Thirty-seven peach varieties (Supplemental Table 1) were selected from the Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Henan Province, China. Peach organs/tissue samples (leaves, styles and pollen) were collected, frozen in liquid nitrogen and stored at −80°C Ultra-low temperature refrigerator for later use.

Peach genomic DNA was isolated from young leaves using the CTAB method (Li et al., 2009), and incubated with RNase I (Invitrogen, CA, USA) at 37°C for 2 h to remove RNA. Total RNA samples were isolated from leaves, styles and pollen using a modified CTAB method (Li et al., 2009) and treated with DNase I (Invitrogen, CA, USA) to remove DNA contamination. RNA was used as template to synthesize first-strand cDNA using the SuperScript reverse transcriptase (Invitrogen, CA, USA) and Oligo-dT primers (According to manufacturer's instructions, Invitrogen, CA, USA).

Peach genomic DNA was used as templates for PCR with the primers listed in Supplemental Table 2. The primers were designed according to the length of the second intron of the S-RNases described previously (Tao et al., 1999). The different S-RNases and S genotypes of peach varieties could be distinguished depending on the size of the amplified fragments.

Pollen cDNA was used as template to clone PperSFBs, PperSLFLs, PperSSK1, PperCUL1, and PperRbx1 and style cDNA was used as template to clone PperS-RNases with the gene specific primers listed in Supplemental Table 2. The PCR products were purified and individually ligated to the pMD19-Tsimple vector (TaKaRa). The constructed vectors were transformed into E. coli competent DH5α cells (Transgene biotech, Beijing, China). Each gene selected 3 positive clones for sequencing.

cDNA samples synthesized from total RNA from leaves, styles and pollen of the 37 peach varieties included in this study were used as templates to analyze tissue-specific expression of PperS-RNases, PperSFBs, and PperSLFLs. Gene specific primers were designed and listed in Supplemental Table 2, and the β-actin gene was used as an internal control for constitutive expression with the following thermal cycling condition: a denaturation step at 94°C for 5 min followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 60 s, and then 72°C for 10 mins.

64 CDSs of S-locus F-box genes (Supplemental Table 3) from Malus domestica, Pyrus pyrifolia, Pyrus bretschneideri, P. avium, Prunus dulcis, Prunus mume, Prunus salicina, Prunus armenica were used to construct phylogenetic trees with the F-box genes specifically expressed in pollen cloned from 37 peach varieties in this study. The deduced amino acid sequences of Skp1-like proteins and cullin-like proteins from Arabidopsis thaliana, Antirrhinum hispanicum, Prunus tenella, Petunia integrifolia, Pyrus bretchneideri, M. domestica, P. avium, Prunus persica. P. integrifolia, Vitis vinifera, Nicotiana tabacum, Prunus tomentosa, and P. mume were aligned by CLUSTALW (Supplemental Table 3). Based on the alignment, phylogenetic trees were constructed using MEGA 6.0 program (Tamura et al., 2013) with the neighbor-joining method (Saitou and Nei, 1987) and the bootstrap test replicated 1,000 times.

Yeast transformation and activity of β-galactosidase assays were performed following the manufacturer's instructions (Clontech, CA, USA). The partial CDSs of PperS-RNases removed signal peptides and the full-length CDSs of PperSSK1, PperCUL1, and PperPA1 were cloned into pGBKT7 vector (Clontech), whereas the full-length CDSs of PperSFBs, PperSLFLs, PperSSK1, and PperRbx1 were cloned into pGADT7 vector (Clontech).

In order to explore how PperSFB protein differentiates self PperS-RNase from non-self S-RNases, normal PperSFB2 was divided into four parts: box domain, box-V1, variable regions V1-V2 and hypervariable regions HVa-HVb. The four parts of PperSFB2 was cloned into pGADT7 vector. Y2H assay was performed to observe the interactions between different portions of PperSFB2 and all PperS-RNases cloned in this study.

For the Y2H assay, AH109 cells containing both AD and BD plasmids were grown on SD/-Leu/-Trp medium for 3 d at 30°C. Ten independent clones for each combination were streaked on SD/-adenine/-His/-Leu/-Trp medium and grown for 3–4 d at 30°C. Then yeast grown on SD/-adenine/-His/-Leu/-Trp medium was stained with X-α-gal (TaKaRa Bio). To quantify the interaction strength, β-galactosidase activity assay was performed using o-nitrophenyl-β-D-galactopyranoside (Sigma Aldrich) as a substrate.

