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

Songmei Shi,  Qiguo Gao,  Jing Zeng,  Xiaohuan Liu,  Quanming Pu,  Guixi Liu,  Hecui Zhang,  Xiaohong Yang and  Liquan Zhu

Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education/College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China

Acta Biochim Biophys Sin 2016, 48: 777–787; doi: 10.1093/abbs/gmw075

Self-incompatibility (SI) is an important mating system to prevent inbreeding and promote outcrossing. ARC1 and Exo70A1 function as the downstream targets of the S-locus receptor kinase and play conservative roles in Brassica SI signaling. Based on the sequence homology, Exo70A1 is divided into four subdomains: leucine zipper (Leu128-Leu149), hypervariable region (Ser172-Leu197), SUMO modification motif (Glu260-Ile275), and pfamExo70 domain (His271-Phe627). ARC1 contains four domains as follows: leucine zipper (Leu116-Leu137), coiled-coil domain (Thr210-Val236), U-box (Asp282-Trp347) motif, and ARM (Ala415-Thr611) domain. Bioinformatics analysis, yeast two-hybrid screening and pull-down assays show that leucine zipper and coiled-coil motifs of ARC1116–236 are required for the interaction with Exo70A1, while the addition of ARM motif results in loss of the interaction with Exo70A1. Meanwhile, the N-terminal of Exo70A1 without any domains shows a weak interaction with ARC1, and the level of LacZ expression increases with addition of leucine zipper and reaches the maximum value with hypervariable region and SUMO modification motif, indicating that hypervariable region and SUMO modification motif of Exo70A1172–275 is mainly responsible for the binding with ARC1, whereas pfamExo70 domain has little affinity for ARC1. Lys181 located in the Exo70A1 hypervariable region may be the ubiquitination site mediating the interaction between ARC1 and Exo70A1. Therefore, both the leucine zipper with coiled-coil structure of ARC1116–236, and the hypervariable region and SUMO modification motif of Exo70A1172–275 are the core interaction domains between ARC1 and Exo70A1. Any factors affecting these core domains would be the regulators of ARC1 mediating ubiquitin degradation in self-incompatible system.

The interaction domains of ARC1-Exo70A1 were detected in the yeast two-hybrid system

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