The Research Of The Regulation Mechanism Of Chloroplast Protein Degradation Induced By Potato Virus Y In Tobacco

Potato virus Y（PVY）is one of the main viruses harmful to tobacco,causing great losses to tobacco leaf producing areas every year.Chloroplasts play an important and complex role in the infection of plant viruses.Most plant viruses affect the formation and development of chloroplasts.Rbc S（Rubisco small subunit）that locate chloroplasts regulate Rubisco enzyme activity and play an important role in plant response to biotic and abiotic stresses.Taking Nb Rbc S and PVY as the main research objects,the effects of Nb Rbc S on virus infection and the effects of virus-infected tobacco on Nb Rbc S expression were studied.This study reveals the role of Rbc S protein in tobacco virus infection,helps to analyze the molecular mechanism of the game between PVY and host,and lays a theoretical foundation for PVY prevention and control and antiviral molecular breeding.The main research contents are as follows:1.VIGS technique was used to silence Nb Rbc S and 35S promote overexpress Nb Rbc S.It was found that the expression levels of PVY,TMV and CMV increased significantly after silencing Nb Rbc S.While over-expressed Nb Rbc S could inhibit the replication and proliferation of PVY,TMV and CMV.However,q RT-PCR and Western Blot analysis showed that PVY,TMV and CMV all down-regulated the expression of Nb Rbc S.These results indicated that Nb Rbc S could inhibit the proliferation of viruses,but viruses also evolved anti-inhibition mechanisms to promote the degradation of Nb Rbc S,but the degradation mechanism is unclear.2.The degradation pathway of Nb Rbc S induced by PVY was analyzed.Pull-down and Western Blot analysis showed that the ubiquitin level of Nb Rbc S was up-regulated by PVY infection.After treatment with the ubiquitination inhibitor MG132,the down-regulation of Nb Rbc S decreased,indicating that PVY infection promoted the ubiquitination degradation of Nb Rbc S.In addition,the expression of autophagy labeled protein ATG8 was up-regulated after PVY infection,indicating that the activity of autophagy in cells was increased.Moreover,autophagy inhibitor 3-MA treatment and silencing autophagy-related genes Nb ATG6 and Nb ATG7 reduced the down-regulation of Nb Rbc S expression indicating that PVY infection can degrade Nb Rbc S protein through autophagy.The results showed that PVY infection promoted the ubiquitination pathway and autophagy pathway degradating the Nb Rbc S protein together.3.The morphology of chloroplasts after PVY infection was observed by transmission electron microscopy.Some chloroplasts were found to be abnormal in morphology,enlarged in volume and separated into vacuoles,which was similar to the chloroplast microautophagy phenomenon.The fusion expression vector of TIP and GFP（locating vacuolar membrane）and the fusion expression vector of Rbc S and RFP were constructed.The chloroplasts with abnormal morphology and structure were also observed by laser confocal microscopy to be enveloped by vacuolar membrane labeled by TIP-GFP,which did not find this phenomenon in healthy cells,The results indicated that further PVY infection induced chloroplast microautophagy in the host.4.ROS is a key inducer of chloroplast microautophagy.The O^2-and H₂O₂ in PVY infected tobacco leaves were stained by NBT staining and DAB staining.It was found that PVY infection caused slightly upregulated ROS in leaves,suggesting that ROS may mediate chloroplast microautophagy degradation.The interaction between Nb Rbc S and autophagy-related proteins Nb ATG7,Nb ATG6 and Nb ATG8f was verified by yeast two-hybrid.Nb Rbc S and Nb ATG7 interreacted in the yeast two-hybrid system,but not with Nb ATG6 and Nb ATG8f.In conclusion,Nb Rbc S can inhibit the infection and replication of PVY,TMV and CMV,but the virus evolved a counter-inhibition mechanism to actively regulate the down-regulation of Nb Rbc S expression,and the expression level of Nb Rbc S may be related to the formation of mosaic symptoms.PVY infection promoted the degradation of Nb Rbc S proteins through autophagy and ubiquitination pathways.Chloroplast microautophagy induced by PVY infection may be one of the concrete pathways leading to Nb Rbc S degradation.