Single-Molecule Analysis Of PEPR1 Dynamics And Endocytosis In Living Arabidopsis Cells

The endogenous elicitor peptide Pepl receptor PEP-RECEPTOR1(PEPR1)plays fundamental roles in Pepl signaling in Arabidopsis thaliana,but the molecular mechanisms underlying the effects of Pepl on PEPR1 internalization and assembly state remain unclear.The present investigation used variable angle total internal reflection fluorescence microscopy(VA-TIRFM)and single particle tracking(SPT)to analyze the endocytosis and dynamics properties of EGFP-tagged PEPR1 in Arabidopsis thaliana.The main results are as follows:(1)Using laser scanning confocal microscope(LSCM)observation,it was revealed that PEPR1-EGFP was expressed in roots,hypocotyls and leaves.Furthermore,the plasmolysis experiment showed that PEPR1-EGFP was not localized at the cell wall.VA-TIRFM analysis showed the diffusion coefficients and motion ranges of PEPR1-EGFP particles in leaf epidermal cells significantly increased under Pepl treatmeent,indicating that the activation with Pep1 dramatically led to faster diffusion of PEPR1.(2)By means of VA-TIRFM and biochemistry,it was revealed that the clathrin mediated endocytosis acts in the internalization of PEPR1 proteins.To address the possible role of clathrin in PEPR1 endocytosis,we examined the effect of Tyrphostin A23(TyrA23),a specific inhibitor of the recruitment of endocytic cargo into the clathrin-mediated pathway.The endocytosis and dynamic changes revealed that internalization of PEPR1 can be affected by TyrA23,suggesting that the PEPR1 was internalized by clathrin mediated endocytosis.The results taken in endocytosis-defective chc2-1 mutants further supported this conclusion.Furthermore,the dual color lines expressing PEPR1-EGFP/CLC-mCherry were generated to further demonstrating that the internalization of PEPR1 is related to clathrin.Colocalization experiments revealed considerable overlap of PEPR1 with endosomal markers respectively,implying that PEPR1 are targeted to these endosomes.The inhibition of protein synthesis by cyclohexamide(CHX)did not interfere with the accumulation of PEPR1-EGFP in brefeldin A body,which colocalized well with FM4-64,illustrating that nonactivated PEPR1 was subjected to constitutive endocytic cycling.More importantly,when treated with pep1,the number of endosomes labeled with PEPR1-GFP and FM4-64 significantly increased in the cotyledon epidermal cells,suggesting that pepl increases the endocytosis of PEPR1.Colocalization experiments showed considerable overlap of PEPR1-EGFP with these markers respectively after Pepl treatment,suggesting that PEPR1 was transferred into EE/MVBs,which likely target the PEPR1 for vacuolar degradation.(3)Through dynamic behavior research and western blot analysis,it was demonstrated for the first time that membrane microdomains are involved in PEPR1 endocytosis.After treated with M?CD,the endocytosis of PEPR1-EGFP was inhibited the motion ranges and diffusion coefficients of PEPR1-EGFP particles were affected.These results suggested that disturbance of membrane microdomains by treatments with M?CD affects PEPR1-EGFP endocytosis.Moreover,in response to Pep1 stimulation,the colocalization of PEPR1-EGFP with AtFlot1-mCherry was increased significantly.In summary,we here provided three novel findings:the behavior of PEPR1 in the plasma membrane of living cells was highly heterogeneous.Clathrin-dependent endocytosis of PEPR1 was a major endocytic pathway and membrane microdomain-associated endocytic pathway was also involved in PEPR1 endocytosis.These results suggest that providing valuable information for further investigation of the regulatory mechanisms of PEPR1 in the plant defense response.