Functional Study Of A Natural Variant Of The RNA Dependent RNA Polymerase 1 From Nicotiana Benthamiana In Anti-viral Defense

RNA dependent RNA polymerase plays a critical role in amplifying silencing signals in plants.It has been reported that RDR1 is required for antiviral defense in plants.Compared with other Nicotianna species,Nicotiana benthamiana displays enhanced susceptibility to the infections of various RNA viruses.This enhanced susceptibility is correlated with a 72-nt(nucleotide)insertion in the open reading frame of the NbRDRl in Nicotiana benthamiana(designated as natural NbRDR1 mutant or NbRDR1m),which presumably disrupts its function(Yang et al.,2004).However,the direct evidence in establishing the causal relationship between the 72-nt insertion in NbRDRlm and the enhanced susceptibility is still lacking.To confirm the postulation,we removed the 72-nt insertion in NbRDR1m and converted it into its functional prototype by fusion PCR approach.Next,the transgenic Nicotiana benthamiana lines transformed with prototype NbRDR1 under a 35S promoter were generated.The true transgenic lines were screened on the agar plates supplied with antibiotics and confirmed by genomic PCR.The expression levels of the NbRDR1 transgene were examined by RT-PCR.Two transgenic lines with relative higher expression of NbRDR1 and one line with relative lower expression were then challenged with tobamovirus,Tobacco mosaic virus(TMV)or nontobamovirus,Turnip mosaic virus(TuMV)and Potato virus X(PVX),all of which were fused with GFP(termed TMV-GFP,TuMV-GFP and PVX-GFP,respectively),to determine the extent of protection provided by NbRDRl expression.The wild type and empty vector control plants were included as negative controls.Our results revealed that the transgenic plants showed enhanced resistance to virus infections compared with wild type and empty vector plants in a viral genus-independent manner.It appeared that over-expression of NbRDR1 significantly reduced both the GFP intensities and the systemic movements of these three GFP tagged viruses.Moreover,more severe viral symptoms were displayed on wild type and vector control plants than on transgenic plants after being challenged with nontobamovirus TuMV-GFP and PVX-GFP.Taken together,our results confirmed that the prototype NbRDR1 plays an important role in antiviral defense and the 72-nt insertion within the NbRDR1m is,at least partially if not fully,responsible for the enhanced susceptibility to different genus of RNA viruses observed in Nicotiana benthamiana.