A new dual-specific incompatibility allele revealed by absence of glycosylation in the conserved C2 site of a Solanum chacoense S-RNase

Abstract

The stylar determinant of gametophytic self-incompatibility (GSI) in Solanaceae, Rosaceae, and Plantaginaceae is an S-RNase encoded by a multiallelic S-locus. The primary structure of S-RNases shows five conserved (C) and two hypervariable (HV) regions, the latter forming a domain implicated in S-haplotype-specific recognition of the pollen determinant to SI. All S-RNases are glycosylated at a conserved site in the C2 region, although previous studies have shown that N-linked glycans at this position are not required for S-haplotype-specific recognition and pollen rejection. Here the incompatibility phenotype of three constructs derived from an originally monoglycosylated S11-RNase of Solanum chacoense, that were designed to explore the role of the HV domain in determining pollen recognition and the role of the N-linked glycan in the C2 region, is reported. In one series of experiments, a second glycosylation site was introduced in the HVa region to test for inhibition of pollen-specific recognition. This modification does not impede pollen rejection, although analysis shows incomplete glycosylation at the new site in the HVa region. A second construct, designed to permit complete glycosylation at the HVa site by suppression of the conserved site in the C2 region, did increase the degree of site occupancy, but, again, glycosylation was incomplete. Plants expressing this construct rejected S 11 pollen and, surprisingly, also rejected S 13 pollen, thus displaying an unusual dual specificity phenotype. This construct differs from the first by the absence of the conserved C2 glycosylation site, and thus the dual specificity is observed only in the absence of the C2 glycan. A third construct, completely lacking glycosylation sites, conferred an ability to reject only S 11 pollen, disproving the hypothesis that lack of a conserved glycan would confer a universal pollen rejection phenotype to the plant.

Fig. 1.

Plants expressing a diglycosylated S₁₁-RNase have a normal S-phenotype. Glc^C2HVa plant lines express an S₁₁-RNase that contains a glycosylation site in the HVa region in addition to the normal and conserved glycosylation site in the C2 region. Western blot analysis of six selected plants shows a range of protein levels detectable using an anti-S₁₁-RNase antibody. The size of the bands in the absence of PNGase (–) is consistant with a mono- and a diglycosylated form, while in the presence of PNGase (+) the sizes are consistent with a non-glycosylated and a monoglycosylated form. L25 plants (S ₁₁ S ₁₂) and the untransformed host G4 (S ₁₂ S ₁₄) are shown as positive and negative controls, respectively. The pollination phenotype of each plant is shown for a representative pollination with S ₁₁ S ₁₂ and S ₁₃ S ₁₄ tester stocks and is reported as the number of fruits set per pollination.

Fig. 2.

Plants expressing the Glc^HVa-RNase have an unusual dual specificity incompatibility phenotype. Western blot analysis of seven plant lines expressing an S₁₁-RNase engineered to contain only a single glycosylation site in the HVa region show a wide range of protein levels (A). L25 plants (S ₁₁ S ₁₂) and the untransformed host G4 (S ₁₂ S ₁₄) are shown as positive and negative controls, respectively, and the pollination phenotype of each plant is shown for a representative pollination with S ₁₁ S ₁₂, S ₁₃ S ₁₄, S ₁₅ S ₁₆, and S ₁₁ S ₁₁ S ₃₁ S ₁₃ tester stocks. The three highly expressing lines are shown with (+) and without (–) PNGase treatment (B).

Fig. 3.

Plants expressing the NoGlc S-RNase have a normal S-phenotype. Western blot analysis of seven plant lines expressing an S-RNase lacking all glycosylation sites shows a range of protein levels. L25 plants are shown as a positive control, and the pollination phenotype of each plant is shown for a representative pollination with S ₁₁ S ₁₂, S ₁₃ S ₁₄, and S ₁₅ S ₁₆ tester stocks.

Fig. 4.

Model for the different S-RNases produced by Glc^C2/HVa and Glc^HVa transgenic lines. Each of the transgenic lines produces two S-RNase forms (spheres), differing from one another with regard to glycosylation at the newly introduced site in the HVa region. The glycan groups are shown as twigs at the protein surface, in either the hypervariable HVa region or the conserved C2 region. A hypothetical interaction with the pollen component (dotted line) is shown for recognition by either the S ₁₁ or S ₁₃ pollen (SLFx and SLFy, respectively).