S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen

Abstract

Self-incompatibility (SI) in Petunia is regulated by a polymorphic S-locus. For each S-haplotype, the S-locus contains a pistil-specific S-RNase gene and multiple pollen-specific S-locus F-box (SLF) genes. Both gain-of-function and loss-of-function experiments have shown that S-RNase alone regulates pistil specificity in SI. Gain-of-function experiments on SLF genes suggest that the entire suite of encoded proteins constitute the pollen specificity determinant. However, clear-cut loss-of-function experiments must be performed to determine if SLF proteins are essential for SI of pollen. Here, we used CRISPR/Cas9 to generate two frame-shift indel alleles of S₂ -SLF1 (SLF1 of S₂ -haplotype) in S₂S₃ plants of P. inflata and examined the effect on the SI behavior of S₂ pollen. In the absence of a functional S₂-SLF1, S₂ pollen was either rejected by or remained compatible with pistils carrying one of eight normally compatible S-haplotypes. All results are consistent with interaction relationships between the 17 SLF proteins of S₂ -haplotype and these eight S-RNases that had been determined by gain-of-function experiments performed previously or in this work. Our loss-of-function results provide definitive evidence that SLF proteins are solely responsible for SI of pollen, and they reveal their diverse and complex interaction relationships with S-RNases to maintain SI while ensuring cross-compatibility.

Figure 1.

Generation of S₂-SLF1 Indel Alleles by CRISPR/Cas9-Mediated Genome Editing. (A) Design of a gRNA specifically targeting S₂-SLF1. A 20-bp sequence (named S₂-SLF1-PS9) of the antisense strand of S₂-SLF1 followed by the PAM motif (TGG) was chosen as the protospacer for CRISPR/Cas9; two mismatches (highlighted in yellow and indicated with asterisks) are found in the corresponding 20-bp region in S₃-SLF1 (highlighted in blue). “Start” indicates the start codon (ATG) on the sense strand (5′ to 3′) of these two genes, and “End” indicates the stop codon (TAG). The positions of the PCR primers specific to S₂-SLF1 (PiSLF2-RT-3For/PiSLF2-RT-4Rev) and those specific to S₃-SLF1 (PiSLF3-Copy1For/PiSLF3-Copy1Rev) are indicated by purple lines. Black triangle indicates the cleavage site of BsrGI in the wild-type S₂-SLF1 sequence. (B) PCR-restriction enzyme digestion screen for edited S₂-SLF1 alleles in 10 transgenic plants. (-): PCR product amplified from genomic DNA of one of the transgenic plants by the S₂-SLF1 specific primers, without digestion by BsrGI. BsrGI (+): BsrGI digestion of the PCR products amplified from genomic DNA of a wild-type S₂S₃ plant and the 10 transgenic plants. Asterisk (*) indicates the ∼220-bp PCR product resistant to, or not subjected to, BsrGI digestion; double asterisks (**) indicate the ∼180-bp BsrGI fragment; triple asterisks (***) indicates the ∼40-bp BsrGI fragment. The plant numbers of those T₀ plants carrying mutant S₂-SLF1 alleles resistant to BsrGI digestion are highlighted in red. (C) Sequences of four indel alleles in the edited region of S₂-SLF1. The sequences shown are those of the antisense strand of S₂-SLF1 (5′ to 3′ from left to right). The black triangle indicates the cleavage site of BsrGI in the wild-type S₂-SLF1 sequence. The open arrow indicates the direction of translation, and the encoded amino acids in the wild-type S₂-SLF1 are shown. The 3-bp in-frame deletion in plant #13 abolishes the codon 5′-CAG-3′ for Gln-210. The 6-bp in-frame deletion in plant #27 abolishes the codon for Gln-210 and disrupts the codon 5′-GTA-3′ for Val-209 and the codon 5′-TTG-3′ for Leu-211. However, as the Val codon is restored as 5′-GTG-3′, only Gln-210 and Leu-211 are deleted from the encoded protein. (D) Sequencing chromatograms of PCR amplicons of S₃-SLF1 from T₀ plant #2/S₂*S₃ and from a wild-type S₂S₃ plant. The sequences are those of the antisense strand from 5′ to 3′ (left to right).

Figure 2.