The pCambia1300 vector was used to construct BiFC fusion vectors, which contained the N- or C-terminal of yellow fluorescence protein (YFP) fragments (YFPN and YFPC), respectively. The full-length CDSs of PperSLFLs without stop codon were cloned into pCambia1300-YFPN vectors, whereas the partial CDSs of PperS-RNase without stop codon and signal peptide were cloned into pCambia1300-YFPC. All the construct vectors were transformed into Agrobacterium tumefaciens GV3101 respectively and co-infiltrated into Nicotiana Benthamiana leaves. YFP fluorescence was observed in epidermal cell layers of N. Benthamiana leaves after infiltrated 5 days using Olympus BX61 fluorescent microscope (Olympus FluoView FV1000).

The box, box-V1, V1-V2, and HVa-HVb frames without the stop codon were cloned into the pCambia1300-YFPN vectors. The recombinant plasmids containing the box-YFPN, box-V1-YFPN, V1-V2-YFPN, or HVa-HVb-YFPN fusion gene and PperS1-RNase-YFPC, PperS2-RNase-YFPC, PperS2m-RNase-YFPC, or PperS4-RNase-YFPC without signal peptides fusion genes and the control plasmid with YFPN and YFPC were co-transformed into maize (Zea mays Linn.Sp.) protoplasts respectively according to Ren et al. (2011). YFP fluorescence was observed using Olympus BX61 fluorescent microscope (Olympus FluoView FV1000). The primers used were listed in Supplemental Table 2.

His-tagged proteins were purified as previously described (Meng et al., 2014). The CDSs of the PperS-RNases without signal peptide were cloned into the pEASY-E1 vector (From TransGen Biotech Company) and transformed into the E. coli strain BL21 plysS (DE3) (From TransGen Biotech Company). The cells were inoculated into LB medium containing 100 μg/ml ampicillin and placed in a shaker for about 3 h with 37°C and 200 rpm. When the cell suspension OD₆₀₀ reached to about 0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into medium with the final concentration 0.2 mM to induce protein production. The cell suspension was incubated in the shaker for 10–12 h with 16°C and 180 rpm. His-tagged fusion proteins were purified using Ni-NTA His binding resin (Novagen, USA) as previously described (according to the manufacturer's instructions of Ni-NTA His Bind Resins, Novagen). The full-length coding sequences of pollen-expressed PperSFB1m, PperSFB2m, PperSFB2, PperSFB4m PperSLFL1, PperSLFL2, and PperSLFL3 were cloned into pMAL-c5x vector, which is designed to generate maltose-binding (MBP) fusion proteins. Similarly, the PperCUL1, PperSSK1, and PperRbx1 were cloned into pGEX4T-1 vector, which is designed to produce glutathione S-transferase (GST) fusion proteins. All the GST-fusion proteins and MBP-fusion proteins were purified using glutathione resin and maltose resin as previously described, respectively (Yuan et al., 2014).

In vitro ubiquitination analysis was performed as previously described (Yang et al., 2009; Yuan et al., 2014). Each 50 μl reaction mixture containing 50 mM Tris (pH 7.4), 10 mM MgCl₂, 2 mM dithiothreitol (DTT), 5 mM HEPES, 2 mM adenosine triphosphate (ATP), 0.05% Triton X-100, 10 mM creatine phosphate, 1 unit of phosphokinase, 10 μg ubiquitin, 50 nM E1 (UBA6, Petunia hybrida), 1 mM PMSF, 850 nM E2 (UBH6, P. hybrida), and aliquots (2 μg) of the recombinant proteins GST-PpSSK1, GST-CUL1, GST-Rbx1, His-S-RNase and one kind of MBP-SLFL fusion protein were incubated at 30°C for 2 h. For detection of PperS-RNases by immunoblot, rabbit anti-S-RNase immunogloblin Gs (lgGs, made by Beijing ComWin Biotech Company) were used as primary antibodies at a dilution of 1:2,000, and goat conjugated anti-rabbit lgG (Bio-Rad) was used as a secondary antibody at a dilution of 1:10,000. For detection of MBP-PperSLFL proteins and GST fusion proteins by immunoblot, mouse anti-MBP and anti-GST monoclonal antibody (Bio-Rad) were used as primary antibodies at a dilution of 1:3,000, and goat conjugated anti-mouse lgG (Bio-Rad) was used as a secondary antibody at a dilution of 1:10,000. The reaction mixtures without PperS-RNase or PperSLFL proteins were negative controls.

