The Evolution of Selfing in Arabidopsis thaliana

Tang et al., 2007

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

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

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

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

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

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

Specificity determinants and diversification of the Brassica self-

incompatibility pollen ligand Chookajorn et al., 2004

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

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

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

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

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

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

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