Ornithine decarboxylase genes contribute to S-RNase-independent pollen rejection

Abstract

Unilateral incompatibility (UI) manifests as pollen rejection in the pistil, typically when self-incompatible (SI) species are pollinated by self-compatible (SC) relatives. In the Solanaceae, UI occurs when pollen lack resistance to stylar S-RNases, but other, S-RNase-independent mechanisms exist. Pistils of the wild tomato Solanum pennellii LA0716 (SC) lack S-RNase yet reject cultivated tomato (Solanum lycopersicum, SC) pollen. In this cross, UI results from low pollen expression of a farnesyl pyrophosphate synthase gene (FPS2) in S. lycopersicum. Using pollen from fps2-/- loss-of-function mutants in S. pennellii, we identified a pistil factor locus, ui3.1, required for FPS2-based pollen rejection. We mapped ui3.1 to an interval containing 108 genes situated on the IL 3-3 introgression. This region includes a cluster of ornithine decarboxylase (ODC2) genes, with four copies in S. pennellii, versus one in S. lycopersicum. Expression of ODC2 transcript was 1,034-fold higher in S. pennellii than in S. lycopersicum styles. Pistils of odc2-/- knockout mutants in IL 3-3 or S. pennellii fail to reject fps2 pollen and abolish transmission ratio distortion (TRD) associated with FPS2. Pollen of S. lycopersicum express low levels of FPS2 and are compatible on IL 3-3 pistils, but incompatible on IL 12-3 × IL 3-3 hybrids, which express both ODC2 and ui12.1, a locus thought to encode the SI proteins HT-A and HT-B. TRD observed in F2 IL 12-3 × IL 3-3 points to additional ODC2-interacting pollen factors on both chromosomes. Thus, ODC2 genes contribute to S-RNase independent UI and interact genetically with ui12.1 to strengthen pollen rejection.

Figure 1

Genetic map of chromosome 3 introgression lines and pollen tube growth of WT and fps2 mutant LA0716 pollen on pistils of IL 3-2, IL 3-3, and IL 3-4. Only pistils of IL 3-3 reject fps2 pollen, which places the ui3.1 locus in the genomic region unique to the IL 3-3 introgression. A genetic map based on S. lycopersicum × S. habrochaites (“L × H” map) shows the approximate location (gray rectangle) of the ui3.1 locus reported by Bernacchi and Tanksley (1997). A genetic map from F₂  S. lycopersicum × S. pennellii (“L × P”) shows the approximate positions of each introgressed segment based on the EXPEN 2000 genetic map (https://solgenomics.net). Arrows indicate the position reached by the longest pollen tube. Scale bar: 1 mm.

Figure 2

RT-PCR analysis of ui3.1 candidate gene expression. A, Qualitative RT-PCR analysis of transcript abundance in leaves, pollen, and styles of S. lycopersicum (L) and S. pennellii (P). B, RT-qPCR analysis of ODC2 mRNA levels in S. lycopersicum and S. pennellii leaves, styles, and ovaries. Data are presented as the mean ± se (n = 3).

Figure 3

Neighbor-joining tree of ornithine decarboxylases and related protein sequences. The predicted amino acid sequences are from SpODC2a (Sopen03g029020), SpODC2b (Sopen03g029030), SpODC2c (Sopen03g029040), SpODC2d (Sopen03g029050), SlODC2 (Solyc03g098300), SlODC3 (Solyc03g098310), SpODC3 (Sopen03g029060), GmODC (NP_001238629.1), DsODC (X87847), SlODC1 (Solyc04g082030), SpODC1 (Sopen04g035680), GmADC (AAD09204.1), and SlADC (NP_001234649.2). Sp = S. pennellii, Sl = S. lycopersicum, Ds = Datura stramonium, Gm = Glycine max. ADC, arginine decarboxylase; ODC, ornithine decarboxylase. The scale indicates the average number of amino acid substitutions per site.

Figure 4

Genetic and phenotypic analysis of WT and odc mutants in IL 3-3. A, TIDE analysis of SpODC2 gene sequences in WT and several independent T₂ generation CRISPR-Cas9 mutants (odc2). B, Pollen tube growth of fps2 pollen on pistils of WT and mutant IL 3-3. Arrows indicate the position reached by the longest pollen tube. WT IL 3-3 rejects pollen of fps2 mutants, whereas several independent T₂ generation odc2 mutants accept fps2 pollen. Mutations (T₁ generation) in the related gene, ODC3, do not affect pollen compatibility. Scale bar: 1 mm. C, fps2 pollen tubes grow to less than 50% of the style length after 48 h on WT IL 3-3 and odc3 mutants, but reach the ovaries on mutant odc2 pistils. Data are presented as the mean ± se (n = 5).

Figure 5

Genetic and phenotypic analysis of WT and odc2 mutants in S. pennellii LA0716. A, TIDE analysis of SpODC2 sequences. B, Pollen tube growth of fps2 mutant pollen on WT and mutant LA0716. Arrows indicate the position reached by the longest pollen tube. Scale bar: 1 mm. C, fps2 pollen tubes grow to ca. 75%–85% of the style length on WT LA0716, but reach the ovaries on odc2 mutants. Data are presented as the mean ± se (n = 5).

Figure 6

Genetic and phenotypic analysis of WT and odc2 mutant F₁ (VF36 × LA0716) hybrids. A, TIDE analysis of SpODC2 gene sequences in two independent mutant F₁’s. B, Pollen tube growth of fps2 mutant pollen on pistils of F₁ (VF36 × LA0716 WT) and F₁ (VF36 × LA0716 odc2). Arrows indicate the position reached by the longest pollen tube. Scale bar: 1 mm.

Figure 7

Growth of S. lycopersicum M-82 pollen tubes on pistils of heterozygous double introgression line hybrids combining introgressions on chromosomes 3 and 12 from S. pennellii. M-82 pollen is rejected on WT IL 12-3 × IL 3-3 hybrids, but not IL 12-3 × IL 3-3 (odc2) mutants. An F₂ plant homozygous for both introgressions (right) rejects M-82 pollen earlier in the style compared with the heterozygotes. Scale bar: 1 mm. Arrows indicate the position reached by the longest pollen tube in each pistil.

Figure 8

Growth of M-82 pollen tubes on pistils of IL 12-3 × IL 3-3, IL 12-2 × IL 3-3, or IL 12-4 × IL 3-3 hybrids. Arrows indicate the position reached by the longest pollen tubes. Scale bar: 1 mm. M-82 pollen are rejected only by IL 12-3 × IL 3-3 pistils, which places the ui12.1 locus in the bin unique to IL 12-3. The previous approximate location of ui12.1 estimated by Bernacchi and Tanksley (1997) in S. lycopersicum × S. habrochaites cross (L × H map) is shown in gray. The map positions of the IL breakpoints are from an F₂  S. lycopersicum × S. pennellii (L × P map) by Alseekh et al. (2013). The position of a putative pollen factor locus, ui12.2, is also shown.