We collected 37 peach varieties which represent local cultivars in 18 provinces/municipalities in China (Supplemental Table 1). These are thought to represent the evolutionary paths of peach from the original regions in central China (Tibet, Yunnan and Guizhou provinces) to the northwest of China (Shanxi province), then to the southwest of China and finally to the coastal and Xinjiang provinces (Cao et al., 2014; Supplemental Figure 1). Only four S-haplotypes S1, S2, S2m, and S4, were detected from 36 peach varieties except “Guang He Tao” by PCR and sequencing (Supplemental Figure 2). The four S-haplotypes have been reported (Tao et al., 2007). The S genotype of 18 varieties, including “Da Hong Pao,” were S2S2 genotype and 9 varieties, including “Hunchun Tao,” were S1S2 genotype, while 3 varieties were S2S4 genotype (“Feicheng Bai Li 10,” “Feicheng Bai Li 17,” and “Feicheng Hong Li 6”) (Supplemental Table 1). The PperS2-RNase in 6 varieties was observed to contain a nucleotide substitution (G–A), which resulted in the conversion of the sixth conserved cysteine residue to a tyrosine in the Prunus C5 domain (Figure 1A). The mutated PperS2-RNase was named as PperS2m-RNase, which has been reported (Tao et al., 2007). S2-RNase and S2m-RNase were also identified in the original species “Guang He Tao,” indicating that the mutation of S2-RNase had occurred before the formation of peach cultivars. The 4 PperS-RNases were specifically expressed in pistil, supporting their roles of pistil determinants (Figure 1D).

The mutated PperSFBs cloned from all varieties in this study were the same as previously reported mutations (Tao et al., 2007), and all the PperSFBm genes were terminated prematurely. However, we noticed that the sequence of inserted 155 bp fragment in PperSFB1m was exactly the same as the sequence of 155 bp fragment upstream of the insertion point. Similarly, the sequence of 5 bp insertion in PperSFB2m was also the same as the 5 bp upstream of the insertion point. In addition, the sequences of 351 bp at both ends of the inserted 4,949 bp fragment in PperSFB4m were also totally identical (Figure 1B). The repeat sequences of inserted fragment of PperSFB4m have been reported (Hanada et al., 2014). In addition, except the mutated SFB2m, a canonical SFB2 gene was cloned from “Guang He Tao,” indicating that peach mutations occurred prior to peach introduction and acclimatization by human and the canonical SFB2 was eliminated during the selection process. Moreover, the expression of PperSFBs was pollen-specific, supporting their role of pollen determinants (Figure 1D).

According to the sequences of S-locus F-box-likes genes (SLFLs) in peach genome (Genome Database for Rosaceae, http://www.rosaceae.org/), the primers were designed to clone the six SLFL genes. Finally, only three SLFL genes were specifically expressed in pollen (Figure 1D), and we named them as PperSLFL1, PperSLFL2, and PperSLFL3, respectively. PperSLFL1 located at about 47 kb downstream of PperS2-RNase and the translation direction was opposite to that of PperS2-RNase; PperSLFL2 located at about 26 kb downstream of PperS2-RNase and PperSLFL3 located at about 1.3 kb upstream of PperS2-RNase, and the translation direction of PperSLL2 and PperSLFL3 was the same as that of PperS2-RNase. The three PperSLFL genes did not have introns (Figure 1C). The identity of the predicted amino acid sequences of PperSLFL1, PperSLFL2, and PperSLFL3 was 52.45%. All the PperSLFL proteins contained the basic F-box domain and the FBA domain (Supplemental Figure 3). The phylogenetic tree analysis showed the three PperSLFL genes clustered with other Prunus SLFL genes, which has been reported (Aguiar et al., 2015). The phylogenetic tree had two large lineages. The Prunus SFB genes did not cluster with the pollen S genes of Pyrus and Malus and clustered into a separate lineage. Prunus SLFL genes clustered with Pyrus and Malus SFBB genes in another lineage. This suggested that the evolutionary relationship between Prunus SLFL genes and SFBB genes of Pyrus and Malus was closer than that of between Prunus SLFL genes and Prunus SFB genes (Supplemental Figure 4A).

The full-length coding sequence (CDS) of the candidate SSK1 gene (PperSSK1) was cloned from “Hunchun Tao” (S1S2) pollen and subsequently identified in the other 36 peach varieties with specific primers (Supplemental Table 2). The canonical Skp1 protein comprises 150–200 amino acid residues and contains a Skp1-POZ domain at the N terminus and a Skp1 domain at the C terminus. The deduced amino acid sequence of PperSSK1 comprised 177 residues and contained the Skp1-POZ and the Skp1 domain. In the phylogenetic tree, PperSSK1 clustered into a lineage with PtSSK1 and PavSSK1 (Supplemental Figure 4B). In addition, the other two subunits of the SCF complex, PperCUL1 and PperRbx1, were cloned with pollen cDNA of “Hunchun Tao” (S1S2) as template. The deduced PperCUL1 protein containing 744 amino acid residues and phylogenetic analysis showed that it clustered with PavCULB, which have been shown to be a component of the SCF complex (Matsumoto and Tao, 2016) (Supplemental Figure 4C). PperRbx1 protein contained 117 amino acid residues and had an H2 loop figure domain at the C-terminus, which is necessary for ubiquitin ligase activity.

