Luciferase Complementation Imaging Assay Of Autophagy-Related Genes In Arabidopsis

Autophagy plays a greater variety of physiological roles in plants. Atg8 is an important protein in autophagy, and nowadays, Atg8-GFP fusion protein is useful in monitoring autophagy. In the previous study,35 proteins which interacted with AtAtg8e were gained, using yeast-two-hybrid assay to screen the cDNA library of Arabidopsis.In this study, luciferase complementation imaging assay (LCI assay) was used to test these protein-AtAtg8 interactions in Nicotiana benthamiana. First,37 N-LUC constructs were generated, including 35 candidates and AtAtg4a, AtAtg4b, consisting with the 9 C-LUC clones of AtAtg8 gene family. Then the LCI experiments were done. Among all the 35 candidate proteins, the fluorescence for two proteins, E58 and E121, was very intense; while for seven proteins, E13, E16, E20, E52, E89, E116 and E134, showed less intense, but others seemed to be slight or had no signal. These results indicated the two proteins, E58 and E121, had significant interaction with Atg8e; E13,E16, E20, E52, E89, E1 16 and E134, had strong interaction with AtAtg8e. E58 and E121, also interacted with other AtAtg8 proteins. It indicated that these AtAtg8 proteins might have different functions.