Use of Domain-Swapping to Identify Candidate Amino Acids Involved in Differential Interactions between Two Allelic Variants of Type-1 S-Locus F-Box Protein and S3-RNase in Petunia inflata
Use of Domain-Swapping to Identify Candidate Amino Acids Involved in Differential Interactions between Two Allelic Variants of Type-1 S-Locus F-Box Protein and S3-RNase in Petunia inflata
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DOI：
10.1093/pcp/pcx176
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发表时间：
2018-02
影响因子：
4.9
通讯作者：
Lihua Wu;Justin S. Williams;Ning Wang;Wasi A Khatri;Daniele San Rom[REPLACEMENT CHARACTER]n;T. Kao
中科院分区：
文献类型：
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作者：
Lihua Wu;Justin S. Williams;Ning Wang;Wasi A Khatri;Daniele San Rom[REPLACEMENT CHARACTER]n;T. Kao
膨胀矮牵牛具有自交不亲和（SI）机制，该机制涉及S-RNase和多态性S位点上的多个S位点F-box（SLF）基因。对于给定的 S 单倍型，每个 SLF 被认为与其一些非自身 S-RNase 相互作用，但不与其自身 S-RNase 相互作用。在这项工作中，我们研究了 SLF1、S2-SLF1 和 S3-SLF1 的等位基因对，它们有 44 个氨基酸不同，并且与 S3-RNase 表现出不同的相互作用。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与 S3-RNase 相互作用，每个嵌合蛋白的三个功能域之一都交换了。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 域中的 16 个。然后，我们通过将 FD3 分为两个子结构域和四个迷你结构域（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为微型结构域 A 中的 4 个和微型结构域 D 中的 4 个。S3-RNase 和 S2-SLF1 之间相互作用的分子模型显示，这八个中的三个位于相互作用表面，并且所有三个在 S1-SLF1 和 S6a-SLF1 中都是保守的，根据体内转基因测定，这两个氨基酸都与 S3-RNase 相互作用。使用三种嵌合蛋白进行体内转基因测定，以确定 FD3 是否单独含有 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。
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.