). All extraction
procedures were conducted in the presence of protease inhibitors (10 mg/mL, leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 1 mg/mL pepstatin-A and 10 mg/mL aprotinin) on ice, unless
stated otherwise, and all samples were stored at -80°C. Style crude protein extracts were obtained
by homogenizing 25 stigmas in 5 mL of extraction buffer (50 mM potassium phosphate, pH 7.0,
and 1% Triton X-100). The homogenate was then centrifuged at 4000g for 10 min to remove
cellular debris.
Immunolocalization in apple pollen tubes
After being treated by ‘Ralls Janet’ apple style crude protein extract, crude protein+Taxol for 1 h,
the pollen tubes were fixed by PBS buffer with 4% paraformaldehyde, 10% sucrose and 0.1%
Triton (pH 6.8) for 1 h. Then they were washed with PBS three times for 5 min each. After being
washed, cell walls of pollen tubes were digested by addition of 1% (w/v) cellulose, 0.4%
macerozyme and 0.5% (w/v) pectinase in PEM buffer (50 mM PIPES, 2 mM MgCl2 and 2 mM
EGTA) for 30 min at 37 oC in order to facilitate penetration of antibodies. Samples were then
attached to glass cover slips that contain polylysine. Then they were blocked in 10% (v/v) BSA for
1 h. After blocking, they were incubated with S-RNase antibodies (1:100) for 3 h at room
temperature, then washed three times for 5 min each using PBS. Fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG antibody (1:20) were used to incubate the sample for 1 h.
Finally, they were washed three times for 5 min each in PBS, covered with a drop of 10% (v/v)
glycerol in PBS and observed with Olympus BX61 (4).
S-RNase activity analysis
We purified the recombinant S-RNase using his-tag. In order to ensure that the recombinant
S-RNase had the same activity as the style secreted S-RNase, we used 100 mg of ‘Ralls Janet’
apple style to get crude protein which contained the secreted S-RNase (Plant total protein
extraction kit). His-tagged S-RNase (15, 25, 50 µg/mL) and secreted S-RNase (50, 75, 100 µg/mL)
were added respectively into the pollen medium and the pollen growth was measured, the results
show that both (his-tagged S-RNase and secreted S-RNase) can inhibit the pollen tube growth.
This was repeated three times.
Ribonuclease activity was measured with torula yeast RNA as the substrate. The reaction buffer
(490 mL) of 0.1 M imidazole-hydrochloric acid (HCl) (pH 7.0) containing 0.1 M KCl and 2 mg
RNA, was incubated at 37 oC for 10 min. The enzyme solution (10 mL) was then added, and the
reaction incubated at 37 oC. After 20 min, 100 mL of stop solution (25% perchloric acid, 0.75%
(w/v) lanthanum acetate) were added. After 30 min of incubation on ice, the solution was
centrifuged at 14,000 g for 5 min. The absorbance of the supernatant at 260 nm was measured
with a Hitachi U-2000 spectrophotometer. One unit of RNase activity was designated as the
amount of RNase which gave an increase in absorbance of one at 260 nm, in 1 min of reaction
time (5).
Labeling of S1-RNase with fluorescein isothiocyanate (FITC) and S2-RNase with rhodamine
B (RBITC)
Labeling of S1-RNase with FITC or S2-RNase with RBITC was carried out according to
Pepperkok et al. (2000) (6). The buffers of the protein solution (0.5 mg/ml) were substituted by 25
mM bicine, 0.1 mM EDTA, pH 8.0, using centrifuge concentrators (Centricon; Amicon). Labeling
was allowed to proceed for 30 min at room temperature, and then suppressed by addition of 5 mM
glycine for 15 min. Excess dye was removed by passing each protein solution through a PD-10
column (Pharmacia) equilibrated with 25 mM potassium phosphate buffer, pH 6.8, 0.5 mM EDTA,
l mM DTT, 10% glycerol.
Bimolecular fluorescence complementation (BiFC) competition assay between MdABCF and
S-RNase
The pCAMIBA1300 were used to construct vectors for BiFC, containing either N- or C-terminal
yellow fluorescence protein (YFP) fragments (pYFPn and pYFPc) (7). MdABCF, -TN2, -T, N1,
-NT fragment were cloned into the pCAMIBA1300-YFPn vector and S1-RNase, non-S-RNase,
RNase I were cloned into the pCAMIBA1300-YFPc vector to make either an N-terminal or
C-terminal fusion protein. All possible pairwise combinations of these constructs were
transformed into Agrobacterium EHA105. One positive colony of strain was inoculated into 5 mL
of YEP liquid medium and incubated with shaking (200 rpm) at 28°C until the OD600 reached 2.0.
And then MES buffer (10 mM MES-KOH, pH 5.2, 10 mM MgCl2, 100 µM acetosyringone) was
used to resuspend the strain. Then YFPc and YFPn constructs were co-infiltrated into young apple
leaves of a tissue cultured plants. Fluorescence was visualized in epidermal cell layers of the
leaves after 5 days of infiltration, using an Olympus BX61 fluorescent microscope (Olympus
FluoView FV1000).
The BiFC competition assay was based on Kodama and Hu (2012) (8). A competition assay used
non-fused protein MdABCF (S-RNase) as the competitor. We co-infiltrated MdABCF-YFPn,
S-RNase-YFPc and MdABCF (S-RNase) with the proportion 1:1:1 or 1:1:2 into young apple
leaves of tissue cultured plants. When the competitor MdABCF protein is present, the interaction
between MdABCF-YFPn and S-RNase-YFPc fusions is subjected to the competition by MdABCF
(S-RNase), leading to a reduction in BiFC efficiency.
Western-blot was used to MdABCF and S-RNase expression in the infiltrated apple leaves. Crude
protein was extracted from leaves after 5 days infiltration using plant total protein extraction kit
(CWbiotech). Western blot was performed according to standard methods. S1-RNase and
MdABCF antibody were used in this analysis, and β-tublin antibody was used as control.
References
1. Huang CF, Yamaji N, Mitani N, Yano M, Nagamur Y, Ma JF (2009) A Bacterial-Type ABC
Transporter Is Involved in Aluminum Tolerance in Rice. The Plant Cell 21: 655–667.
2. Goler-Baron V, Assaraf YG (2011) Structure and function of ABCG2-rich extracellular vesicles
mediating multidrug resistance. PLoS One 6: e16007.
3. Meng X, et al. (2009) Ectopic expression of S-RNase of Petunia inflate in pollen results in its
sequestration and non-cytotoxic function. Sex Plant Reprod 22: 263–275.
4. Poulter NS, Staiger CJ, Rappoport JZ, Franklin-Tong VE (2010) Actin-Binding Proteins
Implicated in the Formation of the Punctate Actin Foci Stimulated by the Self-Incompatibility
Response in Papaver. Plant Physiol. 152: 1274-83.
5. Norioka S, et al. (2007) Purification and characterization of a non-S-RNase and S-RNases from
styles of Japanese pear (Pyrus pyrifolia). Plant Physiology and Biochemistry 45: 878-886.
6. Pepperkok P, et al. (2000) Intracellular Distribution of Mammalian Protein Kinase A Catalytic
Subunit Altered by Conserved Asn2 Deamidation. The Journal of Cell Biology. 148: 715–726.
7. Shyu YJ, Hu CD (2008) Fluorescence complementation: an emerging tool for biological
research. Trends Biotechnol 26:622-630.
8. Kodama Y, Hu CD (2012) Bimolecular fluorescence complementation (BiFC): A 5-year update
and future perspectives. BioTechniques 53:285-298.