Omic Analysis Of Anti-TMV Induced By Prokaryotic Expression Product TDP In Tobacco

Tobacco mosaic virus(TMV)is a highly infectious virus with a wide host range,causing serious economic losses.Until now,pesticides were main method of TMV control in the field.Whereas the abuse of chemical pesticides may cause serious environmental pollution.It is of great significance to develop environment-friendly antiviral pesticides from biological organism.TDP,a protein TDP derived from Flammulina velutipes,can enhance the activity of anti-disease defense enzymes and inhibit the replication of TMV in tobacco.The symptoms of mosaic were alleviated and delayed in FTX271(TDP is the precursor of FTX)transferred tobacco after infected with TMV.TDP protein has the potential to be developed into a biological pesticide,but the antiviral mechanism of TDP need be further researched.In this study,protein TDP(484.126 μg/m L)was obtained from the prokaryotic system.Tobacco was inoculated with TMV after spraying TDP.Then the expression of resistance related genes was analysed and compared through quantitative real-time PCR(q RT-PCR).The molecular mechanism anti-TMV of TDP was explored by transcriptome and proteomics analysis in Nicotiana tabacum.TDP was injected into the leaves back of the Nicotiana tabacum K326 through a disposable needle-free syringe,72 h later,a hypersensitive response(HR)was observed,which characterized as tissue necrosis around the injection site.Different groups were setted as the following: the healthy tobacco was sprayed with water 1 m L(CK);TMV was inoculated after spraying with water 24 h(CV);the healthy tobacco was sprayed with TDP(250 μg/m L,1 m L)(T);TMV was inoculated after TDP(250 μg/m L,1 m L)treatment 24 h(TV).Then the genes that encodes nonexpressor of pathogenesis related protein 1(NPR1)、acid pathogenesis related protein 1(PR1a)、Phenylalanine ammonia-lyase(PAL)and Basic pathogenesis related protein 1(PR1b)were tested by q RT-PCR.The results showed that they were upregulated expression in the group TV when compared with others.In addition,the expressions of NPR1 and PR1 a in the group of TV were 5.42 and 11.7 times higher than that of CV after 5 d inoculate with TMV.The reproduction of TMV-CP was significantly suppressed at 7th TDP treatment(P<0.01),and the symptoms of tobacco mosaic were relieved.Then the same leaves were collected after 7 days for the transcriptome and proteome analysis.The results were as follows:1.After RNA sequencing,1777,3149 and 2091 candidates were the differentially expressed genes(DEGs)in the compare groups of CK/CV,TV/CK and TV/CV,respectively(Qvalue≤0.001).In the TV group,the genes encoding pathogen-associated protein(PR),peroxidase(POD),5-epiaristolochene synthase and polyphenol oxidase(PPO)were significantly up-regulated;the pathways such as glutathione 、phenylalanine、isoflavones、steroids、 Mitogen-activated protein kinase(MAPK)were changes significantly(P<0.05);photosynthesis was significantly down regulated in the CV group.However,in the TV group photosynthesis was down regulated to CK but up relative to the CV.The results indicate that TDP may repair photosynthesis which damaged with TMV infection,and activation the pathways of MAPK,PR protein regulated by salicylic acid(SA).Systemic acquired resistance was induced by TDP2.Through 4D lable free quantitative(4D-LFQ)technology analyse,1190,894 and 478 differentially expressed proteins were identified,respectively(P value<0.05).Cutin,suberine and wax biosynthesis and sesquiterpenes and triterpenes pathways related to plant disease resistance were observably enriched in the TV group.Compared with CV,the expressions of chitinase,PAL,Thiamine synthase(THI),catalase and cytochrome P450 in TV group were up-regulated.3.A total of 7,456 genes and proteins were associated by transcriptome-proteome correlation analysis,which have eight regulatory patterns.After TDP protein treatment,the genes and proteins with the same differentially expression trend showed that the Cytochrome P450(CYP 450),PAL and Chitinase(CHI)in phenylpropane pathway were significantly increased(P value<0.05);Phenylpropane derivatives such as flavonoids and chalcones were induced to express,which enhanced the defense ability of tobacco;TDP protein may repair the down-regulation of photosynthesis caused by TMV through up-regulation of Thioredoxin superfamily protein(THr)expression.TDP protein activates salicylic acid signaling pathway which mediated by NPR1 and PRs,resulting the increased expressions of CAT,PR and PAL;TDP protein induces the expression of thioredoxin that can repair the photosynthetic damage with a certain extent causing by TMV-infected,and synergistic CAT scavenging of hydrogen peroxide;with the action of salicylic acid and reactive oxygen species,the cells produce HR signal;CYP450 and PAL were promoted phenylalanine metabolism,and the upregulation of CHI indicates that the synthesis of secondary metabolites of flavonoids were increased.In conclusion,tobacco was obtained SAR with treated by TDP,which can inhibit the infection of the virus and alleviate the symptoms of mosaic,providing a theoretical basis for the application of TDP protein.