Investigating a role forPUB17andPUB16in the self-incompatibility pathway in transgenicArabidopsis thaliana

Abstract

In Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by the S-haplotype specific interactions between the pollen SCR/SP11 ligand and the stigma S Receptor kinase (SRK). In Brassica SI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARC1, is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A. thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between accessions, and ARC1 is not always needed to produce a strong SI response. For A. thaliana C24, only transforming with A. lyrata SCR and SRK confers a strong SI trait, and so here we investigated if ARC1-related PUBs were involved in the SI pathway. Two close ARC1 paralogs, PUB17 and PUB16, were selected, and CRISPR/Cas9 technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into a transgenic A. thaliana SI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences to implicate PUB17 and PUB16 functioning in the transgenic A. thaliana SI-C24 stigma to reject SI pollen.