The F-box protein AhSLF-S2 controls the pollen function of S-RNase-based self-incompatibility

Abstract

Recently, we have provided evidence that the polymorphic self-incompatibility (S) locus-encoded F-box (SLF) protein AhSLF-S(2) plays a role in mediating a selective S-RNase destruction during the self-incompatible response in Antirrhinum hispanicum. To investigate its role further, we first transformed a transformation-competent artificial chromosome clone (TAC26) containing both AhSLF-S(2) and AhS(2)-RNase into a self-incompatible (SI) line of Petunia hybrida. Molecular analyses showed that both genes are correctly expressed in pollen and pistil in four independent transgenic lines of petunia. Pollination tests indicated that all four lines became self-compatible because of the specific loss of the pollen function of SI. This alteration was transmitted stably into the T1 progeny. We then transformed AhSLF-S(2) cDNA under the control of a tomato (Lycopersicon esculentum) pollen-specific promoter LAT52 into the self-incompatible petunia line. Molecular studies revealed that AhSLF-S(2) is specifically expressed in pollen of five independent transgenic plants. Pollination tests showed that they also had lost the pollen function of SI. Importantly, expression of endogenous SLF or SLF-like genes was not altered in these transgenic plants. These results phenocopy a well-known phenomenon called competitive interaction whereby the presence of two different pollen S alleles within pollen leads to the breakdown of the pollen function of SI in several solanaceaous species. Furthermore, we demonstrated that AhSLF-S(2) physically interacts with PhS(3)-RNase from the P. hybrida line used for transformation. Together with the recent demonstration of PiSLF as the pollen determinant in P. inflata, these results provide direct evidence that the polymorphic SLF including AhSLF-S(2) controls the pollen function of S-RNase-based self-incompatibility.

Figure 1.

Molecular Analysis of TAC26 Transgenic Petunia Plants. (A) A schematic structure of TAC26. Probes for AhS₂-RNase and AhSLF-S₂ used are also indicated and HindIII and BamHI sites with the genomic region covered by TAC26. LB, left border; RB, right border of the T-DNA; Hyg, hygromycin resistance gene. (B) to (G) DNA gel blot analysis of the T0 transgenic lines a, b, d, g, m, l, r, and the untransformed control (WT). Leaf DNA (5 μg) was digested with HindIII ([B], [D], and [F]) or BamHI ([C], [E], and [G]) and was blotted and probed with the Hyg fragment ([B] and [C]), AhSLF-S₂ ([D] and [E]) and S₂-RNase ([F] and [G]). (H) DNA gel blot of HindIII-digested leaf DNA was probed with a PhS₃-RNase cDNA fragment. Sizes of the markers ([B] and [C]) and the hybridizing bands ([D] to [H]) are indicated in kilobase pairs.

Figure 2.

Expression Analysis of AhSLF-S₂ and AhS₂-RNase in the Transgenic Petunia Lines. (A) and (B) RT-PCR analysis of RNA isolated from pollens or styles, with (+) or without (–) reverse transcriptase in the synthesis of cDNA. (A) Top panel, RT-PCR analysis of RNA isolated from pollen of the transgenic lines b, g, l, m, and S₃S₃ wild-type petunia plant using specific primers of AhSLF-S₂. TAC26 plasmid (TAC26) was used as a positive control. Bottom panel, RT-PCR analysis of tubulin for loading control. (B) Top panel, RT-PCR analysis of RNA isolated from styles of transgenic lines b, g, l, m, and the wild type using specific primers of AhS₂-RNase, and the TAC26 plasmid (TAC26) was used as a positive control. Middle panel, RT-PCR analysis of RNA isolated from styles of the trangenic lines using specific primers of PhS₃-RNase. Bottom panel, RT-PCR analysis of tubulin for loading control. (C) and (D) Immunoblot detection of AhSLF-S₂ or S-RNases. (C) Top panel, detection of AhSLF-S₂ from total pollen proteins of transgenic lines b, g, l, m, and wild-type S₃S₃ petunia plant and Antirrhinum of S₂S₄ (Ant) by polyclonal antibody against AhSLF-S₂. Bottom panel, detection of tubulin for loading control. (D) Top panel, detection of AhS₂-RNase (∼29 kD) and endogenous S₃-RNase (∼24 kD) from total style proteins of transgenics lines b, g, l, m, wild-type S₃S₃ petunia plant and Antirrhinum (Ant) by polyclonal antibody against AhS-RNases. Bottom panel, detection of tubulin for loading control. Sizes of the detected protein bands are indicated in kilodaltons.

