Identification of one of the efficient motifs on the stability of ATG8B protein in LDS in Marchantia polymorpha

Autophagy is one of the defensive response pathways under stress and non-stress conditions in different organisms. In autophagy, damaged compounds and organelles are sealed into double-membrane vesicles as an autophagosome. Autophagosome has been directed to the vacuole to be degraded by hydrolytic enzymes into initial substances. ATG8 proteins are the hallmark of this pathway. These proteins are involved in autophagosome formation and target cargo location inside the autophagosome. In selective autophagy, cargo receptors play an important role in trapping the cargo inside the autophagosome. There is the ATG8 binding motif called AIM or LIR in Cargo receptors. This motif makes able the cargo receptors interact with LDS, LIR docking site in ATG8. Any modification in the AIM motif or LDS could abolish the interaction between ATG8 and cargo receptors. In this study, ATG8B mutant in LDS has been generated in Marchantia polymorpha for future studies to identify the possible cargo receptors. In this order, the protein sequence of ATG8B has been identified in the marchntia.info database. Aligning ATG8A from Arabidopsis with ATG8B from Marchantia leads to identifying the residues involving in ATG8B folding and LDS formation. Two residues, Lysin 51 and Arginine 70, have been replaced with Alanine residue by site-directed mutagenesis. The GreenGate has been used for fusing the GFP to ATG8B mutant fragments. To consider the same condition with wild-type ATG8B lines presented in the lab, by using the Gateway adapted primers, ATG8B-LDS mutant has been cloned in the expression vector, pMpGWB303. After transforming to Agrobacterium GWB303+pSOUP strain, it has been transformed to the plants. Analyzing the transformed plants in western blot experiments has shown that the only form of modification that can express and fold as a stable protein is Lysine to Alanine modification, however, in the proteins with the mutation of Arginine to Alanine, protein is not stable, and it cannot be expressed.