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 Ssub(3)-RNase in Petunia inflata
2018
Wu, L. (The Pennsylvania State University, PA (USA). Intercollege Graduate Degree Program in Plant Biology) | Williams, J.S. | Wang, N. | Khatri, W.A. | Román, D.S. | Kao, T.
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, Ssub(2)-SLF1 and Ssub(3)-SLF1, which differ in 44 amino acids and show differential interactions with Ssub(3)-RNase. We first used an in vivo transgenic assay to determine whether four chimeric proteins of Ssub(2)-SLF1 and Ssub(3)-SLF1, each with one of the three functional domains swapped, interact with Ssub(3)-RNase. The results narrowed the candidate amino acids for specific interaction of Ssub(2)-SLF1 with Ssub(3)-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 Ssub(3)-RNase and Ssub(2)-SLF1 revealed that three of these eight are at the interaction surface, and all three are conserved in Ssub(1)-SLF1 and Ssub(6a)-SLF1, both of which interact with Ssub(3)-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 Ssub(2)-SLF1 to interact with Ssub(7)-RNase and Ssub(13)-RNase. The results revealed the diversity and complexity of interactions between SLF proteins and S-RNases.
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