Functional Analysis Of HIR3 Gene In Plant Resistance To Rice Stripe Virus Infection

Rice stripe disease,resulted from Rice stripe virus(RSV),causing serious economic losses of rice yield and quality,threatening agricultural production security and destroying agricultural sustainable development,is an important rice viral disease.Deciphering the interaction mechanism between RSV and plant host,and identificating key host factor involved in RSV infection could offer better strategies for controlling rice stripe disease.RSV is transmitted by the small brown planthopper(Lapdelphax striatellus,SBPH)in natural host rice,and it can also mechanically infect experimental host N.benthamiana plants.In the previous work of our laboratory,we analyzed the expression of genes in RSV-infected N.benthamiana using the commercial GeneChip Tomato Genome Array,and found a sequence annotated as HIR3(Accession No.:BI923297)was upregulated two-fold in RSV-infected N.benthamiana.The hypersensitive induced reaction(HIR)gene family is associated with hypersensitive response(HR),participating in plant defense against pathogens.Of all the known HIRs,the function of HIR3 is less studied.The defense roles of HIRs against bacterial and fungal pathogens were well investigated,while the response of HIRs to viral infection was not clear.We here report that HIR3 gene was upregulated in Rice stripe virus(RSV)-infected N.benthamiana.Silencing of NbHIR3s in N.benthamiana resulted in serious RSV symptoms and increased accumulation of RSV RNAs,decreased level of salicylic acid(SA),and was accompanied by down-regulated expression of EDS1,NPR1 and PR1 genes in the SA pathway.While SA treatment enhanced the resistance of NbHIR3s-silenced plants against RSV infection,showing that NbHIR3s are associated with regulation of SA.To explore the relationship between NbHIR3s and SA further,we overexpressed NbHIR3.1 and NbHIR3.2 under Cauliflower mosaic virus(CaMV)35S promoter,respectively.The expression of NbHIR3.1 or NbHIR3.2 induced significant accumulation of SA and hydrogen peroxide(H2O2)in the area of cell death,but not in EDS1-silenced or NahG transgenic N.benthamiana plants,indicating that NbHIR3s induce EDS1-and SA-dependent cell death,playing an important role in host resistance to RSV infection.To further determine the biological function of NbHIR3s,we made stable transgenic lines of N.benthamiana overexpressing either NbHIR3.1 or NbHIR3.2 and examined the effect of overexpression of NbHIR3s on pathogen infection.Results showed that overexpression of NbHIR3s reduces accumulation of RSV RNAs in N.benthamiana,with significantly more SA and the upregulated expression levels of EDS1,NPR1 and PR1 genes.Transgenic plants expressing NbHIR3s proved also to be resistant to two viruses unrelated to RSV,namely turnip mosaic virus(TuMV)and potato virus X(PVX),and bacterial pathogen P.syringae pv.tomato(Pst DC3000).We next investigated whether OsHIR3(Accession No.:Os06g0136000)in rice,the natural RSV host,also had a resistance function against RSV.We cloned the rice OsHIR3 gene and analyzed the cell death induced by OsHIR3 and its role in RSV infection.Similar to NbHIR3,the transient heterologous expression of OsHIR3 induced cell death,accompanied by accumulation of SA and H2O2,but not in EDS1-silenced or NahG transgenic N.benthamiana plants.Overexpression of OsHIR3 reduces accumulation of RSV RNAs in rice,and enhance rice resistance to Xoo,suggesting that OsHIR3 may also mediated SA-dependent basic resistance in rice.Taken together,the results presented here suggest that the HIR3s positively regulate the EDS1-and SA-dependent pathway to induce cell death,playing roles in basal resistance against viral and bacterial pathogens,which provides a deeper insight into the function of HIRs in defense.