The Role Of Clathrin Light Chain-mediated Endocytosis In Arabidopsis Defense Response

Clathrin-mediated endocytosis（CME）is the major route of internalization of plasmamembrane-localized proteins as well as the extracelluar materials,and plays an important role in signal transduction.Clathrin is composed of three clathrin heavy chain（CHC）subunits and three light chain（Cl C）subunits,forming a triskelion structure.There are three genes encoding light chain（CLC1,CLC2 and CLC3）and two genes encoding heavy chain（CHC1 and CHC2）in the Arabidopsis genome.Although there have been reports of the involvement of CME in plant immune responses,the relationship between CLCs and plant defense responses has not been reported previously.On the basis of the occurrance of the spontaneous cell death and premature senescence on the leaves of clc2clc3 double mutant plants in the absence of pathogen attacks,we provided evidence that the defense responses were constitutively activated in the clc2clc3 plants using combined approaches of genetics,molecular biology and cell biology.The molecular mechanism of this constitutive activated defense responses were further explored.We found that:（1）All the hallmarks reflecting the constitutive activation of the defense responses were observed in the clc2clc3 plants,including excessive accumulation of free salicylic acid（SA）and hydrogen peroxide（H₂O₂）,significantly induced expression of pathogenesis-related genes（PR）,increased callose deposition,enhanced sensitivity of stomatal closure in response to flg22 and ABA,and the heightened resistance to（Pseudomonas syrangae pv.DC 3000,Pst DC3000）.Excessive accumulation of SA and H₂O₂was most likely the main cause of activated defense responses and the spontaneous cell death;Consistent with our hypothesis,the genetic analysis indicated that loss-of-function mutations in genes either encoding ICS1/SID2 or Rboh D,two key enzymes responsible for SA biosynthesis and apoplast H₂O₂production,respectively,could successfully rescue the constitutive activated defense responses observed in the clc2clc3 mutant plants.These results indicated that the concomitant presence of both SA and H₂O₂is essential for the constitutively activated defenses responses;（3）The Pro FLS2::FLS2-GFP was introduced into the clc2clc3 background through a cross between clc2clc3 mutant and a transgenic line expressing the Pro FLS2::FLS2-GFP in Col-0 background.The endocytosis of FLS2-GFP induced by flg22 in both Col-0 and the clc2clc3 background was observed under a confocal microscopy.The results showed that the flg22-induced endocytosis of FLS2-GFP was significantly compromised in clc2clc3 mutant relative to Col-0,indicating that CLC2 and CLC3play critical roles in the endocytosis of the plasmamembrane-localized receptor kinase.Based on the published results from others and the results we generated,an H₂O₂-SA positive feedforward model was proposed,which can explain the constitutive activated defense responses in clc2clc3 mutant.