Plant Molecular Biology

, Volume 100, Issue 4–5, pp 367–378 | Cite as

Recognition of S-RNases by an S locus F-box like protein and an S haplotype-specific F-box like protein in the Prunus-specific self-incompatibility system

  • Daiki MatsumotoEmail author
  • Ryutaro Tao


Key message

S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry.


Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1–3 (SLFL1–3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS1, 6-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS6-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.


General inhibitor Prunus Self-incompatibility SFBL SLFL S-RNase 



This work was supported by a Grant-in-Aid (No. 16H06184) for Young Scientists (A) from the Japan Society for the Promotion of Science to D. M.

Author contributions

D. M. designed and conducted the experiments and drafted the manuscript. Both authors edited the manuscript and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2019_860_MOESM1_ESM.eps (1019 kb)
Supplementary material 1 (EPS 1019 kb). Supplementary Fig. 1 Expression analysis of SLF-like and SFB-like homologs in Japanese apricot cv. Nanko pollen: expression levels were calculated from transcriptome data previously reported (DRR002283; Akagi et al. 2016) and expressed as FPKM
11103_2019_860_MOESM2_ESM.eps (1.4 mb)
Supplementary material 2 (EPS 1384 kb). Supplementary Fig. 2 The alignment of the N-terminal regions of the deduced amino-acid sequences of SLFL7 homologs of sweet cherry, peach and Japanese apricot:The MUSCLE program implemented in MEGA ver. 6.0 (Tamura et al. 2013) was used to generate the alignment. Amino acids shared over 75% among the homologs are shaded. The dashed line represents the region corresponding to the F-box motif in other Prunus SLFLs, SFB and SFBLs. Asterisks represent the amino-acid substitution in PavSLFL7A, which was shown to impair its interaction with PavSSK1
11103_2019_860_MOESM3_ESM.eps (873 kb)
Supplementary material 3 (EPS 873 kb). Supplementary Fig. 3 The GST-pulldown assay of PavCul1A and Prunus avium Rbx1-like protein (XP_021828912.1). 3 × HA-tagged PavCul1A and GST-fused Rbx1-like protein were co-expressed in the cell-free system. The protein complexes bound to the glutathione sepharose beads were detected by the immunoblot


