Identification of Interacting Motifs Between Armadillo Repeat Containing 1 (ARC1) and Exocyst 70 A1 (Exo70A1) Proteins in Brassica oleracea

Abstract

In order to identify the functional domains which regulate the interaction between the self-incompatibility proteins armadillo repeat containing 1 (ARC1) and exocyst 70 A1 (Exo70A1) in Brassica oleracea, fragments containing selected motifs of ARC1 (ARC1210, ARC1246, ARC1279, ARC1354) and site-specific mutants with substitutions at possible interaction sites (ARC1354m, ARC1664m) were PCR amplified and inserted into pGADT7, while coding sequences from Exo70A1 (Exo70A185, Exo70A1) were subcloned into pGBKT7. The interactions between the protein products produced by these constructs were then analyzed utilizing a yeast two-hybrid system. Our data indicate that both ARC1210 and ARC1246 interact strongly with Exo70A185 and Exo70A1, while ARC1279, ARC1354, ARC1354m and ARC1664m exhibited a weak interaction, indicating that the recognition sites are located within the 210 N-terminal amino acids of ARC1 and the 85 N-terminal amino acids of Exo70A1. This was further verified by GST pull-down analysis. This supports a model in which the N-terminal leucine zipper of ARC1 and the first 85 N-terminal amino acids of Exo70A1 mediate the interaction between these two proteins. Bioinformatic and phylogenetic analysis demonstrated that these motifs were highly conserved across different species, indicating that the interaction characterized in B. oleracea may operate in a wide array of cultivars.

N-terminal domains of ARC1 are essential for interaction with the N-terminal region of Exo70A1 in transducing self-incompatibility of <italic>Brassica oleracea</italic>

“…7C,D). These were consistent with the previous findings that binding region with Exo70A1 was located at the N-terminal of ARC1 [14,[23][24][25], and the ARM domain mainly mediated the interaction with receptor kinase by phosphorylation-dependent [7,42]. The full-length ARC1 contained an NLS (amino acids 260 to 266), and two NESs (amino acids 286-300 and 328-347).…”

“…Answers to these questions could improve our understanding of the molecular mechanism of ARC1 mediating ubiquitin degradation in the plant SI system. We have recently shown that the N-terminal of ARC1 was sufficient for its interaction with Exo70A1 by the yeast two-hybrid system [23][24][25].…”

EbARC1, an E3 Ubiquitin Ligase Gene in Erigeron breviscapus, Confers Self-Incompatibility in Transgenic Arabidopsis thaliana

“…Further analysis showed that EbARC1 interacted with Exo70A1 ( Figure 6). The same study in Brassica also indicated that ARC1 interacts with Exo70A1, and the ARC1-Ubox domain and the N-terminal of Exo70A1 are the key regions for the interaction [16,39,40], which is consistent with our results ( Figure 6). Exo70A1 is a compatibility factor that is required for normal germination and growth of pollen [41].…”

The N-Terminal UND Motif of the Arabidopsis U-Box E3 Ligase PUB18 Is Critical for the Negative Regulation of ABA-Mediated Stomatal Movement and Determines Its Ubiquitination Specificity for Exocyst Subunit Exo70B1

“…Thus, the functional differentiation of PUB18 and PUB22 in response to ABA might result from the presence or absence of UND. Liu et al (2016) recently reported that the N-terminal domain of Brassica oleracea ARC1, the closest homolog of Arabidopsis PUB17, confers a binding specificity of ARC1 toward Exo70A1.…”