Role Of Autophagy In Infection And Replication Of Tobacco Mosaic Virus

Autophagy is a highly conserved cellular degradation process in eukaryotes, playing a signif icant role in the maintenance of cellular homeostasis. The interaction between autophagy and virus is becoming a hot spot of research in recent years.In this study, TMV-U1 strain and its susceptible host Nicotiana tabacum cv. Bright yellow, were used to elucidate whether Tobacco mosaic virus infection could induce autophagy in tobacco plants. Trans-mission electron microscope(TEM), monodansylcadaverin(MDC) staining, confocal microscopy analysis of enhanced cyan fluorescent protein–tagged tobacco ATG8f(ECFP-ATG8f) and quantitative real-time PCR were used to investigate the formation of autophagic structure, acidic compartments, and the transcript levels of autophagy related genes including ATG3, ATG4, ATG5, ATG6, ATG7, ATG8 a, and ATG18 a in TMV-infected tobacco plants, respectively.In order to explore the function of autophagy in TMV infection and replication, TMV-GFP friction infection system is constructed. After silencing the autophagy genes, the TMV-GFP were used to infect Nicotiana benthamiana. GFP fluorescence intensity in the UV light could reveal the reproduction level of TMV. To make results of trials more convincing, real-time RT-PCR analys is was used to detect relative level of TMV-CP gene. Specific results are as follows:1. The concrete results of transmission electron microscopy(TEM). A number of autophagic structures containing electron-dense material at 72 hours post inoculation(hpi) under TEM were observed. And the size of autophagic structures were about 300 ~ 900 nm.2. Fluorescent dye Monodansylcadaverine(MDC) were used to stain for detecting autophagic structures. MDC-positive autophagic structures were observed in the leaves of TMV-infect plants but rarely in those of the P BS control plants. Quantitative analysis of the showed that MDC-positive structures in leaves of TMV infection is 4~5 fold greater than PBS control.3. Autophagosome marker ATG8f-ECFP was used to monitor autophagy. The cyan spherical or punctate structures were observed in TMV infection leaves, but fewer structures were detected in PBS control leaves, indicating that relative autophagic activity is induced in TMV infection leaves.4. Quantitative real-time PCR was used to detect the transcript levels of autophagy related genes including ATG3, ATG4, ATG5, ATG6, ATG7, ATG8 a, and ATG18 a in TMV-infected tobacco plants, respectively. The expression levels of all those ATG genes, except ATG5, were increased at 48 hpi, and peaked at 72 hpi, and the ATG genes expression abundance at 72 hpi were 2.51, 2.03, 2.97, 2.55, 3.28, 2.62 fold greater than at 0 hpi, respectively. ATG7 expression levels was most obvious, whereas ATG5 expression showed no significant change.5. The fluorescence size of ATG3-silenced, ATG6-silenced, ATG7-silenced plants is more larger than control plants in UV light. q RT-PCR analysis indicated that the TMV-CP expression level of ATG-silencing plants is higher than control plants, indicating that autophagy can prevent virus replication or/and movement to some extent.