Study On The Correlation Between Cucumber Mosaic Virus And Tobacco Cell Autophagy

Autophagy is a highly conserved physiological degradation process of cell components in eukaryotic cells,which plays an important role in coping with nutritional deficiency,disease and senescence.Among the three major viruses,the relationship between Tobacco mosaic virus(TMV),Potato virus Y(PVY)and autophagy of tobacco cells has been studied,whereas it is rarely reported between Cucumber mosaic virus(CMV)and autophagy.Therefore,the relationship between CMV and tobacco autophagy was studied in this paper to determine whether CMV can induce autophagy.In this study,Nb-CFP-ATG8 f transgenic tobacco was constructed firstly to detect tobacco autophagy easily and accurately,in which cyan fluorescence protein(CFP)was combined with autophagy marker gene ATG8 f.The results of carbon(C)or nitrogen(N)deficiency experiments to induce autophagy showed that Nb-CFP-ATG8 f could express CFP stably and effectively,which can be used as a material for the detection of tobacco autophagy.According to the tool of laser scanning confocal microscopy(LSCM),transmission electron microscopy(TEM),Western Blot and qRT-PCR,respectively,we analyzed the result of autophagy flow,microstructures,the protein associated with protein LC3,P62,the gene expression level with the main experimental materials of CMV and Nb-CFP-ATG8 f.The results showed that:1.The results of LSCM showed that,compared with the control plants treated with PBS,autophagosome labeled by the CFP could be observed after CMV infection.Further TEM results showed that the organelles were destroyed or degraded in different degrees after CMV infection,such as mitochondria,chloroplast,nuclear membrane.There is severe vacuolation with the microautophagy and macrophage structures occurred in the cells.The results showed that CMV infection induced organelle degradation and autophagy occurred in host.2.At the same time,Western Blot was used to detect the dynamic changes of LC3 and P62,which were two major markers of autophagy.The results showed that,the ratio of LC3?/LC3 I increased with the time of CMV infection.However,the expression level of P62 showed a reverse trend which was demonstrated that CMV infection could induce changes in the expression of related proteins and induce autophagy of host cells.3.In order to further verify the regulation of host cell autophagy signal transduction pathway by CMV infection,the expression level of autophagy related genes under CMV infection in tobacco cells was detected by qRT-PCR quantitatively.The results of qRT-PCR showed that the expression of CMV was increased at 0 hpi,24 hpi,48 hpi,72 hpi,96 hpi and 120 hpi.The autophagy associated gene expression trend were consistent with the change of CMV expression,such as NtATG1 a,NtATG1b,NtATG3,NtATG4,NtATG8 a,NtATG8b,NtATG8 c,NtATG8d,NtATG9,NtATG10,NtATG13 a,NtATG13b,NtATG18 a,NtATG18c,NtATG18 d,NtATGVTL12b,NtATGVPS34,and all of which were significantly up-regulated with the infection of CMV.The results reveal that the autophagy signal transduction pathway of host cells were induced by CMV,suggesting that autophagy may participate in the process of CMV infection,replication and pathogenesis.