| Soybean Mosaic Virus(SMV)is a prevalent viral pathogen of soybean that causes severe yield losses and seed quality reductions in the world soybean production regions.SMV has different pathogenic types(strains).For example,a total of 22 SMV strains were identified in China based on an unified national SMV strains identification system.Among them,SC3,SC7,SC8,SC9,SC10,SC11,SC13,SC15 and SC18 were high virulent strains.It was reported that the resistance to each SMV strain was generally controlled by one or a few genes and SMV appeared to break the original resistance by high-speed variation.Thus,the antiviral breeding requires a variety with resistance to different strains,or with a broad-spectrum resistance.RNAi(RNA interference)was widely recognized as a general antiviral mechanism in eukaryotic organisms.Application of RNAi technology has generated broad-spectrum resistant to Papaya Ringspot Virus in papaya plants.Therefore,it is also promising to develop soybean varieties with broad-spectrum resistance to SMV using RNAi principle.At the same time,a quick verification technology system is essential to verify functions of the obtained genes.The purpose of this study is to obtain genes that can mediate broad-spectrum resistance to SMV strains,and establish an experimental approach to test the resistance.The basic lines were as follows:(1)utilizing the pathogen-derived resistance(PDR)mechanism to design RNAi genes against different SMV strains.(2)developing two methods for high throughput functional analysis of the designed genes:one way was to identify model plants that could be infected by SMV;another way was to establish a resistance identification technology system using soybean hairy roots.(3)utilizing the designed RNAi resistance genes and the resistance identification systems to verify their functions in relatively broad-spectrum resistance.The main results are as follows:1.A SMV Isolate 4278-1 overcoming the non-host resistance in Nicotiana benthamiana(Nb)To screen SMV strain that could overcome the resistance in non-host tobacco and Arabidopsis,three varieties of tobacco(Nb,N.tabacum and N.rustica)and Arabidopsis(Col-0)were mechanically inoculated with SMV isolates SC3*(subjected to SC3 strain),4278-1(SC7),6067-1(SC15)and 6003-2(SC18)respectively.The results showed that only SMV Isolate 4278-1 infected Nb with viral symptoms of shrink and stunting.This pathosystem was further confirmed by Enzyme-Linked Immunosorbent Assay(ELISA),virus particle morphology under transmission electron microscopy and back-inoculated soybean.In addition,this back-inoculated test showed that its virulence in soybean NN1138-2 was not weakened,and its pathotype was not changed.The incubation period of Isolate 4278-1 on the host soybean and non-host Nb plants was similar,but the induced symptoms were different.In view of this isolate infecting Nb plants,this system could be used to examine the resistance effect of subsequently cloned RNAi genes.2.Molecular characteristics of SMV Isolate 4278-1 overcoming the non-host Nb resistanceIn order to understand the transduction of SMV isolate 4278-1 in Nb plants,we harvested each leaf from infected Nb plants after inoculated for 7,14 and 21 days,and measured viral contents in each leaf using Quantitative Real-time RT-PCR(qRT-PCR)analysis.The results showed that the inoculated virus could move to uninoculated leaves and cause systemic infection in Nb plants.Whole genome sequencing of this isolate showed that it was consisted of a 9994 nucleic acids(nt)single-stranded RNA while other SMV isolates are generally 9588 nt.Isolate 4278-1 shared overall high amino acids sequence identity with other SMV strains(>89%).However,P1 protein exhibited much lower identity(43.8-45.4%)with known SMV,but was higher with Watermelon Mosaic Virus(WMV,69.6%).The phylogenetic and recombinant analysis suggested that two recombination events happened between SMV and WMV at the P1 gene and 5'-untranslated region(UTR),located from 1-476 nt and from 1145-1349 nt,respectively.Thus,the isolate 4278-1 could infect the host soybean and overcome the resistance in non-host Nb by recombinants.The results of breaking the non-host resistance might provide a new opportunity to reveal the mechanism of plant-virus interactions.3.Expression of a segment of SMV in plants confers high resistance to SMV46 SMV whole genome sequences searching from National Center For Biotechnology Information(NCBI)were analyzed homology comparison by BioEdit software(muscle algorithm),and 5 relatively conserved fragments(named as S1?S5)were slected,whose locations in SMV genome were S1(2595-3056 nt),S2(5601-6013 nt),S3(6930-7426 nt),S4(8261-8582 nt),S5(8919-9274 nt),respectively.Then,we generated 5 constructs containing reverse repeat sequences of the segments(S1?S5)selected.At the same time,we amplified CP gene in SMV genome,and constructed its overexpression vector.The above six clones were transiently expressed in Nb plants,and then inoculated with SMV Isolate 4278-1 virus or infectious clone pGreen-SMV-GUS.The results showed that all the genes in the study had positive effect to resist SMV,but the resistant levels were significantly different.Among them,S1 segment showed the strongest resistance to SMV,while S2 segment showed the weakest resistance.Thus,we suggested that S1 fragment can be transformed to soybean cultivars to improve their antiviral ability.4.S1 fragment mediates broad-spectrum resistance in soybean hairy rootsUsing soybean hairy roots,we showed that a wide range of SMV strains could infect soybean hairy roots,suggesting that the soybean hairy root systems can be used to verify the function of antiviral genes.To further learn whether RNAi played an important role in resistance to SMV in hairy roots,we amplified a silencing suppressor gene(p19)from Tomato bushy stunt virus and expressed it in soybean hairy roots.After SMV infection,the viral contents in hairy roots expressing p19 gene were significantly higher than that the control groups(WT and EV)in all the time points tested,suggesting that RNAi played an important role in the resistance to SMV in the hairy roots.Next,S1 fragments were expressed in the hairy roots.The viral contents were gradually increased upon inoculation.Importantly,viral accumulation rate in hairy roots expressing S1 fragment was significantly lower than that in the control groups(WT and EV).Compared to 10,20 and 25 days after inoculation(dpi),the hairy roots exhibited the most difference in viral contents between the control group and hairy roots expressingSl segment at 15 dpi.Therefore,we chose this time point to determine the resistant levels to other SMV stains.The results showed that the antiviral activities mediated by S1 fragment were strong to all the tested SMV strains,although little differences in the resistance levels were slightly different.The results suggested that S1 fragment possessed a broad-spectrum resistance to SMV and can be used to develop transgenic soybean for anti-SMV breeding. |
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