Study On The Mechanism Of Tobacco Necrosis Virus A Promoting The Secretion Of Extracellular Vesicles In Plants

Extracellular vesicles(EVs)are membrane-enclosed vesicles secreted directly from the cell plasma membranes or released by fusion of Multivesicular Bodies(MVBs)with the plasma membrane.EVs are about 30 to 5 000 nm in size and can selectively encapsulate proteins,lipids,nucleic acids,and glycoproteins.Animal EVs play an important role in defending the pathogens,mediating homeostasis,regulating physiological functions,and causing autoimmune diseases.Plant EVs are essential for the transportation of small RNAs(s RNA)and mediating pathogen-plant interaction.The subject of this thesis mainly focuses on the role of plant EVs during plant virus infection.To explore EVs' role during plant virus infection,a model system utilizing the Tobacco necrosis virus A Chinese isolate(TNV-A)and its host Nicotiana benthamiana was used.Then this study focused on the function of EVs from N.benthamiana in packaging TNV-A genomic RNA or s RNA and in facilitating the viral spread.First,EVs in the intercellular spaces of N.benthamiana leaves or isolated from the apoplastic fluid were observed by transmission electron microscopy.They have a diameter of 25?250 nm.Two EVs markers GFP-AtPEN1 and AtTET8-GFP,and a MVBs protein GFP-AtVPS4 were tested for their ability in labeling the N.benthamiana EVs.GFP-AtPEN1 was found to be a suitable marker for N.benthamiana EVs.The iodixanol density gradient centrifugation assay revealed that GFP-AtPEN1 labeled N.benthamiana EVs are mainly distributed in the fraction containing about 20% iodixanol,of which the density is similar to previously reported Arabidopsis thaliana EVs.Furthermore,the TNV-A-infected plant leaves were observed by a transmission electron microscope(TEM)for the presence of EVs.I found that TNV-A promoted the secretion of EVs.Then,EVs in the leaves infected by the virus were isolated and observed under a transmission electron microscope.These TEM images revealed that TNV-A increased the secretion of EVs by about 10 times.Transiently expressed EVs marker proteins GFP-AtPEN1,AtTET8-GFP,most of the endosomal sorting complex required for transport(ESCRT)proteins,RAB5 that localizing on the MVBs accumulation was found increased in TNV-A infected leaves comparing to the healthy plants.Furthermore,the secretion of EV marker proteins GFP-AtPEN1 / AtTET8-GFP,and two putative EV markers AtVPS25-GFP / AtVPS20.1-GFP increased during TNV-A infection.Later on,several dominant-negative mutants of ESCRT and RAB proteins were tested for their ability to inhibit TNV-A induced EV secretion.The results showed that several mutants,including AtVPS4-K178 A,AtVPS4-E232 Q,and AtRAB6AT23 N,could inhibit GFP-AtPEN1 labeled EV secretion,suggesting an MVBs origin of TNV-A induced EVs.Next,s RNA Sequencing was performed on the RNA extracted from TNV-A induced plant EVs,and revealed that these EVs contain TNV-A derived s RNAs.The virus-induced EVs were also subjected to iodixanol density gradient centrifugation followed by detection of viral components' presence.The results suggested that TNV-A full-length RNA can be enclosed by EVs,while the viral particles are not associated with EVs.Then I aimed to explore the viral gene responsible for the EVs induction.The results showed that TNV-A could promote the secretion of plant EVs in the absence of MP(Movement protein)and CP(Coat protein).However,the expression of viral proteins alone could not induce EVs secretion,suggesting that the viral replication process promotes EV secretion.This study further explored the role of EVs in viral infection.First,purified TNVA-induced EVs were infiltrated into N.benthamiana leaves.A lower level of viral CP accumulation and RNA replication could be detected.Also,TNV-A-induced EVs could attenuate TNV-A systemical infection,but not the local infection,when co-inoculated with the viral particles.Further,inhibition of virus-induced EV secretion by expression of a dominant-negative mutant of ESCRT protein AtVPS4-K178 A significantly inhibited viral spread from the initially infected cell but did not inhibit viral replication within the already infected cells,indicating the role of EVs in promoting cell-to-cell movement during TNV-A infection.Overall,this study determined that GFP-AtPEN1 can be used as a marker for extracellular vesicles secreted from N.benthamiana leaves.This work showed that TNV-A promoted the secretion of plant EVs,which contain TNV-A positive-sense and negative-sense genomic RNAs,but not the viral particles.The TNV-A-induced EVs possibly have an MVB origin,due to that ESCRT proteins participate in the formation of virus-induced EVs.Functional studies of TNV-A-induced EVs suggested that EVs promote local TNV-A infection and probably inhibit systemic viral infection.