The Molecular Mechanism Of Glutathione Regulating Systemic Resistance Against Tobacco Mosaic Virus Infection

Glutathione(GSH)is an important antioxidant in plants,which is the key to maintaining an intracellular redox balance.The active sulfhydryl group of GSH is the reason why it can combine with some drugs and toxins and have the function of detoxification.Meanwhile,GSH also plays a key role in the storage and transportation of reduced sulfur,the synthesis of proteins and nucleic acids,and the regulation of enzyme activities.Studies have shown that GSH is involved in the process of plant resistance to biological stress(fungi,bacteria,viruses)and abiotic stress(salt stress,drought stress,high temperature stress,oxidative stress,etc.).However,the molecular mechanisms by which GSH regulates systemic plant resistance in response to viral infection are unclear.y-Glutamyl cysteine synthase gene(NbECS)and glutathione synthase gene(NbGS)are two key enzyme genes in the synthesis pathway of GSH.First,we cloned full length sequences of NbECS and NbGS from the Nicotiana benthamiana.The results of phylogenetic tree showed that NbECS was most closely related to tobacco NtGSHl and tomato SlGSH1,and NbGS was most closely related to NtGSH2 and SlGSH2.Bioinformatics analysis showed that among the 522 amino acids encoded by NbECS gene,Leu and Gly were higher,and among 375 amino acids encoded by NbGS gene,Leu and Ser were higher.NbECS protein is presumed to be a stable hydrophilic protein,while NbGS protein is an unstable hydrophilic protein.Neither NbECS nor NbGS have signal peptides nor transmembrane.There are many phosphorylation sites of NbECS and NbGS amino acid sequences,especially Ser sites.The Ser site is mostly the guarantee to activate the protein activity,mainly the enzyme activity,the result is consistent with that NbECS and NbGS genes are key genes of enzymes.Both NbECS and NbGS proteins contained more ?-helix and random coil.NbECS protein is a monomer protein,the whole structure is nearly spherical,the edge is composed of many helical regions,the catalytic residues play a role in the center of the whole structure,through the concave hole on the ball to combine with other amino acids.The NbGS protein is a homologous dimer with A and B chains,and the catalytic active residues are very similar in sequence and structure to the homologous genes in other species.In order to further study the molecular mechanism of GSH regulating systemic resistance against Tobacco mosaic virus(TMV)infection,we conducted a series of experiments.Firstly,we detected the expression of NbECS and NbGS genes and the content of GSH in the TMV infected tobacco.qRT-PCR results showed that TMV infection induced the expression of NbECS and NbGS genes,and the GSH content also increased significantly.Then,through external application of GSH and OTC(L-2-Oxothiazolidine-4-carboxylic Acid,GSH synthesis promoter)solution,the increase of GSH content reduced the accumulation of TMV-induced reactive oxygen species(ROS),electrolyte leakage,malondialdehyde(MDA)content and the accumulation of TMV virus.However,external application of BSO(Buthionine sulfoximine,GSH synthesis inhibitor)solution reduced the content of GSH,increased TMV-induced oxidative damage,and the accumulation of TMV virus.Furthermore,virus-induced gene silencing(VIGS)was used to silence NbECS and NbGS genes in N.benthamiana respectively.The reduced GSH content,increased the TMV-induced ROS accumulation,electrical conductivity,MDA content,and the accumulation of TMV virus.We also used agrobacterium-mediated transient overexpression technology to overexpress NbECS and increase the GSH level in N.benthamiana.Similarly,TMV-induced ROS accumulation,electrolyte leakage,MDA content,and the accumulation of TMV virus were decreased significantly.Therefore,these results indicate that increasing the level of GSH in N.benthamiana can reduce the accumulation of ROS caused by TMV infection,the oxidative damage to cell membrane,and the accumulation of TMV virus,to enhance the systemic resistance of N.benthamiana to TMV,while inhibiting the synthesis of GSH will weaken the systemic resistance of plants to TMV.In order to further study the relationship between GSH and SA-mediated defense pathways,we examined the expression of key SA synthesis and signaling genes NbICS1(synthesis pathway),NbNPR1(signaling pathway),and SA-mediated pathogenesis-related protein genes NbPR1,NbPR2,and NbPR5 in N.benthamiana.The results showed that the expressions of NbICS1,NbNPR1,NbPR1,NbPR2 and NbPR5 could be enhanced by external application of GSH,OTC solution and transient overexpression of NbECS.However,the expression of NbICS1.NbNPR1,NbPR1.NbPR2 and NbPR5 were inhibited by external application of BSO solution and silencing of NbECS and NbGS genes Therefore,GSH may regulate the synthesis of SA,activate the SA-mediated signaling pathway,promote the expression of SA-mediated defense genes,to improve the systemic resistance of N.benthamiana to TMV.In summary,GSH improves resistence of N.benthamiana to TMV infection by activating SA-mediated signaling pathway and inducing the generation of systemic resistance of N.benthamiana.Our results can provide theoretical support for improving plant disease resistance,breeding resistant varieties,and improving crop yield and quality.