Endocytosis Of CNGC2 And Its Role In Disease Resistance In Arabidopsis

In the process of evolution,organisms have formed complex nutrient absorption and transport systems including ion channels,ion pumps and carriers.In the early days,scholars have done a lot of work to understand the molecular properties and functions of these channel proteins.In recent years,experiments based on electrophysiology and heterologous expression analysis have demonstrated that the role of the cyclic nucleotide-gated channel(CNGC)is a non-selective cation channel.Plant CNGCs are a relatively large family of genes,and the presence of CNGC has been found in some plants such as Arabidopsis thaliana,Hordeum vulgrare,Oryza sativa,and Zeamays.Their function is not fully understood.CNGC2,as a member of the IV subgroup B of the plant CNGCs gene family,is currently lacking in the study of the endocytic process of this protein,and further research is needed on whether it participates in plant disease resistance.In this study,the Arabidopsis CNGC2 gene was cloned andan endogenous promoter-driven GFP-tagged CNGC2 fusion expression vector was constructed to obtain CNGC2-GFP transgenic Arabidopsis plants.Using variable angle-total internal reflection fluorescence microscopy(VA-TIRFM),fluorescence lifetime and fluorescence resonance energy transfer(FLIM-FRET),fluorescence correlation spectroscopy(FCS),fluorescence coss-correlation spectroscopy(FCCS)techniques in combination with molecular biology methods,studied the distribution of Arabidopsis thaliana CNGC2-GFP on the plasma membrane,exercise patterns,endocytosis,and the relationship between CNGC2 and plant resistance.The main results are as follows:(1)Examination of callose deposition using aniline blue staining revealed that callose deposition was increased in the mutant cngc2 compared with wild-type after activation with lipopolysaccharide(LPS)treatment.Based on the results of this experiment,it was speculated that CNGC2 was associated with plant disease resistance.(2)Using VA-TIRFM technology,the dynamic characteristics of CNGC2 in Arabidopsis seedling leaves were analyzed.The results showed that CNGC2 was unevenly distributed on the plasma membrane.Using MATLAB software analysis,it was further found that CNGC2 had four motion patterns,among which orientation is the main mode of exercise.(3)In the case of cycloheximid(CHX)inhibiting intracellular new protein synthesis,treatment of Arabidopsis seedlings with brefeldin A(BFA)in combination with FM4-64 revealed that CNGC2-GFP formed BFA bodies in the cytoplasm.It indicates that there is constitutive endocytosis of CNGC2 at resting state,and CNGC2 has an endocytic process.After treatment with lipopolysaccharide(LPS),endocytic vesicles appeared in Arabidopsis epidermal cells,and the density of CNGC2 protein on the cell membrane was decreased by FCS.It indicated that the activated CNGC2 protein accelerated in the activated state,and ligand-induced endocytosis existed in CNGC2.(4)Inhibitor treatment experiments demonstrated that clathrin is involved in the regulation of the endocytic process of CNGC2.The clathrin inhibitor TyrA23 inhibited endocytosis and protein degradation of CNGC2.It was was further revealed that the membrane-mediated endocytic pathway is involved in the endocytosis of CNGC2.Treatment of Arabidopsis seedlings with sterol extraction reagent M?CD(methyl-?-cyclodextrin)uncovered that M?CD caused inhibition of CNGC2-GFP endocytosis,suggesting that sterol-rich membrane structure is involved in CNGC2 endocytosis.(5)Using the non-invasive micro-test technique(NMT),the Ca2+ flow in the overexpressing CNGC2,WT and cngc2 was detected after activation of LPS treatment.Subsequently,the expression of disease-resistant genes after LPS treatment was detected for wild-type and mutants,respectively.The results showed that the mutants showed a decrease in Ca2+internal flow and an up-regulation of disease-related gene expression.In addition,the interaction between CNGC2-GFP and CaM-mCherry can be detected by combining FCCS technology and FLIM-FRET technology,and the interaction between the two is enhanced after LPS treatment.The above results indicate that CNGC2-GFP is involved in plant disease resistance.In summary,this study used high resolution fluorescence imaging technology combined with molecular biology research methods to analyze the dynamic characteristics and endocytosis of CNGC2 protein.Studies have revealed that the movement of CNGC2 protein on the plasma membrane is highly dynamic;CNGC2 protein has multiple endocytic pathways,and the clathrin-mediated endocytic pathway plays an important role in the endocytic process of CNGC2-GFP,and the membrane microdomains is also involved in the endocytosis of CNGC2-GFP on the plasma membrane;studies on the relationship between Ca2+flux,resistance-related genes,and the relationship between CNGC2 protein and calmodulin protein CaM4 indicate that CNGC2 is involved in plant disease resistance.These results lay the foundation for further revealing the endocytic pathway of CNGC2 and elucidating the mechanism by which CNGC2 participates in plant disease resistance.