Dynamics and Endocytosis of Flot1 in Require CPI1 Function.

Membrane microdomains are nano-scale domains (10-200 nm) enriched in sterols and sphingolipids. They have many important biological functions, including vesicle transport, endocytosis, and pathogen invasion. A previous study reported that the membrane microdomain-associated protein Flotillin1 (Flot1) was involved in plant development in ; however, whether sterols affect the plant immunity conveyed by Flot1 is unknown. Here, we showed that the root length in sterol-deficient () mutants expressing Flot1 was significantly shorter than in control seedlings. The cotyledon epidermal cells in mutants expressing Flot1 were smaller than in controls. Moreover, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) and single-particle tracking (SPT) analysis demonstrated that the long-distance Flot1-GFP movement was decreased significantly in mutants compared with the control seedlings. Meanwhile, the value of the diffusion coefficient Ĝ was dramatically decreased in mutants after flagelin22 (flg22) treatment compared with the control seedlings, indicating that sterols affect the lateral mobility of Flot1-GFP within the plasma membrane. Importantly, using confocal microscopy, we determined that the endocytosis of Flot1-GFP was decreased in mutants, which was confirmed by fluorescence cross spectroscopy (FCS) analysis. Hence, these results demonstrate that sterol composition plays a critical role in the plant defense responses of Flot1.