Intercollege Graduate Degree Program in Plant Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Plant Cell Physiol. 2018 Feb 1;59(2):234-247. doi: 10.1093/pcp/pcx176.
Petunia inflata possesses a self-incompatibility (SI) mechanism, which involves S-RNase and multiple S-locus F-box (SLF) genes at the polymorphic S-locus. For a given S-haplotype, each SLF is thought to interact with some of its non-self S-RNases, but not with its self S-RNase. In this work, we studied an allelic pair of SLF1, S2-SLF1 and S3-SLF1, which differ in 44 amino acids and show differential interactions with S3-RNase. We first used an in vivo transgenic assay to determine whether four chimeric proteins of S2-SLF1 and S3-SLF1, each with one of the three functional domains swapped, interact with S3-RNase. The results narrowed the candidate amino acids for specific interaction of S2-SLF1 with S3-RNase to the 16 in domain FD3. We then examined seven additional chimeric proteins by dividing FD3 into two subdomains and four mini-domains (A, B, C and D). The results further narrowed the candidate amino acids to four in mini-domain A and four in mini-domain D. Molecular modeling of interactions between S3-RNase and S2-SLF1 revealed that three of these eight are at the interaction surface, and all three are conserved in S1-SLF1 and S6a-SLF1, both of which interact with S3-RNase based on the in vivo transgenic assay. Three of the chimeric proteins were used for the in vivo transgenic assay to determine whether FD3 alone contains the amino acids required for S2-SLF1 to interact with S7-RNase and S13-RNase. The results revealed the diversity and complexity of interactions between SLF proteins and S-RNases.
喇叭水仙具有自交不亲和（SI）机制，该机制涉及多态 S 座上的 S-RNase 和多个 S 座 F-box（SLF）基因。对于给定的 S 单倍型，每个 SLF 被认为与某些非自身 S-RNase 相互作用，但不与自身 S-RNase 相互作用。在这项工作中，我们研究了 SLF1 的等位基因对 S2-SLF1 和 S3-SLF1，它们在 44 个氨基酸上存在差异，并表现出与 S3-RNase 的不同相互作用。我们首先使用体内转基因测定来确定 S2-SLF1 和 S3-SLF1 的四个嵌合蛋白是否相互作用，每个嵌合蛋白具有三个功能域之一的交换。结果将 S2-SLF1 与 S3-RNase 特异性相互作用的候选氨基酸缩小到 FD3 中的 16 个。然后，我们通过将 FD3 分为两个亚域和四个小域（A、B、C 和 D）来检查另外七个嵌合蛋白。结果进一步将候选氨基酸缩小到 A 小域中的四个和 D 小域中的四个。S3-RNase 和 S2-SLF1 之间相互作用的分子建模表明，这八个中的三个位于相互作用表面上，并且所有三个都在 S1-SLF1 和 S6a-SLF1 中保守，根据体内转基因测定，这两种都与 S3-RNase 相互作用。使用三个嵌合蛋白进行体内转基因测定，以确定 FD3 本身是否包含 S2-SLF1 与 S7-RNase 和 S13-RNase 相互作用所需的氨基酸。结果揭示了 SLF 蛋白和 S-RNase 之间相互作用的多样性和复杂性。
