Signalling And Regulatory Roles Of Tryptophan Metabolites In Cotton Immune System To Verticillium Dahliae

Higher plants produce a huge array of low-molecular weight compounds that protect plants against biotic and abiotic stresses,and most of these defensive compounds are derived from amino acid precursors.Although cotton is rich in secondary metabolites,few metabolites have been identified as playing a role in cotton resistance to V.dahliae.In our previous study,some genes putatively responsive to a broad range of pathogens in cotton were identified,from which,we isolated a tryptophan synthesis-related gene GbTSA1.Therefore,we studied the function of tryptophan synthesis related genes in cotton resistance to V.dahliae,and the main results were as follows:1.Expression pattern analysis of GbTSA1Gene expression analysis showed that GbTSA1 was constitutively expressed in all tested tissues(root,stem,leaf,petal,anther,0 d ovule,5 d fiber)of G.barbadense;The induced expression pattern showed that GbTSA1 was greatly suppressed at 1 h and 6 h post V.dahliae infection,but highly activated at 24 h,and the expression of GbTSA1 was also induced by salicylic acid treatment;Sub-cellular localization showed that GbTSA1 was localized in the chloroplast.2.Knock-down of GbTSA1 improves cotton resistance to V.dahliaeTo explore the functions of GbTSA1,the over-expression and RNA interference constructs of GbTSA1 were generated and transformed into cotton,and we finally got four overexpression lines(OS-2,OS-3,OS-5,OS-8)and one RNAi lines(si-1)of GbTSA1.The si-1 seedlings showed lesion-mimic phenotypes accompanied by a high expression level of PR genes and accumulation of SA.It was difficult to get enough samples from the only one transgenic plants si-1 for experiments,so virus-induced gene silencing was employed to knock-down its expression levels,and GbTSA1-silenced seedlings showed a phenotype similar to that of si-1.Further studies showed that knock-down of GbTSA1 improved cotton resistance to V.dahliae,while overexpression of GbTSA1 had little influence on the resistance of cotton to V.dahliae.3.Improved resistance in GbTSA1-silenced seedlings is SA-dependentTo investigate the causes of cell death in si-1 and TRV:TSA1 seedlings,VIGS constructs of GbICS1,the key gene in SA synthesis,and GbNPR1,the key regulator in the SA signaling pathway were built,and infiltrated into plants using Agrobacterium tumefaciens strain GV3101 as vector.Two weeks after the infiltration of combinations with GbICS1 or with GbNPR1,the lesion-mimic and resistance phenotypes of GbTSA1-silenced plants were recovered.These results demonstrate that the activated plant immune system in GbTSA1-silenced plants is dependent on SA synthesis.4.Indole activates the plant defense response and SA synthesisTo explore the possible reasons of lesion-mimic phenotype and elevation of SA synthesis in GbTSA1-silenced plants,the whole tryptophan synthesis pathway was studied and we concluded that the lesion-mimic phenotype was putatively gene specific and tryptophan independent.Furthermore,TRV:TSB1 seedlings showed an even more severe necrotic phenotype than TRV:TSA1 seedlings.Yeast two-hybrid assays demonstrated that GbTSA1 and GbTSB1 can interact with each other to catalyze the final step of tryptophan synthesis;Metabolome analysis showed that indole and indolic metabolites were highly accumulated in GbTSA1-or GbTSB1-silenced plants;Transcriptomic analysis showed that exogenous indole promotes the expression levels of GbSARD and GbWRKY28 genes involved in SA synthesis and the defense response;A dual-luciferase reporter assay confirmed that GbSARD and GbWRKY28 could bind the promoter of GbICS1 and activate the expression of GbICS1.To further validate whether the higher expression of GbSARD and GbWRKY28 is the reason for the activation of the SA pathway in TRV:TSA1 and TRV:TSB1 seedlings,we knocked down the expression of GbSARD in TRV:TSA1-and TRV:TSB1-silenced plants,the stunted and lesion-mimic phenotypes of TRV:TSA1 and TRV:TSB1 plants were almost rescued.Similarly,indole application strongly enhanced cotton resistance to V.dahliae.These results showed that knock-down of GbTSA1 or GbTSB1 led to the accumulation of indole and indolic metabolites,which up-regulate the expression of GbSARD and GbWRKY28.As a consequence,the constitutively activated SA synthesis and signaling pathways enhanced the resistance of cotton to V.dahliae.5.Anthranilate compounds were highly accumulated in TRV:TRP1 plantsIn addition,cell death and spontaneous lesions were also appeared on GbTRP1 silenced seedlings and RNAi transgenic plants.However,the content of SA was significantly decreased in TRV:TRP1 plants.Metabolome analysis showed that anthranilic metabolites were highly accumulated in GbTRP1-silenced plants.The causes of cell death in TRV:TRP1 and GbTRP1-RNAi plants and the relationship between anthranilic metabolites and SA pathway remained to be discovered.