Supplemental Data
The SKP1-like Protein SSK1 is Required for Cross-Pollen
Compatibility in S-RNase-Based Self-Incompatibility
Lan Zhaoa,b,1, Jian Huanga,1, Zhonghua Zhao a,b,1, Qun Lia, Thomas L. Simsc and
Yongbiao Xuea,2
a
Laboratory of Molecular and Developmental Biology, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences and National Center for Plant
Gene Research, Beijing 100101, China
b
c
Graduate University of Chinese Academy of Sciences, Beijing 100049, China
Department of Biological Sciences and Plant Molecular Biology Center, Northern
Illinois University, DeKalb, IL 60115-2861, USA
1
These authors contributed equally to this work
2
Address for correspondence to ybxue@genetics.ac.cn.
This file includes:
Supplemental Figures 1 to 10
Supplemental Tables 1, 2 and 3
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Supplemental Figure 1. Molecular identification of four PhSLFs
(A) An amino acids sequence alignment of PhSLFs, PiSLFs and their paralogs
(PiA134s). PhSLFs were cloned from SI P. hybrida of four S hyplotypes using a
homology-based PCR approach. PiSLFs were identified as the pollen S in SI response
(Sijacic et al. 2004). Residues of over 80% similarity are shaded in black, those of
60-80% similarity in dark gray and 30-60% similarity in light gray. Numbers show
the position of amino acid residues starting from Met. The PhSLFs and PiSLFs share
high sequence similarities of over 90%.
(B) An unrooted neighbor-joining phylogeny tree of the SLFs and their paralogs was
constructed by MEGA 4.1 with default parameter values. Bootstrap values are given
at branch nodes. PhSLFs and PiSLFs are also closely related to each other and formed
one single clade in the phylogenetic tree, whereas they were diverged from paralogs
of PiSLFs, PiA-134-S1, -S2 and -S3.
(C) RNA expression analysis of PhSLFs. The RNA levels of PhSLF-S1, S3L and SV in
SI P. hybrida of their corresponding S hyplotypes were examined by quantitative
RT-PCR using pollen, style and leaf tissues. The results showed that the PhSLFs are
pollen-specific genes.
Together, these results show that PhSLFs are indeed the orthologs of PiSLFs, which
have been demonstrated to be the pollen S gene (Sijacic, et al. 2004), and they could
encode the pollen S genes in P. hybrida.
Supplemental Figure 2. PhSSK1 accumulates in the mature pollen. Western blot
analysis of PhSSK1 during anther development. PS I-VI indicate six developmental
stages of P. hybrida flower as represented by the lengths of flower buds of ~2.0 cm,
~3.0 cm, ~4.0 cm, and 5.5 cm, respectively. PS V-VI indicate the flower opening
stages, and in PS V the anthers still not open, while in PS VI anthers open and pollen
begin to shed. Total proteins of anther or pollen were probed by the anti-PhSSK1
antibody. Tubulin was used as a loading control.
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Supplemental Figure 3. Amino Acid sequences and the predicted secondary
structures of SSK1 proteins.
For SSK1 proteins, their secondary structures were predicted through PredictProtein
website (http://www.predictprotein.org/). The secondary structure of HsSKP1 has
been reported and is shown together with SSK1 proteins (Schulman et al. 2000).
Supplemental Figure 4. An unrooted neighbour-joining phylogeny tree of
PhSSK1, AhSSK1 and Skp1-like proteins. Fifty-seven deduced Skp1-like proteins
are from budding yeast (ScSkp1, AAC49492), human (HsSkp1, AAH20798),
Arabidopsis (21 ASKs) (Zhao et al. 2003), rice (26 Skp1-like proteins predicted by
TIGR, http://rice.plantbiology.msu.edu), Petunia (3 PiSKs) (Hua and Kao 2006), and
other higher plants (FAP1, FAP2, FAP3,(Ingram et al. 1997); NbSkp1, AAO85510;
BnSkp1, AAF82795). The neighbour-joining phylogeny tree of these Skp1-like
proteins was generated by MEGA 4.1 with default parameter values. PhSSK1 and
AhSSK1 form a monophyletic clade as indicated in a dotted line box. Bootstrap
values are given at branch nodes.
Supplemental Figure 5. Schematic representation of the PhSSK1 RNAi vector
(pBI101-LAT52-PhSSK1-RNAi).
Lat52 promoter was obtained from S. McCormick (Twell et al. 1991).
Supplemental Figure 6. Expression analyses of PhSSK1 in pollen of the T0
transgenic lines.
(A). Quantitative RT-PCR analysis of the PhSSK1 RNA levels of the six T0 transgenic
lines. PhSKP1-2 was used as a negative control.
(B). Western blot analysis of the PhSSK1 expression in the T0 lines. The PhSSK1
protein level of the T0 lines was examined by western blot analysis using total pollen
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proteins from five T0 lines (B, J2, K4, H6 and M8) (top panel). Tubulin was used as a
loading control. The relative PhSSK1 protein amounts in the transgenic lines
compared with wild-type pollen were calculated by Quantity One (Bio-Rad) (bottom
panel).
