Identification Of Negative Sense RNA Viruses Infecting Kiwifruit And Study On Interactions Of Proteins Of These Viruses

Plant negative sense RNA(NSR)viruses infect important crops and horticultural plants.Some NSR viruses are vectored by arthropods,which plays roles in disease spreading.The genome of plant NSR viruses consist of one or more negative sense single-stranded RNA(-ssRNA)segments.The terminal sequences of viral genomic RNAs are conserved in each genus and complementary to each other,forming a panhandle structure.NSR viruses have virions with doublemembranes consisted of viral proteins and host components.The species of NSR viruseshave increased greatly and the associations of some viruses with plant disease have been revealed as the development and application of high-throughput sequencing strategies.China is the original place of kiwifruit(Actinidia.spp)has plentiful kiwifruit germplasms.In this study,NSR viruses were identified from kiwifruit plants and molecularly characterized.On the bases,the subcellular locations and interaction relationships of proteinsencoded by three NSR viruses were analyzed.The results areimportant for understandingprotein functions of these viruses.The obtained main results are presented as follows:(1)By using small RNA sequencing and RT-PCR,for the first time,two viruses namely,tomato necrotic spot associated virus isolate Ac(TNSa V-Ac)and Impatiens necrotic spot virus isolate Ac(INSV-Ac),in genus Orthotospovirus,were identified from two kiwifruit samples respectively collected from Guizhou and Yunnan provinces.The full-length genome of TNSa V-Ac and INSV-Ac were composed of three RNA segments(L,M and S).The three RNA segments of TNSAV-Ac and INSV-Ac contained 8908 nt,4772 nt,2991 nt,8776 nt,4969 nt and 2991 nt respectively.It showed 98.5%-100% identity at the amino acid(aa)level with the corresponding proteins of TNSa V-GZT isolates.The aa sequences of five proteins encoded by INSV-Ac shared highest identities range from 99.5% to 100% with five corresponding proteins encoded by INSV-Pepe isolate.(2)A novel emaravirus,provisionally named Actinidia emaravirus 2(Ac EV-2),was identified from a kiwifruit tree showing leaf mottle and chlorosis symptoms by employing the emaraviral conserved primer pair 5H/3C and degenerate primer set AF/CR.This is the second emaravirus infecting kiwifruit plants.The genome of Ac EV-2 consisted of at least six RNAs(RNAs 1-6)with sizes of 7079 nt,2252 nt,1387 nt,1514 nt,1744 nt and 1233 nt,respectively.In order,from RNA1 to RNA6,these opening reading frame(ORFs)encoded the RNA-dependent RNA polymerase(P1),a putative glycoprotein precursor(P2),a putative nucleocapsid protein(P3),a putative movement protein(P4),P5 and P6.The P2 was digested by enzyme to produce two mature glycoproteins Gn and Gc.P1-P4 proteins of Ac EV-2 shared the highest aa sequence identities of 62.2%-77.3% with the corresponding proteins of fig mosaic emaravirues(FMV)and pigeonpea sterility mosaic emaravirus 2(PPSMV-2).P5 and P6 of Ac EV-2 showed the limited identities to the corresponding proteins of PPSMV-2 with identities of 44.2% and 39.2%,respectively.Ac EV-2 was phylogenetically close to PPSMV-2 and FMV,but distinct from Ac CRa V,an emaravirus previously identified from kiwifruit plants in our study group.Virus detection disclosed that a total of 28 positive samples were detected in 136 kiwifruit samples collected from 5 provinces(or city)by RT-PCR,with a frequency rate of20.6%,the relationship between the virus and symptoms was not clear.(3)Subcellular location of protein Gn,Gc,P3,P4 and P5 encoded by Ac CRa V and protein Gn,Gc,P3,P4,P5 and P6 encoded by ACEV-2 were compared and analyzed by using agrobacterium-mediated transient expression system of in epidermal cells of Nicotiana benthamiana leaves.It was found that P3 proteins encoded by two viruses formed cytoplasmic agglomerates,and P4 from two viruses located at plasmodesmata(PD).While other proteins of two viruses exhibited different subcellular localization characteristics.Bimolecular fluorescent complimentary(Bi FC)assays disclosed that the P5 of Ac CRa V could recruit viral four proteins,including Gn,Gc,P3 and P4 to cytoplasm forming viral replication complexes-like structures,suggesting that P5 of Ac CRa V could be involved in virus replication,packaging and movement by interacting with other four proteins,The P5 and P6 of Ac EV-2 interacted with Gc and MP in the nucleus and cell periphery.which may be related to virus packaging and movement.(4)A novel cytorhabdovirus,tentatively named Actinidia virus D(Ac VD),was identified from kiwifruit samples collected from Hubei Province by re-examining of the HTS data of six kiwifruit samples in previous research of our laboratory.The genome of Ac VD consisted of 13,589 nt and is organized into ORFs in its antisense strand.In order,from ORF1 to ORF7,these ORFs encoded nucleocapsid protein(N),phosphoprotein(P),P3,matrix protein(M),glycoprotein(G),P6 and RNA-dependent RNA polymerase(L),respectively.Thus,genomic structure of the virus is3'-N-P-P3-M-G-P6-L-5'.The 3' and 5' untranslated regions of the virus were and 354 nt,respectively.3' and 5' untranslated regions of the virus were 159 nt and 354 nt,respectively.The ORFs were separated by conserved intergenic junctions.The genome sequence of Ac VD was 44.6%–51.5% identical to that of reported cytorhabdoviruses.The proteins encoded by Ac VD shared the highest sequence identities range from 27.3%(P6)to 44.5%(L)with counterparts of reported cytorhabdoviruses.Phylogenetic analyses revealed that Ac VD clustered within the cytorhabdovirus group with the closest phylogenetic relation to Wuhan insect virus 4(Wh IV-4).In order to have a deep understanding of the functions of Ac VD proteins.The subcellular locations of the viral proteins N,P,P3,M,G and P6 were determined by using the agrobacterium-mediated transient expression system in Nicotiana benthamiana leaves.The M protein of Ac VD localized on the microtubules of the cell.This was the first that M protein had this location characteristic among M proteins of rhabdoviruses.Bi FC assays showed that three proteins N,P and M could self-interact,protein N interacte with P and P3 to form viral replication complexes in the cytoplasm,which is an important characteristic of cytorhabavirus,and protein M changed the subcellular location of four proteins N,P,P3 and G,and transported them to the microtubules.In addition,five paired proteins interacted in nucleus.Ac VD is the first cytorhabdovirus naturally infecting kiwifruit plants,and gives us information to better understand the protein functions of the cytorhabdvirus.