Figure 3.

Molecular Analysis of DNA from T1 Progeny of the TAC26 Transgenic Petunia Lines. (A) to (C) Genomic DNA gel blot analysis of the T1 progeny (b1 to b3), T0 transformant b, and the untransformed control. Leaf DNA digested by HindIII was blotted and probed with the Hyg fragment (A), AhSLF-S₂ (B), and AhS₂-RNase (C), respectively. (D) to (F) DNA gel blot analysis of the T1 progeny (l1 to 5, 7, and 8), T0 transformant, and the untransformed control. Leaf DNA digested by HindIII was blotted and probed with the Hyg fragment (D), AhSLF-S₂ (E), and AhS₂-RNase (F), respectively. Sizes of the hybridizing fragments are indicated in kilobase pairs.

Figure 4.

Expression Analysis of AhSLF-S₂ and AhS₂-RNase in the Transgenic Petunia T1 Progeny. (A) and (B) RT-PCR analysis of RNA isolated from pollen or style, with (+) or without (−) reverse transcriptase in the synthesis of cDNA. (A) Top panel, RT-PCR analysis of RNA isolated from pollen of the transgenic T1 lines l1, 2, 3, 4, 5, 7, and 8, lines b1 to 3, and wild-type plant using AhSLF-S₂–specific primers with TAC26 plasmid as a positive control. Bottom panel, RT-PCR analysis of tubulin for loading control. (B) Top panel, RT-PCR analysis of RNA isolated from styles of the transgenic progeny using AhS₂-RNase–specific primers. Middle panel, RT-PCR analysis of RNA isolated from styles of the transgenic progeny using specific primers of PhS₃-RNase. Bottom panel, RT-PCR analysis of tubulin for loading control. (C) and (D) Immunoblot detection of AhSLF-S₂ or S-RNases. (C) Top panel, detection of AhSLF-S₂ from total pollen proteins of the transgenic lines l1 to 5, 7, and 8, b1 to 3, wild-type plant, and Antirrhinum (Ant) by polyclonal antibody against AhSLF-S₂. The bottom panel is tubulin for loading control. (D) Top panel, detection of AhS₂-RNase (∼29 kD) and endogenous S₃-RNase (∼24 kD) from total style proteins of the transgenic lines l1 to 5, 7, and 8, b1 to 3, wild-type plant, and Antirrhinum (Ant) by polyclonal antibody against S-RNases. Bottom panel, detection of tubulin for loading control.

Figure 5.

Molecular Analysis of AhSLF-S₂ Transgenic Petunia Plants. (A) A schematic diagram of pBIAhSLF-S₂. RB and LB, right and left borders of the T-DNA; Km^r, kanamycin resistance (neomycin phosphortransferase) gene. Several restriction sites also are indicated. (B) and (C) DNA gel blot analysis of the transgenic lines WQ-1, WQ-2, WQ-3, WQ-4, and WQ-5 and the untransformed control (WT). Leaf genomic DNA was digested with HindIII (B) or EcoRI (C) was blotted and probed with AhSLF-S₂. Leaf DNA of an S₂S₄ Antirrhinum line (Ant) was as a positive control. (D) Leaf DNA digested by EcoRI from the transgenic lines was blotted and probed with PhS₃-RNase.

Figure 6.

