Analyses of in vitro protein-protein interaction of ACT7 or ACT2 and AIPI-2 from Arabidopsis thaliana and in vivo analysis of AIPI-2 expression

Abstract

Cell polarity is defined as the polar organization of cellular components along an axis. The coordination of cell polarity within a plane of a single tissue layer is termed planar polarity. An example for planar polarity in plants is the hair formation towards the lower (basal) ends of hair-forming cells in the root epidermis of Arabidopsis thaliana. The ACTIN2 (ACT2) and ACT7 genes are involved in the establishment of planar polarity in the root epidermis. Actin filaments are dynamic structures that undergo a constant turnover including polymerization and depolymerization. In presence of actin-interacting protein 1 (AIP1), depolymerization of actin filaments mediated by actin-depolymerizing factor (ADF/Cofilin) is dramatically increased. In comparison to many other organisms, Arabidopsis encodes two AIP1 copies. AIP1-1 is expressed in reproductive tissues and AIP1-2 in all tissues. Despite its conservation throughout eukaryotes, relatively little is known about the developmental roles of AIP1. Here, we investigate the interaction of Arabidopsis AIP1-2 and actin isofoms and their role during planar polarity. To this end, we used in vitro glutathione-S-transferase (GST) pull-down approaches to study the interactions of AIP1s and actins. These revealed that both recombinant GST-AIP1-1 and GST-AIP1-2 purified from E. coli extracts precipitated actins from Arabidopsis root cell suspension protein extracts. This indicated that Arabidopsis AIP1 homologs are able to interact with actins at the protein level. Furthermore, GST-AIP1-2 precipitated bacterially expressed 6xHistidine (6xHis)-tagged ACT2 (6xHis-ACT2) and 6xHis-ACT7. This strongly suggested that the interaction does not strictly require other plant proteins. Moreover, we generated transgenic lines expressing up to two copies of native AIP1-2, two copies of AIP1-2-Venus and two copies of AIP1-2-mCherry to analyze the effect of AIP1-2 overexpression on root hair positioning. In preliminary analyses, it appeared that root hairs in lines carrying these six copies of AIP1-2 were shifted more apically than in the wild type indicating AIP1-2 as a regulator of planar polarity. Transgenic lines expressing an ER-resident mCherry construct under control of the native AIP1-2 5‟ and 3‟ genomic regions were generated to analyze the AIP1-2 expression pattern in the root. Highest promoter activity was observed in hair cell files, suggesting that cell file-specific enrichment of AIP1-2 protein did, at least partially, depend on cell file-specific promoter activity. Promoter activity was detected from the elongation zone onwards indicating that AIP1-2

2 transcript is not expressed in the meristem. Taken together, this study shows that Arabidopsis AIP1s interact with actins, and that interaction of AIP1-2 with ACT7 and ACT2 does not require other plant proteins in vitro. AIP1-2 is transcriptionally upregulated in hair cell files and its overexpression may shift root hair positioning apically, suggesting a regulatory role for AIP1-2 in planar polarit