Supporting Information Legends
Figure S1. Yeast two-hybrid assay of interactions between two allelic variants of SLF1
and two allelic variants of S-RNase.
The sequences for the full-length S2-SLF1, full-length S3-SLF1, truncated S2-SLF1
containing only the F-box domain, and truncated S2-SLF1 containing only the C-terminal
region (CTR; lacking the N-terminal 49 amino acids) were cloned into bait vector
pGBKT7. The sequences for the full-length mature S2-RNase and S3-RNase (without
their respective leader peptides) were cloned into prey vector pGADT7. Three colonies of
yeast Y2HGold carrying a pair of bait and prey constructs were streaked out on selective
plates lacking adenine, histidine, leucine and tryptophan, but containing X-α-Gal and
aureobasidin, to examine growth and -galactosidase activity. A representative colony is
shown for each pair. Yeast colonies co-expressing SV40 large T-antigen (T) and p53 (53)
were used as positive control, and colonies that co-expressed SV40 large T-antigen and
lamin (lam) were used as negative control. The colonies on each plate are numbered and
the identity of each colony is shown in the table.
Figure S2. Fluorescence images of pollen tube growth in pistils of a wild-type S2S3 plant
20 hours after pollination with pollen from (a) a wild-type S2S3 plant, (b) an S2S3
transgenic
plant
homozygous
for
the
S2-SLF1:GFP
transgene
(LAT52P:S2-
SLF1:GFP**/S2S3), and (c) an S2S3 transgenic plant carrying the S2-SLF1(∆295312):GFP transgene (LAT52P:S2-SLF1[∆295-312]:GFP/S2S3).
Pollen tubes were stained with aniline blue and monitored by an epifluorescence
microscope. Pollen tubes in (a), (c) are arrested in the upper segment of the pistil,
characteristic of self-incompatible pollination, suggesting that expression of S2SLF1(∆295-312):GFP in S3 pollen does not cause breakdown of self-incompatibility. A
large number of pollen tubes in (b) have grown through the entire pistil, consistent with
the previous finding that expression of S2-SLF1:GFP in S3 pollen caused breakdown of
self-incompatibility (Sijacic et al., 2004).
Figure S3. Alignment of the 18-amino-acid sequence of S2-SLF1 containing a degron
with the corresponding sequences of 16 other SLF proteins of S2-haplotype and all 17
SLF proteins of S3-haplotype.
The amino acid residue numbers refer to those of S2-SLF1. The four amino acids marked
in red are identical among all 34 SLF proteins, and the three amino acids marked in green
are highly conserved but not identical among all these SLF proteins. The overall amino
acid identities between these 34 SLF proteins range from 44.7 to 87.7% (Williams et al.,
2014).
Figure S4. Immunoblotting analysis to identify the additional degradation motif of S2SLF1 in yeast.
Protein levels of the truncated forms (each denoted by residue numbers of the first and
last amino acids of S2-SLF1 it contains), the deleted form (denoted by a ∆ sign followed
by the residue numbers of the first and last amino acids of the region deleted), and the
lysine-to-arginine mutated forms (denoted by the residue number of the specific lysine, K,
that is replaced with arginine, R). All forms of S2-SLF1 were fused with a Gal4 DNAbinding domain (BD) and a Myc-tag. Anti-Myc antibody was used to assess whether
these fusion proteins were produced in yeast cells. A duplicated membrane,
immunoblotted using anti-actin antibody, serves as control for equal loading. For the
protein not detected by anti-Myc antibody, an asterisk is placed to the left side of its
would-be position based on its predicted molecular mass.
Table S1. Raw Ct values for Real-Time PCRs.
Table S2. List of peptides that match each of the proteins identified by mass
spectrometry from co-immunoprecipitation of S2-SLF1(∆295-312):GFP.
Table S3. List of PCR primers and their sequences.
Table S4. Determination of PCR efficiencies for Real-Time PCR primers.
Methods S1. Supporting methods.
Sijacic, P., Wang, X., Skirpan, A.L., Wang, Y., Dowd, P.E., McCubbin, A.G., Huang, S. and Kao,
T.H. (2004) Identification of the pollen determinant of S-RNase-mediated self-incompatibility.
Nature, 429, 302-305.
Williams, J.S., Der, J.P., dePamphilis, C.W. and Kao, T.-h. (2014) Transcriptome analysis reveals the
same 17 S-locus F-box genes in two haplotypes of the self-incompatibility locus of Petunia inflata.
Plant Cell, 26, 2873-2888.