Interaction Between Two Self-Incompatible Signal Elements, EXO70A1 and ARC1

Abstract

ARC1 and EXO70A1 are important signal elements of self-incompatibility in Brassica. To characterize the interaction of ARC1-EXO70A1 during the course of self-incompatibility, the coding sequences of ARC1 and EXO70A1 were cloned from Brassica napus L. and B. oleracea L. var.acephala. Sequence analysis showed that ARC1 consisted of 663 amino acids in B. oleracea and 661 amino acids in B. napus, with a 45-amino-acid difference between them. Sequence alignment showed 95.9% similarity, with 93.9% exact match between BoARC1 and BnARC1. Only a 6-amino-acid difference was detected between BoEXO70A1 and BnEXO70A1, with 99.4% similarity and 98.9% exact match according to further sequence alignment. The homology between EXO70A1 alleles was higher than that between ARC1 alleles. Yeast 2-hybrid results indicated that a strong interaction existed between ARC1 and EXO70A1, which could activate the expressions of 4 reporter genes (ADE2, HIS3, AUR1-C, and MEL1) in diploid yeast. However, there was very weak interaction between EXO70A1 and a 316-C-terminal-deletion mutant of ARC1 (ARC1_N), which only activated the expressions from 3 reporter genes (ADE2, AUR1-C, and MEL1). This indicated that the interaction interface between ARC1 and EXO70A1 might not reside within the Armadillo (ARM) repeat domains of ARC1. The N-terminal domains of ARC1 play an essential role in the interaction of ARC1 with EXO70A1. The influence of the differences in amino-acid composition between BoARC1 and BnARC1 on the interaction between ARC1-EXO70A1 was not detected with a yeast 2-hybrid system, which may indicate that the binding interface between ARC1 and EXO70A1 was not altered by sequence differences between the 2 proteins in these Brassica species.