Mechanism Of Melatonin Enhancing The Resistance Of Upland Cotton To Verticillium Wilt

Plants growing in the soil are constantly threatened by the aggression of a wide range of pathogens in the surrounding environment.Due to the long-term co-evolution and mutual selection with pathogens,plants have evolved a sophisticated and multilayered surveillance system to perceive and distinguish different invaders.Verticillium Wilt caused by Verticillium dahliae is the most destructive disease,which is known as the “cancer” of cotton.V.dahliae is a soil-borne plant pathogen that invades the waterconducting xylem vessels of susceptible plant species to cause vascular wilt disease.The typical symptoms of Verticillium wilt in cotton plants include leaf chlorosis and wilt,leaf defoliation,vascular tissue browning,and plant death.Its wide spread and destructive effect has led to huge economic losses for the cotton industry.Verticillium wilt is therefore a critical issue for the cotton industry and will require new research for the development of effective disease control measures.Melatonin,also known as pineal voxel,is a biologically active molecule that widely exists in plants.Because of its versatile biological functions,melatonin has attracted widespread attention in recent years.Plant melatonin alleviates abiotic stresses from cold,heat,drought,salt,to heavy metals.Studies have implicated melatonin in plant immunity,but the underlying mechanisms remain unclear.In this study,transcriptional sequencing(RNA-Seq)and virus-induced gene silence(VIGS)were used to explore the mechanism of melatonin in mediating cotton resistance to V.dahliae.The main results were as follows:After pretreatments with different melatonin concentrations(0 ?M,10 ?M,25 ?M,50 ?M,and 100 ?M),cotton seedlings were inoculated with V.dahliae.It was found that various concentrations of melatonin pretreated cotton seedlings exhibited different resistance to V.dahliae.The symptoms,including wilt,chlorosis,and dark brown streaks in stems,were much more severe in the control than in pretreated seedlings.To determine whether exogenous melatonin acted as a fungicide and protected the pretreated cotton seedlings against the disease,V.dahliae was cultured on PDA medium with different melatonin concentrations.There were no significant differences in the diameters of V.dahliae mycelium and sclerotium among the treatments,suggesting instead that melatonin might act as a regulator to enhance cotton host resistance against V.dahliae.The melatonin levels in different tissues were higher in the resistant cultivars than in the susceptible cultivars.Although melatonin was induced in all of the cultivars after V.dahliae inoculation,the induction was more pronounced in the resistant cultivars.Using AtSNAT(AT1G32070)and AtCOMT(AT5G54160)as queries,BLAST analyses were performed to identify their homologs in the cotton genome.Functional complementation experiments were performed by transforming the putative SNAT(Gh_D02G1063)and COMT(Gh_D12G2714)genes into Arabidopsis snat and comt mutants,respectively.The melatonin-deficient phenotypes of the mutants were both complemented by the introduced sequences.Endogenous melatonin levels were reduced by suppressing GhSNAT and GhCOMT expression using VIGS.The VIGS cotton seedlings were inoculated with V.dahlia.The disease infection in TRV:SNAT and TRV:COMT plants was more severe than in the TRV:00.Most significantly,TRV:SNAT+COMT plants were almost dead.Co-silenced TRV:SNAT+COMT plants with extremely low endogenous melatonin content were more susceptible than TRV:SNAT and TRV:COMT plants during V.dahliae infection,indicating a dosage effect of endogenous melatonin on cotton resistance to V.dahliae.In this study,the RNA-Seq and metabolite analysis of melatonin pretreated cotton seedlings showed that melatonin up-regulated the expression of phenylpropanoid pathway related genes after inoculation with V.dahliae,induced lignin accumulation in cotton plant vascular tissue,followed by enhancing the ability to defense the invasion and proliferation of V.dahliae.VIGS assays confirmed the involvement of phenylpropanoid pathway and ligninin melatonin-mediated cotton resistance,as lignin induction was inhibited in melatonin-suppressed cotton seedlings during V.dahliae infection.Moreover,melatonin pretreated cotton seedlings with genes silenced by VIGS were able to restore the response of the phenylpropane pathway and lignin to V.dahliae.Therefore,melatonin could enhance the induced tissue structure resistance of cotton to V.dahliae via up-regulating the expression of phenylpropanoid pathway related genes and accelerating the synthesis of lignin.Melatonin pretreatment could increase the expression of genes involved in mevalonate and gossypol pathways,and further accelerate the biosynthesis of gossypol.Gossypol can significantly inhibit a wide spectrum of growth of pathogens.Suppressing melatonin level in cotton seedlings by VIGS could inhibit the response of gossypol to the invasion of V.dahliae,and compromise the resistance to pathogen.Melatonin pretreatment of the VIGS cotton seedlings restored the response of the mevalonate and gossypol pathway to V.dahliae invasion.Hence,melatonin could improve the induced biochemical resistance of cotton to V.dahliae by up-regulating the expression of genes involved in mevalonate and gossypol pathways and enhancing gossypol accumulation.