Study On The Antiviral Function Of Transcription Factor NbWRKY18

WRKY is a type of plant specific transcription factors(TFs)with highly conserved WRKY and zinc finger domains.The TFs specifically bind to the W-box motif in the promoter region of target genes and regulate the expression of target genes.WRKY family included numerous members and widely distributed in the plant.According to their conserved domains and introns,WRKY can be divided into three categories,?,?,?,respectively.Previous studies in our laboratory showed that Nicotiana benthamiana WRKY18(NbWRKY18)gene may be involved in the viral infection,but its functions remain unclear.In order to explore the biological function of NbWRKY18,the gene was first cloned and sequence alignment and phylogenetic analysis showed that NbWRKY18 contained a highly conserved WRKY structure(WRKYGQK)and zinc finger structure(C₂H₂ type),and clustered into IIa branch.Then NbWRKY18 was subcloned into a prokaryotic expression vector for inducible expression of its recombinant protein in E.coli and preparation of the specific antibody against the protein.Subcellular localization analysis showed that NbWRKY18 was localized to the nucleus.Quantitative assay by q RT-PCR showed that the expression pattern of NbWRKY18 was not only affected by high salinity and drought stress,but also induced by the infection of four plant viruses and salicylic acid(SA).The results supported the hypothesis that NbWRKY18 gene might be involved in viral infection.In order to further explore the relationship between NbWRKY18 and viral infection in plant,the gene was under the control of a constitutive promoter and transformed into N.benthamiana by Agrobacterium-mediated method to obtain transgenic plants overexpressing NbWRKY18(35S:NbWRKY18).Meantime,the virus-induced gene silencing(VIGS)technology was used to obtain plants silencing NbWRKY18(TRV:NbWRKY18).The Agroinfiltration method was used to respectively inoculate the infectious c DNA clones of Tomato Mosaic Virus(To MV)and Turnip Mosaic Virus(Tu MV),which were fused with a gene encoding green fluorescent protein(GFP).Fluorescence observation of virus-infected plants showed that the fluorescence intensity of GFP in the transgenic plants(35S:NbWRKY18)was weaker than that in control plants,while the fluorescence intensity of GFP in the silenced plants(TRV:NbWRKY18)was stronger than that in control plants.It was speculated that the transcription factor NbWRKY18 might have antiviral function.Further analysis with Northern blot and Western blot showed that the transcription and translation from both To MV and Tu MV infectious clones in the transgenic plants(35S:NbWRKY18)were lower than those in the control,while the transcription and translation from both To MV and Tu MV infectious clones in the silenced plants(TRV:NbWRKY18)were higher than those in the control,which supported from the molecular level the viewpoint that the transcription factor NbWRKY18 may have antiviral function.In order to explore the antiviral mechanism of the transcription factor NbWRKY18,high performance liquid chromatography-mass spectrometry(HPLC-MS)was applied to determine the content of 3 kinds of hormones in transgenic(35S:NbWRKY18),silenced(TRV:NbWRKY18)and control plants.The results showed that the content of salicylic acid(SA)was significantly higher in transgenic(35S:NbWRKY18)plants than that in the control plants before and after virus inoculation,and the contents of jasmonic acid(JA)and abscisic acid(ABA)were lower than those in the control;however,the hormone contents in silenced(TRV:NbWRKY18)plants display a opposite pattern,in which the content of SA was lower than the control and the contents of JA and ABA was higher than the control.These findings indicate that the transcription factor NbWRKY18 may positively regulate the antiviral response by influencing the content of hormones and participating in the hormone signaling pathways in plants.