EHD1-positive endocytic PM-recycling vesicles contribute to cell plate formation and root development in arabidopsis

Recycling of internalized endocytic proteins to the plasma membrane (PM) occurs at the trans-Golgi network (TGN) in plant cells where the secretory and endocytic protein trafficking pathways converge. Little is known about the nature and identity of PM-recycling vesicles in plants. A family of proteins containing the Epidermal growth factor receptor substrate 15-Homology Domain (EHD) are markers of recycling, and EHD bearing tubules function in slow-recycling of internalized proteins from sorting endosome to the PM in animal cells. To define the PM-recycling pathway in plants, we cloned the Arabidopsis homolog of mammalian EHD1 and generated a green fluorescent protein (GFP)-fusion and expressed it in tobacco BY-2 cells and Arabidopsis plants. Cytosolic punctate structures labeled by GFP-EHD1 fusion or endogenous EHD1 are distinct from the Golgi apparatus and prevacuolar compartment (PVC), but partially colocalize with a TGN marker. EHD1-positive structures do not lie on the endocytic pathway as judged by FM4-64 labelling. Pharmacological studies revealed that Brefeldin A, but neither Concanamycin A nor Wortmannin, inhibits EHD1 PM-recycling. During interphase, EHD1 and a Rab11 homolog RabA1g partially colocalize as cytosolic punctae, but highlight the developing cell plate during meiosis. Using an inducible RNA interference (RNAi) approach, we showed that EHD1 knockdown by RNAi could hamper root development and cell plate formation in Arabidopsis