Laboratory of Pomology, Faculty of Agriculture, Yamagata University, Tsuruoka, 997-8555, Japan.
Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
Plant Mol Biol. 2019 Jul;100(4-5):367-378. doi: 10.1007/s11103-019-00860-8. Epub 2019 Apr 1.
S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry. Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1-3 (SLFL1-3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.
S-RNase 被证明主要被甜樱桃亲和花粉管中的 S 座位 F-box 样蛋白和 S 单倍型特异性 F-box 样蛋白识别。自交不亲和（SI）是一种生殖障碍，它排斥自花粉并抑制自受精以促进异交。在茄科和蔷薇科中，基于 S-RNase 的配子体 SI（GSI）由 S-RNase 和 F-box 蛋白（s）分别作为雌蕊和花粉 S 决定因素组成。亲和花粉管被认为可以解毒内化的细胞毒性 S-RNase 以维持生长。S-RNase 解毒由花粉 S 决定因素、S 座位 F-box 蛋白（SLFs）形成的 Skp1-cullin1-F-box 蛋白复合物（SCF）完成，在茄科中。在李属中，一般抑制剂（GI），而不是花粉 S 决定因素 S 单倍型特异性 F-box 蛋白（SFB），被假设可以解毒 S-RNase。最近，SLF 样蛋白 1-3（SLFL1-3）被认为是 GI 的候选者，尽管其他蛋白在 GI 中起主要作用的可能性仍然存在。为了鉴定其他 GI 候选者，我们从甜樱桃中分离出另外 4 种花粉表达的 SLFL 和 SFB 样（SFBL）蛋白 PavSLFL6、PavSLFL7A、PavSFBL1 和 PavSFBL2。与 4 种 PavS-RNase 的结合实验表明，PavSFBL2 与 PavS-RNase 结合，而其他蛋白与任何东西都不结合。PavSFBL2 被证实可以在体外形成 SCF 复合物。使用重组 PavS-RNase 作为诱饵进行的共免疫沉淀实验，以及对花粉提取物进行的质谱分析，鉴定出了 PavSLFLs 和 PavSFBL2、M 座位编码谷胱甘肽 S-转移酶（MGST）、DnaJ 样蛋白和其他少量蛋白的 SCF 复合物成分。这些结果表明，SLFLs 和 SFBLs 可能在李属植物特异性 S-RNase 基 GSI 中作为主要 GI 发挥作用。
