Screening And Its Action Mechanism Of A New Compounded Antiviral Agent

CytPM-COS is a novel type of antiviral agent,which is compounded from Cytosinpeptidemycin of our lab research and plant defense activator Chitosan oligosaccharide.CytPM-COS has many functions of preventing,treating and inducing and improving the control efficiency.To explore the mechanism of CytPM-COS,we researched CytPM-COS effects on TMV RNAs and proteins,and CytPM-COS effect on oxygen species,chlorophyll content and disease resistance related genes.The main research results as follows:1.The control efficiencies of 10 agents on TMV were tested on different hosts including Nicotiana glutinosa and Nicotiana tabacum K326.The Chitosan oligosaccharide treatment exhibited(or showed)the best control efficiency.The control efficiency of Chitosan oligosaccharide treatment was 42.10% on N.tabacum K326.While the lesion inhibition rate of Chitosan oligosaccharide was 56.78% on TMV at N.glutinosa.The optimum concentration of Chitosan oligosaccharide and Cytosinpeptidemycin were screened based on their control efficiencies against TMV,on Nicotiana benthamiana,N.glutinosa and pepper,respectively.The novel antiviral agent was compounded based on the screened concentration called CytPM-COS.Control efficiencies and lesion inhibition rates of CytPM-COS against TMV were tested in three hosts,the control efficiency on TMV in N.tabacum K326 was 78.78%,TMV lesion inhibition rate in N.glutinosa reached 91.88%,the pepper TMV control efficiency reached 58.24%.The control efficiency of CytPM-COS on virus is better than either Cytosinpeptidemycin or Chitosan oligosaccharide.2.The anti-virus mechanism of CytPM-COS were studied on TMV and the host.On TMV,the mechanism was studied by Northern blot detection of Cyt PM-COS effects on accumulations of TMV RNA sense and antisense strands in BY-2 tobacco cells,CytPM-COS could effectively inhibit TMV sense and antisense strands RNA accumulations,inhibiting the proliferation of TMV in BY-2.TMV CP accumulation was detected in BY-2 cell system and N.benthamiana by Western blot.The results showed that CytPM-COS treatment significantly reduced TMV CP accumulation.In addition,applying CytPM-COS twice most significantly inhibited TMV CP accumulation.By constructing TMV CP,TMV MP,TMV p126 fused with GFP respectively,transferring vectors into Agrobacterium tumefaciens and subsequently infiltrating N.benthamiana,changed of subcellular localization of CP,MP and p126 were obeserved in N.benthamiana.CytPM-COS treatment affected the location of TMV CP in N.benthamiana,and the concentration of CP in the cell wall was higher in the endoplasmic reticulum.With the treatment of CytPM-COS,CP aggregated more on the endoplasmic reticulum,and the amount of binding in the cell wall decreased.CytPM-COS had no significant effect on the location of TMV MP and TMV p126.CytPM-COS could induce resistance of host,which was another important mechanism of action.CytPM-COS could induce increased of the active oxygen content which reached a peak in BY-2 cells treated with CytPM-COS after 3 hours,the OD(600nm)value of 0.264,which was 2.06 times than OD value in control.CytPM-COS could increase chlorophyll content when K326 was infected.The chlorophyll content of control was 0.285.The chlorophyll content was 0.381 mg/g by treatment of CytPM-COS.Quantitative detection of resistance-related genes was by qPCR,the treatment shown resistance genes PR5,NPR1,HSP70,HSC70,MAPKKK,WRKY70,WRKY53,were upregulated.HSP70 expression was upregulated 31.60 times,PR5 was upregulated 16.15 times;NPR1,WRKY53 were increased 8.51 times and 8.06 times,respectively.It is indicated that the CytPM-COS could induce the host resistance to disease.In short,CytPM-COS inhibits the accumulation of TMV RNA and CP,and influence the localization of TMV CP.It could also induce the increase of reactive oxygen species and chlorophyll content in host plants,as well the expression of resistance related genes is upregulated.3.In the field,the control efficiency of CytPM-COS on tobacco virus disease was 40.17%~40.33%,which was significantly higher than Ningnanmycin,Cytosinpeptidemycin and Chitosan oligosaccharide;the control efficiency of CytPM-COS on pepper virus disease was 34.03%~37.03%,which was significantly higher than Ningnanmycin,Cytosinpeptidemycin and Chitosan oligosaccharide;the control efficiency of CytPM-COS on tomato virus disease was 34.53%~36.73%,which was significantly higher than Ningnanmycin,Cytosinpeptidemycin and Chitosan oligosaccharide.Above all,it indicates that the CytPM-COS has good control efficiency on tobacco,pepper and tomato virus diseases.