The Research On The The Synergistic Mechanism Of TSWV And HCRV Co-infection

Orthotospoviruses are most of important virus in agricultural production.The range of damage caused by Orthotospoviruses has been expanding year by year.Yunnan province has become the region with the most types and severe outbreaks of orthotospoviruses in China,and is also important area for the prevention and control of this virus disease.TSWV and HCRV are representative species of Orthotospoviruses.The kind of virus can infect various plants,with characteristics such as wide occurrence area,large host range,rapid virus recombination and mutation,high frequency of compound infection,and great harmful.The co-infection is the key factor in expanding the host range and the epidemic trend.There are many co-infection phenomena in production,TSWV and HCRV has co-infected on crops such as tobacco and tomato,and caused serious economic losses.However,the specific synergistic mechanism between the viruses is still unclear.In order to explore the mechanism of synergistic effect on co-infection,and to provide new ideas and targets for the prevention and control of Orthotospoviruses.In this study,under the co-infection of TSWV and HCRV,we explored the molecular mechanism of orthosporusvirus after co-infection the Nicotiana benthamiana by High-throughput sequencing,RT-q PCR、VIGs、transient expression and so on.The main results are as follows:(1)Detection of virus accumulation and physiological in indexes.Using buffer solution as controls,after single infection with TSWV,HCRV,as well as co-infection with HCRV&TSWV,on the 1st,7th,and 14 th days,it was found that the virus accumulation of Nicotiana benthamiana was increased compared with the control group.Meanwhile,Physiological indexes were detected and found that the activity of defense enzymes-POD、PPO、SOD、β1,3-GA was increased,but the content of chlorophyll a,chlorophyll b,total chlorophyll and carotenoids was induced.(2)Through transcriptome analysis,after HCRV and TSWV were separately infected Nicotiana benthamiana,significant transcriptional pattern changes occurred after 14 days of infection;HCRV infection leaded to significant changes with related genes of secondary metabolic pathways,photosynthesis;TSWV infection inhibited significant changes in related genes of secondary metabolic pathways and JA biosynthesis pathways.But HT co-infection can inhibit the expression of host resistance genes significantly.HT co-infection mainly affected the photosynthesis and basal nutrient metabolism of tobacco,inhibited the expression of JA biosynthesis,MAPK,secondary metabolism,and PR gene,and weakened host resistance and escaped host immune recognition.(3)During the HT co-infection process,395 mi RNAs were upregulated.28 key mi RNAs were identified to be involved in regulating host resistance and inhibiting the expression of JA biosynthesis,MAPK,and secondary metabolism by inducing mi RNA expression.Among of 28 mi RNAs,five specifically expressed mi RNAs were identified,and there were novel-m0693-5p,novel-m0782-3p,novel-m0915-3p,novel-m1488-5p and novel-m1992-3p.These mi RNAs can inhibit the expression of downstream potential targets AOC3,CDPK18,CDPK4,LOX,SAM,WRKY1,WRKY6,and WRKY22,and reduce the host resistance.(4)Based on ps RNATarget website prediction and transcriptome data,it was determined that Nb SAM was a potential target gene for mi R1992-3p,and Nb WRKY6 was a potential target gene for novel-m0782-3p.It was confirmed that novel-m1992-3p could target and bind to Nb SAM,while novel-m0782-3p can target and bind to Nb WRKY6 by the 5’-RLM-RACE technology.And it was confirmed that the tobacco resistance reduced significantly by silencing Nb SAM and Nb WRKY6 with VIGs,and downregulation of PR1 gene was detected in silenced plants.(5)Further verification through VIGS technology showed that silencing Nb PR1 could reduce tobacco resistance to HCRV and TSWV significantly.This indicated that Nb PR1 was a host dependent key pathogenesis-related protein resistant to HCRV or TSWV infection.In summary,co-infection induced the expression of mi RNA of plants and inhibited the expression of downstream target resistance genes,and decreased plant resistance.And it provided new insights and important basis for the synergistic mechanism of TSWV and HCRV co-infection,and provided new ideas and targets for the prevention and control of Orthotospoviruses.