  1. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Čech M et al (2018) The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46:W537–W544CrossRefPubMedPubMedCentralGoogle Scholar
  2. Aguiar B, Vieira J, Cunha AE, Fonseca NA, Iezzoni A, Nocker S et al (2015) Convergent evolution at the gametophytic self-incompatibility system in Malus and Prunus. PLoS ONE 10:e0126138CrossRefPubMedPubMedCentralGoogle Scholar
  3. Akagi T, Henry IM, Morimoto T, Tao R (2016) Insights into the Prunus-specific S-RNase-based self-incompatibility system from a genome-wide analysis of the evolutionary radiation of S locus-related F-box genes. Plant Cell Physiol 57:1281–1294CrossRefPubMedGoogle Scholar
  4. Babu V, Rajan V, D’Silva P (2009) Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors. Funct Integr Genomics 9:433–446CrossRefGoogle Scholar
  5. Boivin N, Morse D, Cappadocia M (2014) Degradation of S-RNase in compatible pollen tubes of Solanum chacoense inferred by immunogold labeling. J Cell Sci 127:4123–4127CrossRefPubMedGoogle Scholar
  6. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chen J, Wang P, de Graaf BHJ, Zhang H, Jiao H, Tang C et al (2018a) Phosphatidic acid counteracts S-RNase signaling in pollen by stabilizing the actin cytoskeleton. Plant Cell 30:1023–1039CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen Q, Meng D, Gu Z, Li W, Yuan H, Duan X et al (2018b) SLFL genes participate in the ubiquitination and degradation reaction of S-RNase in self-compatible peach. Front Plant Sci 9:227CrossRefPubMedPubMedCentralGoogle Scholar
  9. de Nettancourt D (2001) Incompatibility and incongruity in wild and cultivated plants. Springer, BerlinCrossRefGoogle Scholar
  10. Deshaies RJ, Joazeiro CAP (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78:399–434CrossRefPubMedGoogle Scholar
  11. Dobriyal N, Tripathi P, Sarkar S, Tak Y, Verma AK, Sahi C (2017) Partial dispensability of Djp1’s J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1. Cell Stress Chaperones 22:445–452CrossRefPubMedPubMedCentralGoogle Scholar
  12. Entani T, Iwano M, Shiba H, Che FS, Isogai A, Takayama S (2003) Comparative analysis of the self-incompatibility (S) locus region of Prunus mume: identification of a pollen-expressed F-box gene with allelic diversity. Genes Cells 8:203–213CrossRefPubMedGoogle Scholar
  13. Entani T, Kubo K, Isogai S, Fukao Y, Shirakawa M, Isogai A et al (2014) Ubiquitin–proteasome-mediated degradation of S-RNase in a solanaceous cross-compatibility reaction. Plant J 78:1014–1021CrossRefPubMedGoogle Scholar
  14. Goldraij A, Kondo K, Lee CB, Hancock CN, Sivaguru M, Vazquez-Santana S et al (2006) Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana. Nature 439:805–810CrossRefPubMedGoogle Scholar
  15. Hiratsuka S, Zhang S, Nakagawa E, Kawai Y (2001) Selective inhibition of the growth of incompatible pollen tubes by S-protein in the Japanese pear. Sex Plant Reprod 13:209–215CrossRefGoogle Scholar
  16. Igic B, Kohn JR (2001) Evolutionary relationships among self-incompatibility RNases. Proc Natl Acad Sci USA 98:13167–13171CrossRefPubMedGoogle Scholar
  17. Kakui H, Kato M, Ushijima K, Kitaguchi M, Kato S, Sassa H (2011) Sequence divergence and loss-of-function phenotypes of S locus F-box brothers (SFBB) genes are consistent with non-self recognition by multiple pollen determinants in self-incompatibility of Japanese pear (Pyrus pyrifolia). Plant J 68:1028–1038CrossRefPubMedGoogle Scholar
  18. Kubo K, Entani T, Tanaka A, Wang N, Fields AM, Hua Z et al (2010) Collaborative non-self recognition system in S-RNase-based self-incompatibility. Science 330:796–799CrossRefPubMedGoogle Scholar
  19. Kubo K, Paape T, Hatakeyama M, Entani T, Takara A, Kajihara K et al (2015) Gene duplication and genetic exchange drive the evolution of S-RNase-based self-incompatibility in Petunia. Nat Plant 1:14005CrossRefGoogle Scholar
  20. Kubo K, Tsukahara M, Fujii S, Murase K, Wada Y, Entani T et al (2016) Cullin1-P is an essential component of non-self recognition system in self-incompatibility in Petunia. Plant Cell Physiol 57:2403–2416CrossRefPubMedGoogle Scholar
  21. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefPubMedPubMedCentralGoogle Scholar
  22. Li W, Chetlat RT (2014) The role of a pollen-expressed cullin1 protein in gametophytophytic self-incompatibility. Genetics 196:439–442CrossRefPubMedGoogle Scholar
  23. Li S, Sun P, Williams JS, Kao T (2014) Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata. Plant Reprod 27:31–45CrossRefPubMedGoogle Scholar
  24. Li S, Williams JS, Sun P, Kao T (2016) All 17 S-locus F-box proteins of the S2- and S3-haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility. Plant J 87:606–616CrossRefPubMedGoogle Scholar
  25. Liu W, Fan J, Li J, Song Y, Li Q, Zhang Y et al (2014) SCFSLF-mediated cytosolic degradation of S-RNase is required for cross-pollen compatibility in S-RNase-based self-incompatibility in Petunia hybrida. Front Genet 5:228PubMedPubMedCentralGoogle Scholar
  26. Luu DT, Qin X, Laublin G, Yang Q, Morse D, Cappadocia M (2000) S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility. Nature 407:649–651CrossRefPubMedGoogle Scholar
  27. Matsumoto D, Tao R (2012) Isolation of pollen-expressed actin as a candidate protein interacting with S-RNase in Prunus avium L. J Jpn Soc Hortic Sci 81:41–47CrossRefGoogle Scholar
