Characterization and Functional Analysis of NET3C and VAP27-1 Interacting Proteins in Plants

In plants, the cortical ER network connects to the plasma membrane via the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins (e. g. NET3C, VAP27-1 and SYT1) and the cytoskeleton. However, little is known about the mechanism of regulation of the cytoskeleton at the plant EPCS, and its biological functions remain poorly understood. From previous protein-protein interaction screens, hundreds of candidate proteins that potentially interact with NET3C or VAP27-1 were identified, and three proteins, KLCR1, AtBRO1 and SINE2 were selected for further study. Firstly, microtubule binding proteins, KLCR1 and IQDs were identified to interact with NET3C and form an intermediate component of the plant EPCS. The NET3C-KLCR1-IQDs protein complex mediates links between actin filaments and microtubules, and their overexpression enhances their association. The expressions of NET3C and IQDs also have a direct influence to the ER morphology, with more ER polygonal and cisternal structures formed when their expression level is high. The loss of function of KLCR1 results in disorganization of both actin filaments and microtubules, which causes left handed helical growth of roots and defects in the shape of cotyledon pavement cells. AtBRO1 is another protein that has been confirmed to interact with NET3C. It localized to punctate structures and associated with the ER network and actin filaments in N. benthamiana leaf epidermal cells. Reverse-genetic analyses suggested the knockout AtBRO1 expression is lethal, and defective development was observed in about a quarter of seeds in siliques from heterozygous plants. Additionally, we also identified a protein complex which is comprised of VAP27-1, NET3A and SINE2. This protein complex links the nuclear envelope with the ER network and actin filaments and is likely to regulate the morphology of ER network and nuclear envelope during mitosis in plants. In summary, our results revealed novel NET3C and VAP27-1 complexes that are important for the ER morphology, cytoskeleton structures and plant development