Identification and characterization of BoPUB3: a novel interaction protein with S-locus receptor kinase in Brassica oleracea L

Abstract

Armadillo repeat containing 1 (ARC1) is phosphorylated by S-locus receptor kinase (SRK) and functions as a positive regulator in self-incompatibility response of Brassica. However, ARC1 only causes partial breakdown of the self-incompatibility response, and other SRK downstream factors may also participate in the self-incompatibility signaling pathway. In the present study, to search for SRK downstream targets, a plant U-box protein 3 (BoPUB3) was identified from the stigma of Brassica oleracea L. BoPUB3 was highly expressed in the stigma, and its expression was increased with the stigma development and reached to the highest level in the mature-stage stigma. BoPUB3, a 76.8-kDa protein with 697 amino acids, is a member of the PUB-ARM family and contains three domain characteristics of BoARC1, including a U-box N-terminal domain, a U-box motif, and a C-terminal arm repeat domain. The phylogenic tree showed that BoPUB3 was close to BoARC1. The synteny analysis revealed that B. oleracea chromosomal region containing BoPUB3 had high synteny with the Arabidopsis thaliana chromosomal region containing AtPUB3 (At3G54790). In addition, the subcellular localization analysis showed that BoPUB3 primarily localized in the plasma membrane and also in the cytoplasm. The combination of the yeast two-hybrid and in vitro binding assay showed that both BoPUB3 and BoARC1 could interact with SRK kinase domain, and SRK showed much higher level of β-galactosidase activity in its interaction with BoPUB3 than with BoARC1. These results implied that BoPUB3 is a novel interactor with SRK, which lays a basis for further research on whether PUB3 participates in the self-incompatibility signaling pathway.

Keywords: S-locus receptor kinase; expression analysis; gene cloning; protein interaction; subcellular localization; synteny analysis.