  28. Matsumoto D, Tao R (2016a) Distinct self-recognition in the Prunus S-RNase-based gametophytic self-incompatibility system. Hortic J 85:289–305CrossRefGoogle Scholar
  29. Matsumoto D, Tao R (2016b) Recognition of a wide-range of S-RNases by S locus F-box like 2, a general-inhibitor candidate in the Prunus-specific S-RNase-based self-incompatibility system. Plant Mol Biol 91:459–469CrossRefPubMedGoogle Scholar
  30. Muñoz-Sanz JV, Zuriaga E, Badenes ML, Romero C (2017) A disulfide bond A-like oxidoreductase is a strong candidate gene for self-incompatibility in apricot (Prunus armeniaca) pollen. J Exp Bot 68:5069–5078CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y et al (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104:34–41CrossRefPubMedGoogle Scholar
  32. Niu S, Huang J, Zhang Y, Li P, Zhang G, Xu Q et al (2017) Lack of S-RNase based gametophytic self-incompatibility in orchids suggests that this system evolved after the monocot–eudicot split. Front Plant Sci 8:1106CrossRefPubMedPubMedCentralGoogle Scholar
  33. Ono K, Akagi T, Morimoto T, Wünsch A, Tao R (2018) Genome re-sequencing of diverse sweet cherry (Prunus avium) individuals reveals a modifier gene mutation conferring pollen-part self-compatibility. Plant Cell Physiol 59:1265–1275CrossRefPubMedGoogle Scholar
  34. Pratas MI, Aguiar B, Vieira J, Nunes V, Teixeira V, Fonesca NA et al (2018) Inferences on specificity recognition at the Malus × domestica gametophytic self-incompatibility system. Sci Rep 8:1717CrossRefPubMedPubMedCentralGoogle Scholar
  35. Qiao H, Wang H, Zhao L, Zhou J, Huang J, Zhang Y et al (2004) The F-box protein Ah SLF-S2 physically interacts with S-RNases that may be inhibited by the ubiquitin/26S proteasome pathway of protein degradation during compatible pollination in Antirrhinum. Plant Cell 16:582–595CrossRefPubMedPubMedCentralGoogle Scholar
  36. Ramanauskas K, Igić B (2017) The evolutionary history of plant T2/S-type ribonucleases. PeerJ 5:e3790CrossRefPubMedPubMedCentralGoogle Scholar
  37. Sassa H, Kakui H, Miyamoto M, Suzuki Y, Hanada T, Ushijima K et al (2007) S locus F-box brothers: multiple and pollen-specific F-box genes with S haplotype-specific polymorphisms in apple and Japanese pear. Genetics 175:1869–1881CrossRefPubMedPubMedCentralGoogle Scholar
  38. Shirasawa K, Isuzugawa K, Ikenaga M, Saito Y, Yamamoto T, Hirakawa H, Isobe S (2017) The genome sequence of sweet cherry (Prunus avium) for use in genomics-assisted breeding. DNA Res 24:499–508CrossRefPubMedPubMedCentralGoogle Scholar
  39. Sijacic P, Wang X, Skirpan AL, Wang Y, Dowd PE, McCubbin AG et al (2004) Identification of the pollen determinant of S-RNase-mediated self-incompatibility. Nature 429:302–305CrossRefPubMedGoogle Scholar
  40. Steinbachs JE, Holsinger KE (2002) S-RNase-mediated gametophytic self-incompatibility is ancestral in eudicots. Mol Biol Evol 19:825–829CrossRefPubMedGoogle Scholar
  41. Sun P, Kao T (2013) Self-incompatibility in Petunia inflata: The relationship between a self-incompatibility locus F-box protein and its non-self S-RNases. Plant Cell 25:470–485CrossRefPubMedPubMedCentralGoogle Scholar
  42. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tao R, Yamane H, Sugiura A, Murayama H, Sassa H, Mori H (1999) Molecular typing of S-alleles through identification, characterization and cDNA cloning for S-RNases in sweet cherry. J Amer Soc Hort Sci 124:224–233CrossRefGoogle Scholar
  44. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ et al (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515CrossRefPubMedPubMedCentralGoogle Scholar
  45. Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano H (2003) Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed F-box gene with haplotype-specific polymorphism. Plant Cell 15:771–781CrossRefPubMedPubMedCentralGoogle Scholar
  46. Ushijima K, Yamane H, Watari A, Kakehi E, Hauck NR, Iezzoni AF et al (2004) The S haplotype-specific F-box protein gene, SFB, is defective in self-compatible haplotypes of Prunus avium and P. mume. Plant J 39:573–586CrossRefPubMedGoogle Scholar
  47. Vieira J, Fonseca NA, Vieira CP (2008) An S-RNase-based gametophytic self-incompatibility system evolved only once in eudicots. J Mol Evol 67:179–190CrossRefPubMedGoogle Scholar
  48. Walsh P, Bursać D, Law YC, Cyr D, Lithgow T (2004) The J-protein family: modulating protein assembly, disassembly and translocation. EMBO Rep 5:567–571CrossRefPubMedPubMedCentralGoogle Scholar
  49. Williams JS, Der JP, dePamphilis CW, Kao T (2014a) Transcriptome analysis reveals the same 17 S-locus F-box genes in two haplotypes of the self-incompatibility locus of Petunia inflata. Plant Cell 26:2873–2888CrossRefPubMedPubMedCentralGoogle Scholar
  50. Williams JS, Natale CA, Wang N, Li S, Brubaker TR, Sun P, Kao T (2014b) Four previously identified Petunia inflata S-locus F-box genes are involved in pollen specificity in self-incompatibility. Mol Plant 7:567–569CrossRefPubMedGoogle Scholar
  51. Zhao L, Huang J, Zhao Z, Li Q, Sim T, Xue Y (2010) The Skp1-like protein SSK1 is required for cross-pollen compatibility in S-RNase-based self-incompatibility. Plant J 62:52–63CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Laboratory of Pomology, Faculty of AgricultureYamagata UniversityTsuruokaJapan
  2. 2.Graduate School of AgricultureKyoto UniversityKyotoJapan

Personalised recommendations