Identification Of A New Host Of CMV And Studies On The Function Of CMV-RNA3 Intergenic Region

The Metaplexis japonica(Thunb.)Makino plant is a kind of herbaceous perennial vine climber,which is a species of the genus Metaplexis in the family Asclepiadaceae and has a wide distribution in China,commonly founded in important economic crops such as pumpkin,soybean and corn.As a traditional Chinese medicine,Metaplexis japonica plants can play the role of clearing away heat and detoxifying,treating traumatic injury,snake bite and cough,it’ small molecule extraction has anti-tumor effect,and polysaccharide extractions can improve the body’s immunity.Cucumber mosaic virus(CMV)is a model species of Cucumovirus,Bromoviridae.It has a wide range of host and strong variability,and is transmitted by a variety of vector insects,making huge harms.In 2016,some Metaplexis japonica plants growing in the pumpkin field showed severe mosaic symptom,and were suspected to infect with a virus.In order to prevent the spread of the virus-like disease,it is necessary to identify the pathogen.In addition,each of gene,3’UTR,5’UTR and RNA3 intergenic region(IGR)of CMV genome had been systematically studied,but RNA3 IGR was studied in vitro.To illuminate the functions of IGR in vivo,agrobacterium-mediated transient expressiont were carried out in Nicotiana benthamiana.1.Identification of a new CMV host.The double-stranded RNA(dsRNA)and the crude sap were extracted from symptomatic Metaplexis japonica plants leaves,and subjected to PAGE electrophoresis and mechanical inoculation onto N.benthamiana,respectively.The results showed that leaves of N.benthamiana plants inoculated with crude sap began to appear slight curlling and mottle symptoms 3 days post-inoculation(3dpi),suggestting that the disease is contagious.The PAGE electrophoretic profile of dsRNA indicated that four bands with the estimation of 4.0,3.5,2.7,and 1.7 kb in size,respectively were detected from the leaf tissues with virus-like symptoms.These results indicated that the Metaplexis japonica plants were most likely infected with a RNA virus.In order to obtain the nucleic acid information of the RNA virus,degenerate oligonucleotide primer amplification,sequencing and BLASTx analysis of the nucleotide sequence were peformed.The results showed that two of the 13 fragments sent to sequence shared a high identity(95-97%)with the 1a gene and 2a of CMV deposited in GenBank.To obtain the whole genome sequence of CMV infecting Metaplexis japonica(CMV-Met),the degenerate primers of CMV was designed and reverse transcription PCR(RT-PCR)was performed.Products of RT-PCR were ligated into pMD19-T vector,and sequenced.The results showed that the full length of RNA1,RNA2 and RNA3 of CMV-Met were 3358 nt,3023 nt,and 2215 nt,respectively.Phylogenetic trees constructed based on the amino acid sequence of CP and the 5’ UTR nucleotide sequence of RNA3 indicated that CMV-Met was clustered into subgroup IB.2.Preliminary study on the function of CMV-RNA3 intergenic region.In order to explore the functions of the RNA3 intergenic region in vivo,three RNA3 IGR deletion mutants(pCass-Rz-CMV-ΔAll,pCass4-Rz-CMV-Δ3’UTR and pCass4-Rz-CMV-Δ5’UTR)were constructed by gene synthesis and double digestion using the complete sequence of CMV-Fny RNA3,and were transformed into agrobacterium tumefaciens.Inoculation experiments on N.benthamiana plants by Agrobacterium-mediated infectious clones showed that the N.benthamiana plants inoculated with pCass4-Rz-CMV-Δ3’UTR appeared leaf curl,yellowing and growth retardation symptoms being indistinguishable from those observed in N.benthamiana inoculated with wild type(Wt).These symptoms appeared at 6.5 dpi,3.5 days delay in comparison with Wt.While the N.benthamiana inoculated with pCass4-Rz-CMV-Δ5’UTR and pCass4-Rz-CMV-ΔAll did not show any symptoms.Western blot analysis indicated that the N.benthamiana plants inoculated with pCass4-Rz-CMV-Δ3’UTR had a lower coat protein(CP)concentration in comparison with that of those inoculated with Wt,while the N.benthamiana plants inoculated with pCass4-Rz-CMV-Δ5’UTR or pCass4-Rz-CMV-ΔAll did not have CP expression.Northern blot analysis indicated that the accumulation of RNA3 or RNA4 in the N.benthamiana plants inoculated with pCass4-Rz-CMV-Δ3’UTR was obviously lower than that in those inoculated with Wt,whereas RNA3 or RNA4 could not find in either the systemic leaves or the inoculated leaves from the N.benthamiana plants inoculated with pCass4-Rz-CMV-Δ5’UTR or pCass4-Rz-CMV-ΔAll.The results of semi-quantitative experiments against inoculated leaves showed that both the expected bands in Wt and Δ3’UTR groups show a high brightness,indicating that the expression of RNA3 was high and RNA3 replication might be active,while in the N.benthamiana plants inoculated with pCass4-Rz-CMV-Δ5’UTR or pCass4-Rz-CMV-ΔAll,the brightness of the expected bands was very low,indicating that RNA3 were transcribed by cDNAs of the two infectious clones also,but almost no replication was used to accumulate new RNA3.The above results indicate that the IGR 5’UTR is responsible for the initiation of RNA3 replication,and its deletion will completely lose their systemic infectivity;the IGR 3’UTR controls the efficiency of RNA3 replication,and its deletion results in a significant decrease in the expression level of RNA3.Intrestingly,the CP expression of N.benthamiana plants in the Δ3’ UTR group was always low,suggesting that a small amount of CP can cause severe symptoms in the host.The identification of CMV-Met increases the diversity of CMV,indicating that weeds such as Metaplexis japonica can be used as the natural virus pool of CMV and eradicating these weeds will be beneficial to the prevention and control of CMV.It also contributes to the production of high quality medicinal Metaplexis japonica plants,helping relavant disease prevention.In addition,the results of RNA3 intergenic region function studies provide a reference for the construction of different functional vectors for different segments of IGR.IGR 5’UTR can also be used as a new target for genetic engineering to develop antiviral species,making plants produce CMV resistance with the plant post-transcriptional gene silencing(PSTG) principle.