1. The Evolution of Selfing in Arabidopsis thaliana Tang et al., 2007

2. Evolution of flowering plants  Transition from outcrossing to selfing  Occurred independently in numerous lineages  Important for colonization  A. thaliana highly selfing  Closest relative A. lyrata (self-incompatible)  Separated by ~5 million years

3. Role of the S locus in the transition to selfing  Self-incompatibility (SI) locus (S) tightly linked main components:  S-locus receptor kinase (SRK) Encodes female specificity determinants of SI  S-locus cysteine-rich protein (SCR) Encodes male specificity determinants of SI Pollen ligand for SRK

4. SI Model in Brassicaceae  SCR is a ligand for SRK  In self-pollination, SCR protein delivered to stigma, binds to ectodomain of “self” SRK, activates SRK kinase  Triggers signal transduction pathway resulting in inhibition of fertilization  In cross-pollination, SCR protein cannot bind or activate “non-self” SRK  SRK and SCR genes must coevolve to maintain SI  Generation of novel SI specificity requires compensatory mutations in receptor and ligand of same haplotype, so SRK-SCR binding is maintained

5. S-locus characterized by highly divergent haplotypes  SI system in A. thaliana inactivated (SCR and SRK are psuedogenes in Col-0)  Inactivation of S-locus could be key step in evolution Transformation with A. lyrata S-locus alleles restores SI in A. thaliana  Should show reduced variability compared to ancestral locus if inactivation only occurred once

6. Investigating variation at S-locus  PCR of SCR and SRK - failed  Whole-genome resequencing data for 20 accessions with oligonucleotide arrays Designed based on reference sequence Many accessions failed to hybridize  Dideoxy-sequenced BACs of S-locus from 2 accessions C24 Cvi-0

7. Conclusions  Found high variation at S-locus allele  Ancestral balanced polymorphism at S-locus gradually eroding through genetic drift  Selection for inactivity  Multiple evolutionary routes to selfing Transformation with A. lyrata SI alleles does not always restore SI  Species-wide selfing evolved 1 m.y.a. or more

8. Specificity determinants and diversification of the Brassica self- incompatibility pollen ligand Chookajorn et al., 2004

9. Research goals  Use ligands from two haplotypes for structure- function studies of SCR  SCR6 sequence variants made by swapping specific domains between SCR6 and SCR13 variants by in vitro mutagenesis  Used variants to map sequences that determine recognition specificity in SCR  Asses if residues largely conserved in SCRs are important for SCR6 function  Relate results to hypotheses for the evolution of new SI specificities

10. Predicted conservation of overall structure among highly diverged SCR variants  Few residues conserved between most variants  8 cysteines (Cys-1 through Cys-8)  Gly-12 in GlyxCys-2 motif  Tyr-26 in Cys-3xxxTyr/Phe motif

11.  Generated structural models of several SCR variants  found to fit a similar fold recognition despite extreme sequence variance Predicted conservation of overall structure among highly diverged SCR variants

12. Identification of SCR specificity determinants by domain swapping  Regions between Cys-3 and Cys-4; Cys-5 and Cys- 6 are candidate specificity determinants - predicted to be surface-exposed  Generated SCR6-SCR13 chimeras by exchanging various domains  Only SCR6 chimera (5-6) showed modified specificity  Inactive on SCR6 stigmas  Activated SI on S13 stigmas  Four specificity determinants: first 4 residues (TDTQ) from SCR13 C5-C6 region

13. Accessing the flexibility of SCR6 protein  Predict that because the 4 specificity residues from SCR13 function in SCR6 backbone, SCR6 may tolerate many mutations  Generated variants of SCR6 by alanine-scanning mutagenesis of C3-C4 and C5-C6 regions  Substituted conserved residues with Alanine  Found Tyr-26 (Y to A mutation) to be required for SCR6 function  Mutant failed to activate SI on S6 stigmas despite high binding affinity  Uncoupling of SCR binding to SRK from its ability to activate SI response

14. Conclusions  Because of evolutionary adaptability of SCR and the inferred flexibility of SCR-SRK interaction:  Hypothesize that new SI specificities generated through SI intermediates by process that changes pollen and stigma components of S locus haplotype, but preserves allelic recognition  New mutations in SCR favored if they increase activation of SRK allele, or if they reduce false activation of the mismatching SRK alleles

15. Model for generation of new SI specificities through self- incompatible intermediates  Within each functional haplotype, variability in SCR and SRK is tolerated and mutual recognition exists  If, in a subset of alleles (*), SCR and SRK show stronger affinity with each other than with corresponding proteins, then natural selection drives the strengthening of SRK-SCR and SRK*- SCR* interactions  Leads to origination of a novel allele without producing any SC intermediates