Crystal Structure at 1.5-Å Resolution of Pyrus pyrifolia Pistil Ribonuclease Responsible for Gametophytic Self-incompatibility

Abstract

The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S 3 -RNase) responsible for gametophytic selfincompatibility was determined at 1.5-Å resolution. It consists of eight helices and seven ␤-strands, and its folding topology is typical of RNase T 2 family enzymes. Based on a structural comparison of S 3 -RNase with RNase Rh, a fungal RNase T 2 family enzyme, the active site residues of S 3 -RNase assigned were His 33 and His 88 as catalysts and Glu 84 and Lys 87 as stabilizers of an intermediate in the transition state. Moreover, amino acid residues that constitute substrate binding sites of the two RNases could be superimposed geometrically. A hypervariable (HV) region that has an S-allele-specific sequence comprises a long loop and short ␣-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively charged. Four positively selected (PS) regions, in which the number of nonsynonymous substitutions exceeds that of synonymous ones, are located on either side of the active site cleft, and accessible to solvent. These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen.Many flowering plants have a self-incompatibility system that recognizes the self or non-self between the pistil and pollen (tube) after pollination and suppresses growth of the self-pollen tube to prevent self-fertilization (1, 2). Gametophytic self-incompatibility (GSI) 1 is controlled genetically by a single locus (S-locus) with multiple alleles (1, 2). When a pollen grain lands on a stigma of the pistil, a process that discriminates as to whether an S-allele of the pollen matches one of the two Salleles of the pistil takes place. The pollen grain germinates on the stigma and grows into the style toward the embryo. If its S-allele matches one of the two S-alleles of the pistil, pollen tube growth is arrested in the style, and no fertilization takes place. In solanaceous, scrophulariaceous, and rosaceous plants that have GSI, the pistil glycoproteins that cosegregate with the S-alleles have been identified as ribonucleases of the RNase T 2 family (S-RNase) (3). McClure et al. (4) reported that pollen rRNA is degraded after self-pollination but not after crosspollination and suggested that GSI expression is mediated by degradation of the pollen rRNA of self-pollen tubes by S-RNase, leading to depletion of protein biosynthesis and the eventual arrest of tube growth. S-RNase has been confirmed necessary for GSI from results of gain-of-function and loss-of-function experiments on transgenic plants of solanaceous species (5, 6). Transgenic experiments also have shown that the RNase activity of S-RNase is necessary for GSI (7), which in petunia the carbohydrate moiety is not responsible for GSI (8), and that mutant S-RNase, which has lost RNase activity, acts as a dominant negative for GSI (9).Based on these findings, two models have been proposed to explain S-allele-specific inhibition of pollen tube growth; the...

The Molecular and Genetic Bases of S-RNase-Based Self-Incompatibility

“…They were found subsequently to be the hypervariable regions of Antirrhinum S-RNases as well (Xue et al, 1996) and to correspond to two of the four regions of rosaceous S-RNases for which evidence of positive selection has been found (Ishimizu et al, 1998). The crystal structures of a solanaceous S-RNase and a rosaceous S-RNase show that both HVa and HVb regions are exposed on the surface of the protein and accessible to solvent Matsuura et al, 2001). Together, HVa and HVb are considered the most likely candidates for the determinant of S-allele specificity.…”

Structural and Transcriptional Analysis of the Self-Incompatibility Locus of Almond: Identification of a Pollen-Expressed F-Box Gene with Haplotype-Specific Polymorphism

“…Amino acid sequence alignment showed that SFB contained two quite variable regions, as did S-RNase (Ioerger et al, 1991;Ushijima et al, 1998). The hypervariable region of S-RNase is exposed on the surface of the folded protein molecule and plays a pivotal role in the recognition of self-pollen (Matton et al, 1997(Matton et al, , 1999Ida et al, 2001;Matsuura et al, 2001). The two variable regions of SFB at the C terminus were found to be hydrophilic enough to be exposed on the surface (data not shown) and may have important recognition functions analogous to those of the S-RNase.…”

Molecular Basis of Self-(in)compatibility and Current Status of S-genotyping in Rosaceous Fruit Trees

“…Down-regulation of S-RNase expression by the antisense technique showed the essential role of S-RNase in the GSI reaction in apple (Broothaerts et al, 2004). The crystal structure of pear S-RNase revealed a folding topology that is typically found in RNase T 2 family enzymes (Matsuura et al, 2001). Although the biochemical function of rosaceous S-RNase is unknown, a series of immunochemical and radioactive tracer experiments with solanaceous S-RNase indicated that SRNase could function as a cytotoxin to degrade pollen RNA in an incompatible reaction.…”

“…There is a single hypervariable region (Ushijima et al, 1998), which may form the interacting domain for GSI biochemical reactions (Matsuura et al, 2001). The function of S-RNase has been studied genetically and biochemically.…”