Expression of recombinant S-locus F-box-S2 protein and computational modeling of protein interaction at the self-incompatibility locus of Rosaceae
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Self-incompatibility (SI) is a major mechanism that prevents inbreeding in ow-ering plants, which was identi ed in Rosaceae, Solanaceae and Scrophulariace. In these families, SI is gametophytic and retains inter-speci c genetic variations by out-crossing promotion. Self-incompatibility is genetically controlled by an S- locus where both male (pollen) and female (pistil) S-determinants are encoded. The female determinant (SRNase) has been extensively studied, whereas its male counterpart (SLF/SFB) has only recently been characterized as a pollen-expressed protein, which encodes for an F-box domain. However, the exact mechanism of in- teraction between SLF/SFB and SRNase is still largely unclear in Rosaceae. This study takes a closer look at the mechanism of self-incompatibility to gain a clearer understanding of the ligand-receptor binding mechanism of SI using molecular evolutionary analysis, structure prediction and binding speci city characteriza- tion, the outcome of which, will translate into a guideline for future studies. The major aims of this study were to derive an evolutionary pattern for GSI in Rosaceae subfamilies and to further assess the collaborative non-self recognition in Malus domestica Borkh.. The evolutionary analysis suggests a di erence in the evolution- ary pattern of Prunoideae and Maloideae S-genes, hence proposing a di erence in their GSI systems. Furthermore, sites responsible for this divergence are identi ed as critical amino acids in GSI function. To maintain GSI it is expected that the S-genes must be linked and co-evolve as a genetic unit. The results of this study show that these genes have co-existed, while SRNase have experienced a higher rate of evolution compared to SLF, thus rejecting the co-evolution of these genes in Maloideae. Furthermore, positively selected sites of S-locus pistil and pollen genes were identi ed that are likely to be responsible for speci city determination. Di erent numbers of these sites are found for both S-genes, while SRNase holds a larger number of positively selected sites. Additionally a model of speci city is introduced that supports the collaborative non-self recognition in Malus GSI, while critical sites responsible for such speci city are proposed and mapped to the predicted ancestral tertiary structure of SRNase and SLF/SFB. The identi cation of regions determining pollen pistil speci city as well as proposing a Collaborative Non-self Recognition model for Malus domestica Borkh. provide greater in-sight into how pollen-pistil communication system works in Maloideae (Rosaceae subfamily).