Analysis of SI Behavior of T₀ Plants Carrying Either a 1-bp Deletion or a 1-bp Insertion Frame-Shift Allele of S₂-SLF1. (A) Aniline blue staining of pollen tubes in the bottom segment of the style after a wild-type S₃S₃ plant was separately pollinated by pollen from T₀ plants, #2/S₂*S₃ and #35/S₂*S₃, and a wild-type S₂S₃ plant. White arrows indicate where growth of most pollen tubes stopped, in the case of incompatible pollinations. Scale bar = 1 mm. (B) Progeny analysis of crosses using pollen from #2/S₂*S₃ or #35/S₂*S₃ to separately pollinate the wild-type S₇S₇ and S₁₃S₁₃ pistils. n indicates the number of plants in each progeny analyzed.

Figure 3.

Analysis of SI Behavior of S₂S₂ Plants Homozygous for Either Frame-Shift Indel Allele of S₂-SLF1. (A) Results of pollination using pollen from two bud-selfed (BS) progeny plants of #2/S₂*S₃ (#2-BS-#2 and #2-BS-#8), and two BS progeny plant of #35/S₂*S₃ (#35-BS-#8 and #35-BS-#10) to separately pollinate pistils of various S-genotypes. S₂*S₂* indicates that all four BS progeny plants were S₂S₂ and homozygous for the indel allele of S₂-SLF1 inherited from their respective T₀ plants. (Cas9+) and (Cas9-) indicate presence and absence of the Cas9 transgene, respectively, in the BS plants. —: incompatible pollination (no fruit set); +: compatible pollination (fruit set). As-S₃/S₃S₃: a self-compatible transgenic plant not producing any S₃-RNase in the pistil due to expression of an antisense S₃-RNase gene. (B) to (G) Aniline blue staining of pollen tubes in the bottom segment of the style of the pistil from each of the wild-type plants of five different S-genotypes, as indicated, and from a transgenic plant As-S₃/S₃S₃. These plants were separately pollinated with pollen from #35-BS-#8 or #35-BS-#10, as indicated. Scale bar = 0.25 mm.

Figure 4.

The in Vivo Gain-of-Function Assay Used for Determining Interaction Relationships between SLF Proteins and S-RNases. (A) Schematic of the transgene constructs for S₂-SLF9 and S₂-SLF10. (B) Schematic of nine additional transgene constructs, each containing one of the SLF genes indicated (denoted S₂-SLFn). All constructs shown in (A) and (B) were made using Ti plasmid pBI101 as the backbone. RB, right border of the T-DNA; Nos-pro, promoter of the gene encoding nopaline synthase; NPT-II, gene encoding neomycin phosphotransferase II (conferring resistance to kanamycin); Nos-ter, transcription terminator of the gene encoding nopaline synthase; LAT52P: promoter of the pollen-specific LAT52 gene from tomato; GFP, gene encoding green fluorescent protein; LB, left border of the T-DNA. (C) Workflow of the in vivo gain-of-function assay. (D) Graphic illustration of the genetic basis for determining interaction relationships between SLF proteins and S-RNases. The transgene construct for an SLF gene of S₂-haplotype, denoted S₂-SLFn, is introduced into S₂S_x plants, with S_x being an S-haplotype different from S₂. Pollen from the LAT52P:S₂-SLFn:GFP/S₂S_x transgenic plant is used to pollinate a wild-type S₂S_x plant. Among the four genotypes of pollen produced by the transgenic plant, S₂ and S_x should be rejected by the S₂S_x pistil, and S₂ carrying the transgene is expected be rejected, as the S₂-SLF_n transgene is from the same S-haplotype as pollen. Thus, whether or not this pollination is compatible is determined solely by the SI behavior of S_x pollen carrying the transgene. If S₂-SLFn interacts with S_x-RNase to mediate its ubiquitination and degradation in the LAT52P:S₂-SLFn:GFP/S_x pollen tube, then the pollination should be compatible, and all the progeny will inherit the transgene and carry S_x-haplotype. If S₂-SLFn does not interact with S_x-RNase, then the LAT52P:S₂-SLFn:GFP/S_x pollen tube should be rejected in the style and the pollination should be incompatible.

Figure 5.

