Plant Mol Biol (2013) 81:245–257 DOI 10.1007/s11103-012-9995-x Identification of a canonical SCFSLF complex involved in S-RNase-based self-incompatibility of Pyrus (Rosaceae) Chi Xu • Maofu Li • Junkai Wu • Han Guo • Qun Li • Yu’e Zhang • Jijie Chai • Tianzhong Li • Yongbiao Xue Received: 31 July 2012 / Accepted: 5 December 2012 / Published online: 20 December 2012 Ó Springer Science+Business Media Dordrecht 2012 Abstract S-RNase-based self-incompatibility (SI) is an Pyrus bretschneideri from the tribe Pyreae of Rosaceae. intraspecific reproductive barrier to prevent self-fertiliza- Both yeast two-hybrid and pull-down assays demonstrated tion found in many species of the Solanaceae, Plantagin- that they could connect PbSLFs to PbCUL1 to form a aceae and Rosaceae. In this system, S-RNase and SLF/SFB putative canonical SCFSLF (SSK/CUL1/SLF) complex in (S-locus F-box) genes have been shown to control the pistil Pyrus. Furthermore, pull-down assays showed that the SSK and pollen SI specificity, respectively. Recent studies have proteins could bind SLF and CUL1 in a cross-species shown that the SLF functions as a substrate receptor of a manner between Pyrus and Petunia. Additionally, phylo- SCF (Skp1/Cullin1/F-box)-type E3 ubiquitin ligase com- genetic analysis revealed that the SSK-like proteins from plex to target S-RNases in Solanaceae and Plantaginaceae, Solanaceae, Plantaginaceae and Rosaceae form a monoc- but its role in Rosaceae remains largely undefined. Here we lade group, hinting their shared evolutionary origin. Taken report the identification of two pollen-specific SLF-inter- together, with the recent identification of a canonical acting Skp1-like (SSK) proteins, PbSSK1 and PbSSK2, in SCFSFB complex in Prunus of the tribe Amygdaleae of Rosaceae, our results show that a conserved canonical SCFSLF/SFB complex is present in Solanaceae, Plantagina- Chi Xu and Maofu Li contributed equally to this work. ceae and Rosaceae, implying that S-RNase-based self- incompatibility shares a similar molecular and biochemical Accession numbers: HE802069 (PbSLF3-S34), HE802070 (PbSLF6- mechanism. S21), HE802072 (PbSSK1), HE802071 (PbSSK2), and HE802075 (PbCUL1). Keywords SSK Á SLF Á SCF complex Á S-RNase Á Electronic supplementary material The online version of this Self-incompatibility Á Pyrus article (doi:10.1007/s11103-012-9995-x) contains supplementary material, which is available to authorized users. & C. Xu Á M. Li Á H. Guo Á Q. Li Á Y. Zhang Á Y. Xue (&) J. Wu Á T. Li ( ) State Key Laboratory of Molecular Developmental Biology, Laboratory of Fruit Tree Cell and Molecular Breeding, China Institute of Genetics and Developmental Biology, Chinese Agricultural University, Beijing 100193, China Academy of Sciences and National Center for Plant Gene e-mail: [email protected] Research, Beijing 100101, China e-mail: [email protected] J. Chai School of Life Sciences, Tsinghua University, Beijing 100084, C. Xu Á H. Guo China University of Chinese Academy of Sciences, Beijing 100190, China Present Address: M. Li State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 123 246 Plant Mol Biol (2013) 81:245–257 Abbreviations alternative non-canonical SCFSLF complex is suggested and SI Self-incompatibility appears to consist of three subunits: SBP1 (S-RNase-binding SLF S-locus F-box protein1) (Sims and Ordanic 2001), CUL1 and SLF. In this SFB S-haplotype-specific F-box E3 complex, the constitutively expressed SBP1 is proposed SFBB S-locus F-box brother to play a dual role: serving as an adaptor to bridge SLF to SSK SLF-interacting Skp1-like CUL1 similar to Skp1 and also acting like Rbx1 to connect SCF Skp1-Cullin1-F-box the novel E3 complex to an E2-like protein (Hua and Kao SBP1 S-RNase-binding protein1 2006). Although further studies are required to define the precise roles of these two alternative SCF complexes in SI, two additional lines of evidence obtained from both Antir- rhinum and Petunia support that SLFs function as substrate receptors in an SCF E3 ligase manner involving the ubiq- Introduction uitin–proteasome system. First, SLF could interact with S-RNases in a non-S-haplotype-specific manner and appears Self-incompatibility (SI) is an intraspecific prezygotic to exhibit a stronger interaction affinity to non-self S-RNase reproductive barrier found in many flowering plants, which than self one and second, the ubiquitination of S-RNases has prevents inbreeding and promotes outcrossing by rejecting been observed, which could be blocked by proteasomal self (genetically related) pollen but accepting non-self inhibitors (Qiao et al. 2004b; Hua and Kao 2006, 2008; Hua (genetically unrelated) pollen for fertilization (De Nettan- et al. 2007; Kubo et al. 2010). court 2001; Franklin-Tong 2008). In most cases, SI is However, it is unclear whether similar SLF-containing controlled by a single