Y2H assay was performed to detect the interactions between PperSFBs and PperS-RNases. PperPA1 (ppa011133m) (Aguiar et al., 2015), also a T2-RNase of P. persica with no signal peptide, was cloned into pGBKT7 to detect the interactions with S-locus F-box proteins. The results showed that the mutated PperSFBs and normal PperSFB2 interacted with all the PperS-RNases cloned in the study, and these interactions displayed no S allelic specificity, while there was no interaction between PperPA1 and PperSFBs (Figure 2A). Furthermore, the activity of β-galactosidase was detected and the results showed that the intensity of interactions between these combinations was not high and the intensity of interaction between normal PperSFB2 and PperS2-RNase was slightly higher than that of other combinations (Figure 2B). Because of the insertion of the fragments, the proteins encoded by PperSFB1m, PperSFB2m, and PperSFB4m genes were terminated prematurely and the domains at C-terminus was lost in varying degrees. In order to explore the effect of each part of the SFB on S-RNase, we divided the normal PperSFB2 gene into four parts: box, box-V1, V1-V2, and HVa-HVb (Figure 3A). Y2H and BiFC analysis showed that the box region did not interact with all four PperS-RNases, whereas box-V1 and V1-V2 portions of PperSFB2 physically interacted with the four PperS-RNases, and the interactions displayed no S allelic specificity. The yeast coloring time stained by X-α-gal and fluorescence intensity suggested that the interaction intensity of each combination was not high. Interestingly, the HVa-HVb of PperSFB2 only interacted with PperS2-RNase, indicating a potential role in S-RNase-SFB specific recognition (Figures 3B,C).

The three PperSLFL1-3 genes specifically expressed in pollen and contained F-box domains, which led to a problem that whether they play roles in self-incompatibility of peach. Firstly, we detected the interactions between PperSLFL1-3 and PperS-RNases. The Y2H assay showed that PperS-RNases interacted with PperSLFL1-3 with no S allelic specificity, and PperPA1 did not interact with any PperSLFL proteins (Figure 4A). The activity of β-galactosidase analysis suggested that the intensity of interactions between PperSLFL1 and the four PperS-RNases were slightly higher than other various combinations (Figure 4B). To further confirm the results, BiFC experiment was performed in N. Benthamiana leaves, and the results also indicated the interactions between PperSLFL1-3 and the four PperS-RNases with no S allelic specificity (Figure 4C).

The Y2H analyses were performed to investigate the interactions between PperSSK1 and PperSFBs/PperSLFLs, and the interactions between PperCUL1 and PperSSK1/PperRbx1. The results indicated that PperSSK1 interacted with PperSFB2, PperSFB1m, PperSFB2m, and PperSFB4m (Supplemental Figure 5A), and the activity of β-galactosidase confirmed the intensity of the interaction was high (Supplemental Figure 5B). Both PperSSK1 and PperRbx1 interacted with PperCUL1 (Supplemental Figure 5C), and the activity of β-galactosidase quantitatively demonstrated the interactions between them (Supplemental Figure 5D).

We also examined the interactions between PperSSK1 and three other pollen-expressed F-box proteins, PperSLFL1-3. The Y2H results showed that all the three PperSLFL proteins interacted with PperSSK1 (Supplemental Figure 5A). The activity of β-galactosidase quantitatively demonstrated that the interactions between PperSSK1 and PperSLFL1/PperSLFL2 were stronger than the interaction between PperSSK1 and PperSLFL3, and the interaction between PperSLFL1 and PperSSK1 was the strongest (Supplemental Figure 5B).

In order to test whether PperS-RNases could be ubiquitinated by SCF^SLFL in vitro, commercial His-UBA6 was used as the ubiquitin-activating (E1) and His-UBH6 was used as ubiquitin-conjugating enzyme (E2). Purified MBP-PperSLFL1-3, GST-PperSSK1, GST-PperRbx1, and GST-PperCUL1 were used as E3. Firstly, the purified His-PperS-RNase proteins, MBP-PperSFB proteins and MBP-PperSLFL proteins were detected, and the results showed that each protein was detected a single band, indicating that these proteins were pure and the antibody was specific (Figures 5A,B). In vitro ubiquitination results showed that distinct immunoreactive bands with higher molecular masses (between 34 and 130 KDa) were detected above the predicted His-PperS-RNase (26 KDa) bands. The molecular weight of detected proteins above His-PperS-RNase bands was 8, 16 KDa and so on heavier than that of His-PperS-RNases. The molecular weight of ubiquitin added in the reaction system is 8 KDa, and the extra molecular weight was exactly multiples of ubiquitin molecular weight. No band was detected in the negative control reactions without His-PperS-RNase (Figure 6), and the single 26 KDa protein band was detected in the control reactions without MBP-PperSLFL protein (Figure 7). The results indicated that PperSLFL proteins could ubiquitinate PperS-RNases.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.