Assessment of Interaction between S₂-SLF2 and S₇-RNase by in Vivo Gain-of-Function Assay. (A) Aniline blue staining of pollen tubes in the bottom segment of the style after a wild-type S₂S₇ plant was self-pollinated (right) and pollinated with pollen from transgenic plant LAT52P:S₂-SLF2:GFP/S₂S₇ (left). This transgenic plant (a T₁ plant) was obtained by pollinating a wild-type S₇S₇ plant with pollen from a T₀ transgenic plant LAT52P:S₂-SLF2:GFP/S₂S₃. Scale bar = 1 mm. (B) Analysis of 24 T₂ plants resulting from the cross, S₂S₇ x LAT52P:S₂-SLF2:GFP/S₂S₇, shown in (A). T₁ indicates genomic DNA from the T₁ plant LAT52P:S₂-SLF2:GFP/S₂S₇; P indicates plasmid DNA of pBI101-LAT52P:S₂-SLF2:GFP (as positive control for the PCR amplification of the GFP transgene). S₂S₇ indicates genomic DNA from a wild-type S₂S₇ plant (as negative control for the PCR amplification of the GFP transgene). S₂S₂ indicates genomic DNA from a wild-type S₂S₂ plant (as negative control for the PCR amplification of the S₇-RNase gene). S₃S₇ indicates genomic DNA from a wild-type S₃S₇ plant (as negative control for the PCR amplification of the S₂-RNase gene). (C) Chi-square analysis of the S-haplotype inheritance in the 24 T₂ plants analyzed in (B). Chi-square analysis was used to test the null hypothesis of S-haplotype inheritance, 1:1 ratio of S₂S₇:S₇S₇ versus 1:2:1 ratio of S₂S₂:S₂S₇:S₇S₇. (D) Graphic illustration of interpretation of the results of progeny analysis from S₂S₇ x LAT52P:S₂-SLF2:GFP/S₂S₇ shown in (B). The observation that all progeny plants inherited the GFP transgene and none were S₂S₂ indicates that only the transgenic S₇ pollen carrying the LAT52P:S₂-SLF2:GFP transgene can effect fertilization. This result suggests that S₂-SLF2 produced in the transgenic S₇ pollen interacts with and detoxifies S₇-RNase to render the transgenic S₇ pollen compatible with the S₂S₇ pistil.

Figure 6.

Assessment of Interaction between S₂-SLF1 and S₁₂-RNase by in Vivo Gain-of-Function Assay. (A) Analysis of 24 T₂ plants resulting from the cross, S₂S₁₂ x LAT52P:S₂-SLF1:GFP/S₂S₁₂. T₁ indicates genomic DNA from the T₁ plant LAT52P:S₂-SLF1:GFP/S₂S₁₂; S₂S₃ indicates genomic DNA from a wild-type S₂S₃ plant (as negative control for the PCR amplification of the GFP transgene and S₁₂-RNase gene); S₃S₁₂ indicates genomic DNA from a wild-type S₃S₁₂ plant (as negative control for the PCR amplification of the S₂-RNase gene). (B) Chi-square analysis of the S-haplotype inheritance in the 24 T₂ plants analyzed in (A). Chi-square was used to test the null hypothesis of S-haplotype inheritance, 1:1 ratio of S₂S₁₂:S₁₂S₁₂ versus 1:2:1 ratio of S₂S₂:S₂S₁₂:S₁₂S₁₂.

Figure 7.

Analysis of SI Behavior of S₂* Pollen Carrying a 3-bp or a 6-bp In-Frame Deletion Allele of S₂-SLF1. (A) Pollen tube growth in the style after a wild-type S₃S₃ plant was separately pollinated with pollen from T₀ plants #13/S₂*S₃ (carrying a 3-bp deletion allele) and #27/S₂*S₃ (carrying a 6-bp deletion allele). (B) Pollen tube growth in the style after a wild-type S₃S₁₃ plant was pollinated with pollen from T₀ plant #13/S₂*S₃. (C) Pollen tube growth in the style after a wild-type S₁₃S₁₃ plant was pollinated with pollen from a progeny plant, #27-BS-#1/S₂*S₂*, obtained by bud-selfing T₀ plant #27/S₂*S₃. Scale bar = 1 mm in all microscopy images of aniline blue staining in (A), (B), and (C). (D) Effect of a single amino-acid deletion (Q₂₁₀) and a two-amino acid deletion (Q₂₁₀ and L₂₁₁) of S₂-SLF1 on its ability to detoxify S₃-RNase and S₁₃-RNase. Seven amino acids of the wild-type S₂-SLF1 in the region where deletions occur are shown for comparison. + indicates ability to detoxify S₃-RNase and S₁₃-RNase, and — indicates inability to detoxify these two S-RNases.