Arabidopsis Reticulons Inhibit ROOT HAIR DEFECTIVE3 to Form a Stable Tubular Endoplasmic Reticulum Network.

The endoplasmic reticulum (ER) is a network of interconnected tubules and sheets stretching throughout the cytoplasm of plant cells. In Arabidopsis (Arabidopsis thaliana), ROOT HAIR DEFECTIVE3 (RHD3) mediates ER tubule fusion, while reticulon proteins induce ER membrane curvature to produce ER tubules. However, it is unclear if and how RHD3-reticulon interplay during the formation of the interconnected tubular ER network. We discovered that RHD3 physically interacts with Arabidopsis reticulon proteins, including reticulon-like protein subfamily B3 (RTNLB3), on ER tubules and at three-way junctions of the ER. The RTNLB3 protein is widely expressed in Arabidopsis seedlings and localizes to ER tubules. Although the growth of knockout rtnlb3 mutant plants was relatively normal, root hairs of rtnlb3 were shorter than those of wild type. The ER in mature mutant cells was also more sheeted than that in wild type. rhd3 is known to have short roots and root hairs and less branched ER tubules in cells. Interestingly, rtnlb3 genetically antagonizes rhd3 in plant root development and in ER interconnectivity. We show that reticulons including RTNLB3 inhibit the ER fusion activity of RHD3, partly by interfering with RHD3 dimerization. We conclude that reticulon proteins negatively regulate RHD3 to balance its ER fusion activity for the formation of a stable tubular ER network in plant cell growth.