Supplemental Figure 7. Molecular identification of the style S defective line SOSO.
(A) DNA sequence alignment of SO-RNase (M81686) and the S-RNase sequence
denoted as SOSO. To identify a stylar S function defective plant, we searched
commercially available stocks of self-compatible (SC) P. hybrida and found a
self-compatible line with white flowers. To examine whether the SI of its style was
normal or not, the S-RNase of the compatible line of SOSO was identified by a
homology-based PCR cloning approach. The primers used are listed in Supplemental
Table 2. Sequencing alignment showed that this S-RNase was of SO haplotype as
described previously (Robbins et al. 2000). Since S-genotyping of progeny derived
from the crosses between this SC line and plants with other S-genotypes demonstrated
that all progeny carried the S-RNase-SO (see Table 2 and Supplemental Figure 9), this
SC line should be homozygous for its SI genes and thus it was referred to as SOSO in
this study. It was reported that the S-RNase of SO allele was found to be present in
approximately 80% of existing research stocks and commercial cultivars of P. hybrida
and is probably a major contributor to self-compatibility observed in many P. hybrida
varieties (Robbins, et al. 2000), suggesting that S-RNase-SO is a non-functional allele
despite of its RNA expression.
(B) Ribonuclease activity and expression of S-RNases in P. hybrida. (a) In-gel assay
of ribonuclease activity in Petunia. The arrow indicates active S-RNases present in SI
lines. The numbers on the left side indicate the molecular weight (MW) in kD. The
result showed that little S-RNase activity could be detected in the total protein of SOSO.
(b) Western blot analysis of S-RNase-SO antigen and S-RNases in P. hybrida of SC
line (SOSO) and three SI lines (S1S1, S3LS3L and SVSV). Bands marked with asterisk
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represent a non-specific protein detected by the antibody. The numbers on the left side
indicate the molecular weight in kD. Gel stained with coomassieblue was used as a
loading control. Western blot was probed by an anti-S-RNase antiserum, which was
made using S-RNase-S3L as the antigen and also cross-reacted with the S-RNase-SO
antigen expressed in E. coli. The result showed that little S-RNase was detected in the
SOSO styles, indicating that the S-RNase-SO protein is unlikely to be expressed.
Incidentally, an extra prominent higher molecular weight band also was detected in
the stylar proteins of the S3LS3L and SVSV lines but not the S1S1 line. Because S-RNases
are known to be differentially glycosylated (Woodward et al. 1989, Woodward et al.
1992), we postulated that S-RNase-S3L and -SV might be more glycosylated than
S-RNase-S1. In fact, two potential N-glycosylation sites were found in both
S-RNase-S3L and -SV but none in S-RNase-S1 (Supplemental Figure 8). Although
further deglycosylation treatment of stylar extracts is required to confirm this, the
differential glycosylation of S-RNases could explain our western blot results.
Taken together, these results suggested that the SOSO line has a defective stylar S
function due to the lack of S-RNase protein expression and could be a suitable line to
test whether the reduced fertility of cross compatible pollen observed in the PhSSK1
RNAi transgenic lines was related to pollen tube developmental processes and/or SI
responses.
Supplemental Figure 8. Predicted glycosylation sites in PhS-RNase-S3L/SV/S1/SO.
Potential amino acid sites for glycosylation in PhS-RNase-S3L/SV/S1/SO were predicted
by NetNGlyc 1.0 Server and NetOGlyc 3.0 Server and are highlighted in red (Julenius
et al. 2005). The amino acids ‘N’ (asparagine) and ‘T’ (threonine) are possible
N-glycosylation and O-glycosylation sites, respectively.
Supplemental Figure 9. Genotype identification of T2 progeny from the
outcrosses of the SOSO wild type compatible line with the T1 transgenic lines as
pollen donor.
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Twenty individuals are shown for each T2 progeny population. T-DNA, S3L-RNase,
SV-RNase and SO-RNase were detected by genomic DNA PCR using gene-specific
primers. The parents of the each T2 population, the SC wild-type SOSO and the T1
transgenic lines, were used as controls.
Supplemental Figure 10. Genotype identification of T2 progeny derived from the
outcrosses of the S3LS3L wild type plants with the T1 transgenic lines as pollen
donor.
Twenty individuals are shown for every T2 progeny population. T-DNA, S3L-RNase
and SV-RNase were detected by genomic DNA PCR using gene-specific primers. The
parents of the each T2 population, SI wild-type S3LS3L and T1 transgenic lines, were
used as controls.
Supplemental Table 1. A summary of yeast two-hybrid assays between the
Skp1-like proteins and the S-locus F-box proteins of A. hispanicum and P.
hybrida.
Supplemental Table 2. Pollination analyses of seven independent T0 transgenic
lines.
Supplemental Table 3. Primers used in this study.
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