Mechanism Of Involvement Of LecRK Ⅵ.2 Endocytosis In Plant Immune Response

Plant receptor like kinases are a group of structurally conserved proteins,which play an important role in plant immune response and signal transduction.With the application of the broad-spectrum inhibitors of endocytosis,endocytosis-related mutant,and various imaging technologies,the dynamics and endocytosis of receptor like kinases during immune process as well as its role in regulating plant immune response have been reported.However,researches on targeted inhibition of a specific protein endocytosis are still limited.Lectin like receptor kinase VI.2（Lec RK VI.2）is an important member of receptor like kinase involved in disease resistance response.It participates in PTI-mediated mitogen-activated protein kinase（MAPK）activity,callose deposition and gene expression upon treatment with the MAMP flagellin.However,its involvement in the early response of the immune response is unclear.In this study,Lec RK VI.2-EGFP fusion gene vector driven by native and 35S promoter was constructed respectively,and the transgenic plants were obtained.The subsequent functional complementation experiment indicated that Lec RK VI.2-EGFP is functional.On this basis,using laser scanning confocal microscope（LSCM）,variable angle total internal reflection fluorescence microscope（VA-TIRFM）,fluorescence correlation spectroscopy（FCS）and other technologies,combined with multi-disciplinary approaches such as biochemistry and molecular biology,genetics and cell biology,the dynamics and endocytosis of Lec RK VI.2 on the plasma membrane and its response to immune stimulation were analyzed at the single molecule level in living cells.Moreover,based on the site-directed mutation of its endocytosis sorting motif YXXΦ,the transgenic mutant plants expressing site substitution Lec RK VI.2-EGFP fusion proteins were obtained.The endocytosis and the downstream immune response were further studied.The main results are as follows:（1）Observations of transgenic seedlings expressing Lec RK VI.2-EGFP using confocal microscopy revealed that Lec RK VI.2 was widely distributed in various organs of Arabidopsis seedlings.The subcellular localization of Lec RK VI.2 is mainly concentrated at PM,which is unevenly distributed.（2）Single-molecule analysis of Lec RK VI.2 was performed with VA-TIRFM to track the trajectories of Lec RK VI.2 particles over a period of time at PM.Combined with the analysis of dynamic characters of Lec RK VI.2 with or without flg22,it was found that the motion range and diffusion coefficients of Lec RK VI.2 particles increased upon flg22 treatment,and the dwell time was decreased.Detection of the density of Lec RK VI.2 using FCS showed that the density of Lec RK VI.2at PM decreased upon flg22 treatments.Meanwhile,the fluorescence cross-correlation spectroscopy（FCCS）and protein proximity index（PPI）analysis revealed that the PPI value and relative cross-correlation values of Lec RK VI.2-EGFP and the membrane protein REM1.3-m Cherry decreased.Combined with the biochemical analysis which showed that the proportion of Lec RK VI.2 in organellar membrane increased,all the results indicated that Lec RK VI.2 internalized into cytoplasm after flg22 treatment.（3）Observations of BFA incubated CHX pre-treated transgenic seedlings expressing Lec RK VI.2 showed that Lec RK VI.2 accumulated in BFA bodies,illustrating that Lec RK VI.2 undergoes constitutive endocytosis in the steady state.Moreover,after flg22 treatment,the number of endocytic Lec RK VI.2 spots and the diameter of Lec RK VI.2 enriched bodies increased significantly,indicating that the existence of flg22-induced endocytosis of Lec RK VI.2.（4）Dulo-color VA-TIRFM analysis revealed that partial Lec RK VI.2-EGFP particles colocalized with CLC-m Cherry spots.Mass spectroscopy detection found that Lec RK VI.2 complex contains some clathrin mediated endocytosis（CME）related proteins such as CHC.In addition,the use of specific inhibitor of CME,Tyrphostin A23（Tyr A23）dampened the endocytosis of Lec RK VI.2.Hence,Lec RK VI.2 could internalize through CME pathway.（5）By sequence analysis,two putative canonical Tyr-based endocytic motifs were found in the cytoplasmic domain of Lec RK VI.2.Using site-directed mutation,we obtained transgenic Arabidopsis with the Tyr-to-Phe and Tyr-to-Ala substitution in Y³⁵⁷RDL and Y⁴⁴⁰DYI of Lec RK VI.2.The Tyr substitution in Y³⁵⁷RDL resulted in BFA insensitive endocytosis,and BFA insensitive vesicles increased after flg22 treatment.Meanwhile,the total Y³⁵⁷RDL mutant protein and Lec RK VI.2 protein displayed a degradation trend unpon flg22 treatment.Combined with the result of the protein levels in plasma membrane and organellar membrane,as well as the co-localization with TGN marker,it was considered that Y³⁵⁷RDL mutation hindered the endocytosis and its trafficking to plasma membrane.Moreover,the Tyr substitution in Y⁴⁴⁰DYI caused a weakened endocytosis of of Lec RK VI.2.（6）Upon flg22 treatment,Y³⁵⁷RDL and Y⁴⁴⁰DYI mutant proteins still activated mitogen-activated protein kinase（MAPK）activity,which is an early response to PTI.However,the PTI-response transcript NHL10 in Lec RK VI.2Y^357A-EGFP transgenic Arabidopsis was similar to the control,while the Tyr-to-Phe in Y⁴⁴⁰DYI resulted in an accumulation of NHL10.Combining the effects of these two YXXΦmutation on the endocytosis,it was suggested that the endocytosis and trafficking of Lec RK VI.2 participated in the plant immune response.Taken together,this study revealed the function of YXXΦmotif of Lecrk VI.2 during its endocytosis,deepened the mechanism of Lec RK VI.2 endocytosis involved in plant immune response.The results laied a foundation for elucidating the regulatory mechanism of endocytosis on membrane protein function.