polymorphic locus, named the complexes are present in Rosaceae. Although the Solana- S-locus. It contains at least two tightly linked genes, the pistil ceae, Plantaginaceae and Rosaceae all employ S-RNase as S and the pollen S, and the interaction of their protein the pistil S determinant and SLF/SFB as the pollen products determines the self-/non-self-specific recognition S determinant, SLF/SFB identified in Prunus from the tribe between pistil and pollen (Takayama and Isogai 2005; Amygdaleae of Rosaceae appears to function differently, as Zhang et al. 2009; Chen et al. 2010; McClure et al. 2011). the deletion or alteration of SFB in Prunus is thought to lead Among the Solanaceae, Plantaginaceae and Rosaceae, the to self pollen acceptance rather than rejection as found in pistil and pollen S encode a pistil-expressed ribonuclease the Solanaceae and Plantaginaceae (Ushijima et al. 2004; (S-RNase) and a cluster of pollen-expressed S-locus F-box Sonneveld et al. 2005; Hauck et al. 2006a; Tsukamoto et al. (SLF/SFB) proteins, respectively (Anderson et al. 1986; 2006; Vilanova et al. 2006). Therefore, Prunus SFB is Sassa et al. 1992; Ushijima et al. 1998; Lai et al. 2002; proposed to function as a protector to prevent self S-RNase Entani et al. 2003; Ushijima et al. 2003; Yamane et al. from detoxification rather than a destroyer to target non-self 2003; Zhou et al. 2003; Qiao et al. 2004a; Sijacic et al. S-RNase for degradation as observed in Solanaceae and 2004; Sassa et al. 2007; Kubo et al. 2010). Plantaginaceae. Additionally, competitive interaction, a Recent studies have shown that the SLF functions as a well known phenomenon observed in the Solanaceae and component of a multi-subunit SCF (Skp1/Cullin/F-box)- Plantaginaceae whereby the coexistence of two different type E3 ubiquitin ligase, but two alternative SLF-containing pollen S alleles in a pollen results in the pollen SI break- complexes have been proposed. In Antirrhinum hispanicum down, is not always observed in tetraploid Prunus (Entani (Plantaginaceae) and Petunia hybrida (Solanaceae), a et al. 1999; Golz et al. 1999, 2001; Hauck et al. 2006b; Xue SLF canonical SCF complex has been detected, based on the et al. 2009). However, multiple related SFBBs (S-locus identification of a pollen-specific SLF-interacting Skp1- F-box brothers), the pollen S candidates identified in the like1 (SSK1) protein, which could serve as an adaptor to tribe Pyreae (e.g., apple and pear) of Rosaceae, are assumed bridge SLF to CUL1, as observed in the canonical SCF to play a similar role in SI response as SLFs in Solanaceae complexes that are composed of Skp1, Cullin1, an F-box and Plantaginaceae, because the loss-of-function mutants of protein and Rbx1 (Zheng et al. 2002; Huang et al. 2006; Zhao SFBB genes display a collaborative non-self recognition et al. 2010; Chen et al. 2012). Interestingly, SSK1 appears to pattern, similar to Petunia (Sassa et al. 2007; Kubo et al. represent a novel type of Skp1-like proteins, with a distin- 2010; Minamikawa et al. 2010; Kakui et al. 2011; Okada guished feature having a unique disordered coil tail at the et al. 2011). Furthermore, the tetraploids of this tribe exhibit C-terminus following the conventional residues ‘‘WAFE’’ competitive interaction (Crane and Lewis 1942; Adachi found in most plant Skp1-like proteins (Gagne et al. 2002; et al. 2009). Risseeuw et al. 2003). In addition, the SSK1 family appears In this study, we first identified two pollen-specific SLF- to form a monoclade among Skp1-like proteins (Huang et al. interacting Skp1-like (SSK) proteins in Pyrus bretschneideri 2006; Zhao et al. 2010). However, in Petunia inflata,an from the tribe Pyreae of Rosaceae, named PbSSK1 and 123 Plant Mol Biol (2013) 81:245–257 247 PbSSK2, which could serve as adaptors to bridge PbSLF to RT-PCR and RT-PCR/CAPS analysis PbCUL1, together constituting a putative canonical SCFSLF (SSK/CUL1/SLF) complex. Second, we demonstrated that Total RNA was prepared as previously described (Lai et al. SSK proteins interact with SLF and CUL1 in a cross-species 2002) and digested with DNase I (TaKaRa, Dalian, China). manner between Pyrus and Petunia.Furthermore,wefound The cDNA was produced using the SuperScript reverse that SSKs appear to share a single evolutionary origin. transcriptase (Invitrogen, Carlsbad, CA, USA) and a poly- Collectively, our results, together with the recent identifi- dT primer. The RT-PCR was performed to detect the cation of a canonical SCF complex in Prunus (Matsumoto expression patterns of PbS21-RNase, PbS34-RNase, et al. 2012), show that a conserved canonical SCF complex PbSSK1, PbSSK2 and PbCUL1. The RT-PCR/CAPS was occurs in Solanaceae, Plantaginaceae and Rosaceae, sup- conducted to detect the expression patterns of PbSLF6-S21 porting the notion that S-RNase-based self-incompatibility and PbSLF3-S34.