The Mechanism Of GDSL Lipase Regulating Tomato Immune Defense Against TMV Infection

GDSL lipase is a new member of the lipase family,which exists widely in animals,plants and microorganisms.It has a conserved GDSL amino acid sequence(Gly-Asp-Ser-Leu)at the N-terminal of the protein.The catalytic site is at the N-terminal,and Ser is the key catalytic residue.Different from other lipases,it is more flexible in structure for the active site of GDSL lipase.According to the substrate,the conformation of active site will change,which leads to different enzyme activity.At present,the research of GDSL lipase is mainly to reveal structure and physiological function.It is known that GDSL lipase is involved in plant growth and development,seed germination,organ morphogenesis,stress and metabolism.However,there are few studies on the defense response of GDSL lipase to virus,and it is unclear for the molecular mechanism of plant GDSL lipase against TMV infection.Tomato is used as the model system for the study of plant-pathogen interactions,growth and development.In this study,80 GDSL esterase/lipase family genes in tomato were identified and named SIGDSL1-80.These genes were mapped to their positions on chromosomes,and their physical and chemical properties,collinear relationship,gene structure,protein conservative domain,phylogenetic relationship and promoter cis-elements were analyzed.The results showed that the longest protein encode 1662 amino acids and the shortest 171 amino acids.The isoelectric point and molecular weight respectively are 4.529.49 and 19.40-182.73 kDa.Most of the proteins are stable,and approximately 3/4 protein is hydrophilic.Members of the SIGDSL family are divided into 3 subfamilies A,B and C,all of which contain four conserved motifs.Most of the genes contain SGNH plant lipase like domain and SGNH hydrolase superfamily domain,indicating that the family is highly conserved.Through phylogenetic tree analysis and quantitative real-time PCR detection,SIGDSL19 was found to be related to disease resistance of tomato.Therefore,we further studied the SIGDSL19 gene.Firstly,the full-length SIGDSL19 gene was successfully cloned from tomato by RT-PCR.As same as other GDSL lipase genes,SIGDSL19 contains four amino acid residues Ser(S),Gly(G),Asn(N)and His(H),which play an important role in enzyme catalysis.And the four conserved residues are located in four conserved domains Ⅰ,Ⅱ,Ⅲ and V,respectively.Subcellular localization analysis showed that the signal peptide was related to the localization of the protein,and it may play a role in the cell membrane and cytoplasm.It was found that the tissue-specific expression of SlGDSL19 was the highest in tomato leaves.Then SlGDSL19 protein and SlGDSL19 mutant protein with Ser as Ala were expressed and purified by prokaryotic expression system,and the lipase activity of SlGDSL19 was detected.It was found that SlGDSL19 is a kind of lipase and Ser plays a key role in lipase activity.In addition,we also prepared polyclonal antibodies against SlGDSL19 protein,which laid the foundation for the study about the molecular mechanism of SlGDSL19.Then the mechanism of SIGDSL19 regulating plant immune defense against TMV infection was studied by molecular biology methods.The results showed that it is increased for the accumulation of virus,ROS,electrical conductivity and MDA in the plants with SlGDSL19 protein and overexpression of SIGDSL19.And the expression of key genes in SA,JA and related signal pathways were down-regulated.Instead,it is decreased for the accumulation of virus,ROS,electrical conductivity and MDA in silenced SIGDSL19 plants and gene-edited SIGDSL19 transgenic plants.And the content of SA,JA and related signal pathway key gene expression were up-regulated.The result suggests that tomato GDSL lipase gene SIGDSL19 plays a negative regulatory role in defense against TMV infection.The expression of SIGDSL19 decreased in plants can reduce the replication of TMV,the accumulation of ROS,the damage of cell membrane oxidative,and regulate SA,JA synthesis and signal pathways to enhance plant resistance to TMV,However,the expression of SlGDSL19 increased will weaken the resistance of plants to TMV.In conclusion,tomato GDSL lipase gene SlGDSL19 may play a part in resisting TMV infection,and its expression decreased can promote the resistance to TMV.The result provides a basis for the role of SIGDSL19 to biological stress.In addition,it is helpful to cultivate improved varieties and enhance the resistance of crops to virus diseases.