Synergism Between Tobacco Vein Banding Mosaic Virus And Potato Virus X And Transcriptomic Analysis Of Nicotiana Benthamiana Infected By These Two Viruses

Potyviruses and Potato virus X(PVX)often co-infect plants and produce synergistic effect,causing more severe mosaic and even necrosis symptoms.Tobacco vein banding mosaic virus(TVBMV),a distinct member of the genus Potyvirus,can infect solanaceous crops including tobacco,potato and tomato.The incidence of TVBMV rises rapidly in the field.With the development of the high-throughput sequencing technology,the use of transcriptome sequencing technology for plant transcription has gradually become a hot spot.However,reports of using this technique to study the mechanism of synergism of viruses were scarce.In this study,the wild type infectious c DNA clone and attenuated mutant of TVBMV,and the infectious c DNA clone of PVX were used to study the synergism of TVBMV and PVX.The mutations of D198 to K and IQN motif to DEN in HCpro eliminated its RNA silencing suppression activity and attenuated the symptoms of TVBMV in Nicotiana benthamiana plants.Transcriptome sequencing analysis of Nicotiana benthamiana infected with wild type or attenuated mutant TVBMV and PVX were compared.Young leaves of sesame plants showing symptoms of mosaic,etiolation,and puckering were collected for small-RNA deep-sequencing(SRDS)analysis,the complete sequence of TVBMV isolate amplifited from the sesame plant was investigated,and the molecular variation of the isolate was analyzed.The main results read as follows:The synergistic effect of TVBMV and PVX was studied from the aspects of disease symptoms and virus concentration.At 10 days after PVX infection,symptoms of mosaic could be found in the upper systemic leaves of Nicotiana benthamiana plants.At 10 days after co-infectiono of TVBMV and PVX,the symptoms of growth retardation,mosaic and curling could be found.However,at 10 days after the co-infection of attenuated mutant and PVX,only light mosaic could be found in the systemic leaves.At 14 days after co-infection with TVBMV and PVX,the N.benthamiana plants showed growth retardation and necrosis,but the N.benthamiana plants co-infected the attenuated mutant and PVX only showed mosaic similar to that caused by PVX.The combination of TVBMV and PVX enhanced the symptom and significantly inhibited the growth of N.benthamiana.The ELISA results showed that virus accumulation in the leaves of N.benthamiana plants co-infected with TVBMV and PVX increased significantly at 6 days after inoculation to same level of that in the plants inoculated with TVBMV only,while that in plants inoculated with the attenuated mutant and PVX complex infection were maintained at a low level all the time.RNA-Seq technique was used to study the variation of the wild-type TVBMV and attenuated mutants combined with PVX during the infection.The virus was replicated massively in the second days after inoculation,and there was a large amount of virus RNA in the inoculated leaves for 2 days after inoculation and the systematic leaves for 10 days after inoculation,and clean reads could be less than that of the control genomes.GO enrichment analysis showed that the combined infection of TVBMV and PVX resulting in a large number of host gene abnormal expression,interfere with the normal metabolism of the host affects the host protein and enzyme metabolism related genes,genes involved in DNA replication,photosynthesis related genes,so that the host cell group produced a series of biological processes,and the molecular function change.Compared with attenuated mutants combined with PVX,at the initial stage of TVBMV and PVX co-infection,DNA replication pathway was affected with 17 genes up-regulated expression.And then the expression of 33 genes of cell wall synthesis related genes and 61 genes of photosynthesis related genes down-regulated while DCL2 and RDR1 in the RNA silencing pathway up regulation after systemic infection.KEGG enrichment analysis showed that compared with the attenuated mutant and PVX complex infection,wild type TVBMV combined with PVX induced expression of ribosome synthesis related genes to help viral protein synthesis,through inhibition of photosynthesis related metabolic pathways,inhibition of cell wall synthesis related enzymes of metabolic pathways,regulating hormone mediated signal transduction pathway.The SA signaling pathway of plantswas induced to produce a large number of PR proteins in order to prevent virus infection.We conducted a series of field surveys in Zhangqiu of Shandong Province,China,to investigate the viruses infecting sesame.Sesame plants showing symptoms of mosaic,etiolation,shoestring and puckering were found in several fields.Young leaves of symptomatic plants were collected for small-RNA deep-sequencing(SRDS)analysis.BLAST searches identified three potyviruses,Watermelon mosaic virus(WMV),Bean common mosaic virus(BCMV)and Tobacco vein banding mosaic virus(TVBMV).To the best of our knowledge,this is the first report that TVBMV can naturally infect sesame and the crops other than solanaceous crops.Further tests using RT-PCR and antiserum confirmed the sequencing of small RNAs.The complete genome sequence of the TVBMV-Zhangqiu isolate was obtained by the step-by-step amplification and 5 'RACE method.The complete sequence was 9596 nucleotides.TVBMV-Zhang Qiu shared the highest identities with TVBMV isolates YY of 97.89% at the nucleotide level and of 99.06% at the amino acid level.The phylogenetic analysis showed that TVBMV-Zhangqiu isolates clustered to the MC group of China.The recombination analysis showed that TVBMV-Zhangqiu isolate was a potential recombination of SDYS1 and YN isolates occurred in the front of CP and 3'-UTR,which belonged to MC group,and the main parents of the genome were SDYS1.The symptoms of sesame infected with TVBMV-Zhangqiu and TVBMV-HN39 were different.Zhangqiu isolates showed only mild mosaic symptoms after inoculation,while the symptoms of sesame plants infected with HN39 were obviously mosaic and grew slowly.