Soybean Mosaic Virus Disease Resistance Gene Screening And Functional Verification Of GmSN1

Soybean mosaic virus(SMV)infection is a kind of soybean disease which seriously affects the yield and quality of soybean.The pathogen is a virus of the genus potyvirus Y,there are different virus strain classification systems based on either susceptible or resistant on different choices of soybean line groups.Although some SMV disease resistance genes have been reported,their function and mechanism of the resistance have not been fully interpreted.The aim of the present study was to identify and characterize endogenous SMV resistant genes.Two soybean cultivars were used,namely a cultivar R-SMV which resistant to SMV strains SN1 and SN3,and a susceptible cultivar S-SMV.Genechip experiments and differential expressed genes analyses were conducted.A total of 7791 genes were found differential expressed among mock and SMV inoculated R-SMV and S–SMV plants.Because there is no set to repeat,so we put more strictly screening conditions,the different expression genes(DEGs)were more four times instead of the usual 2 times.There were 3226 different expression genes(DEGs;4 folds change)between R-SMV SMV incoluation vs.R-SMV mock;3106 DEGs between S-SMV SMV incoluation vs.S-SMV mock;1509 DEGs between R-SMV mock vs.S-SMV mock;5083 DEGs between R-SMV SMV incoluation vs.S-SMV SMV inoculation.There were 2168 genes up-regulated in at least one comparison,but down-regulated in at least another comparison.We can see that there was relatively less DEGs between the resistant and susceptible cultivars with mock(inoculatedwith PBS buffer)inoculation,while more than three times of DEGs were identified from the SMV inoculation.The DEGs were further processed with AGRIGO,the results showed that genes in“immunization” were found enriched in resistant cultivar,probably because more growth related genes were activated in the R-SMV than in the S-SMV after SMV inoculation;There were evidences indicated that the mobilization and SMV might be hindered in the R-SMV cultivar.Different hormones response patterns were also observed between the cultivars.Putting these results together,it can be speculated that multiple disease resistant genes existed in the R-SMV cultivar.Seventeen candidatedisease resistant genes which containing conservative domains were identified,enhanced SMV resistance were observed in 3 genes(GmCnx1,GmAKT2,and KR3)over-expression transgenic soybean lines.Virus infection induced down-regulation of resistance-related genes,might also account for the susceptibility of some soybean cultivars.Eighteen DEGs which were down-regulated more than 16 times in S-SMV inoculation vs S-SMV mock inoculation were selected to test their virus resistances,which included 7 GASA domain containing genes,4 AUX/IAA family genes,5 LRR domains containing genes,and 2 cytochrome P450 family genes.These genes were cloned and constructed to plant over-expression vectors,and then transformed to Arabidopsis.GmSN1,a GASA domain containing gene,significantly increased the resistance to turnip mosaic virus(TuMV)in the transgenic Arabidopsis.To further elucidate the virus resistance mechanism of the gene,a comparative transcriptome sequencing experiment was conducted.Analyses showed that the GmSN1 transgene-related DEGs were mostly enriched in growth related genes,yet genes involved in cell death were uprelated in the susceptible mock transgenic lines,These transcriptome derived results showed perfect consistence with our previous Genechip analyses.