| Soybean mosaic virus(SMV)is the main pathogen of soybean.After SMV infection,soybean showed the symptoms of yellow spot,curling and seed mottle,which severely affect the normal growth and development of soybean and lead to the decline of yield and quality.A soybean gene GmNH23(Glycine max N homolog 23),which is homologous with tobacco N gene and resistant to both tobacco mosaic virus(TMV)and SMV,has been found in previous work.GmNH23 is a typical TN(TIR-NBS)type R protein.Although the antiviral function of GmNH23 gene has been identified,the downstream signaling pathway and functional related genes of GmNH23 have not been reported.Therefore,in this paper,the functions of related genes involved in GmNH23 disease resistance immune response were screened and studied.Firstly,20 functional related genes were cloned and successfully constructed RNAi recombinant vectors for 16 candidate genes,which were silenced in the leaves of soybean variety Hefeng 55 by Agrobacterium mediated infiltration of the RNAi constructs.Overexpression of GmNH23 inhibited SMV infection.The genes related to the function of GmNH23 gene were screened by the change of SMV resistance mediated by GmNH23.The results showed that silencing of GmEDS1,GmSGT1-1,GmSGT1-2,GmJAR1 and GmSGS3 genes inhibited the resistance of GmNH23 gene to SMV,which indicated that these five genes were the key genes in the downstream signaling pathway related to GmNH23 resistance,while the other 11 genes did not participate in GmNH23 mediated resistance.In addition,the expression of GmSGT1-1 and GmSGS3 was induced at 1 day after SMV infection,indicating that they played a role in the early stage of SMV infection,while the expression of GmEDS1,GmSGT1-2 and GmJAR1 was down regulated at 5 days and up regulated at 10 days after SMV infection,indicating that they played a role in the late stage of SMV infection.The plant hormone salicylic acid(SA)is widely involved in plant disease resistance immune response.The results showed that the expression of SA signaling gene PR1,SA synthesis genes GmICS1,GmPAL1.1,and SA receptor genes GmNPR1.2 and GmNPR1.1,were induced by SMV infection,suggesting that SA may participate in the immune response of soybean against SMV.The overexpression of GmNH23 in 17 wt Nicotiana Benthamiana can inhibit the proliferation of SMV and lead to hypersensitive reactions(HR)such as etiolation and cell necrosis.NahG is the coding gene of salicylate hydroxylase from bacteria,and its overexpression plant NahG-Ox is used as the model SA dificient system.In NahG-Ox plants,the inhibition of GmNH23 on SMV was obviously weakened or even totally abolished,and there was no HR response.The overexpression of 3-hydroxylase and 5-hydroxylase coding genes AtS3 H,AtS5H from Arabidopsis thaliana also inhibited the resistance of GmNH23 to SMV.The treatment of SA analogues,methyl salicylate(MESA)and INA(2,6-dichlorooisonic),effectively inhibited the infection of SMV in Nicotiana Benthamiana.These results indicate that SA is the downstream signaling pathway of GmNH23 and the key hormone for SMV resistance. |