Expression Analysis of AhSLF-S₂ Transgenic Petunia Plants. (A) RT-PCR analysis of RNA isolated from pollen of the AhSLF-S₂ transgenic lines WQ-1, WQ-2, WQ-3, WQ-4, and WQ-5 and wild-type plant, using specific primers of AhSLF-S₂ with (+) or without (−) reverse transcriptase in the synthesis of cDNA. The TAC26 plasmid (lane 13) was used as a positive control (top panel). RT-PCR analysis of tubulin for loading control (bottom panel). (B) Top panel, immunoblot detection of AhSLF-S₂ from total pollen protein of the transgenic plants WQ-1, WQ-2, WQ-3, WQ-4, and WQ-5 and wild-type S₃S₃ petunia by polyclonal antibody against AhSLF-S₂. Bottom panel, detection of tubulin for loading control. Antirrhinum pollen protein (Ant) was used as a positive control. (C) Immunoblot detection of PhS₃-RNase from total style proteins of the transgenic lines of WQ-1, WQ-2, WQ-3, WQ-4, and WQ-5 and wild-type plant by polyclonal antibody against AhS-RNase (top panel). Detection of tubulin for loading control (bottom panel). Antirrhinum style protein (Ant) was used as a positive control.

Figure 7.

Physical Interaction between AhSLF-S₂ and PhS₃-RNase. (A) Yeast cells containing various combinations of BD and AD fusions were tested for their growth on -Leu/-Trp/-His/-Ade dropout media. Plasmid pGBKT₇ with various AD:constructs and plasmid pGADT₇ with various BD:constructs were used as negative controls. (B) The strains were grown further to test for expression of the β-galactosidase reporter gene. (C) A pull-down assay for the physical interaction between AhSLF-S₂ and PhS₃-RNase. Ni-NTA resin and the purified fusion proteins of His-AhSLF-S₂-C were incubated with the style extract of the S₃S₃ line of P. hybrida (Ph Style Extracts). Bound proteins were pulled down with Ni-NTA resin, eluted with the lysis buffer, separated by 12% SDS-PAGE, transferred to membranes, and analyzed by immunoblotting using the anti-S-RNase antibody (top panel). Style extracts from Antirrhinum (S₂S₅) (Ant Style Extracts) were also included as a control. Input style total protein was used as a positive control (bottom panel). Molecular mass markers are indicated in kilodaltons.

Figure 8.

Amino Acid Sequence Alignment of Predicted SLF Polypeptides from Antirrhinum and Petunia and Expression Analysis of Endogenous SLF Genes in Transgenic Petunia Plants. (A) Alignment of the predicted polypeptide sequences from the SLF family. Antirrhinum, AhSLF-S₂ (CAC33010); P. inflata, PiA134-S₁ (AAR15914), PiA134-S₂ (AAR15915), PiA134-S₃ (AAR15916), PiSLF-S₁ (AY500390), PiSLF-S₂ (AY500391), and PiSLF-S₃ (AY500392); P. hybrida, PhSLF-S₃A (AY639403) and PhSLF-S₃B (AY639402). PhC1 and PhC2 indicate the conserved regions that were used to design degenerate primers for cloning PhSLF-S₃A and PhSLF-S₃B. The F-box domain is also indicated. (B) and (C) RT-PCR analysis of RNA isolated from pollen of the TAC26 and AhSLF-S₂ transgenic lines and wild-type plant, using specific primers of PhSLF-S₃A and PhSLF-S₃B with (+) or without (−) reverse transcriptase in the synthesis of cDNA. Tubulin control was the same as in Figures 4A and 6A.

Figure 9.

Schematic Representations of the Phylogenetic Relationships for Predicted SLF and S-RNase Polypeptides. The phylogentic relationships are shown for the S-RNases (top) and SLFs (bottom), respectively. Antirrhinum, AhSLF-S₂, AhSLF-S₄ (CAD56661), AhSLF-S₅ (CAD56664), AhS₂-RNase (CAC33020), AhS₄-RNase (Q38717), and AhS₅-RNase (CAA65318); P. inflata, PiSLF-S₁, PiSLF-S₂, PiSLF-S₃, PiS₁-RNase (S20989), PiS₂-RNase (AAG21384), and PiS₃-RNase (AAA33727); P. hybrida, PhSLF-S₃A and PhS₃-RNase (AJ271065); Prunus dulcis, PdSFB-Sa (BAC65206), PdSFB-Sb (BAC65207), PdSFB-Sc (BAC65201), PdSa-RNase (BAA95317), PdSb-RNase (T12078), and PdSc-RNase (T12076); P. mume, PmSLF-S₁ (BAC66622), PmSLF-S₇ (BAC66623), PmS₁-RNase (BAC56115), and PmS₇-RNase (BAC56116).