Functional Characterization Of Key Genes HvWRKY6 And HvWRKY70 Of Systemic Acquired Resistance

Systemic acquired resistance(SAR)is an inducible form of plant defense that confers broad-spectrum immunity to secondary infections beyond the initial infection ction site.NPR1(Non-expresser of PR genes 1,also known as NIM I and SA11)protein acts as the master regulator of plant SAR.In model plants of Arabilopsis and rice,several WRKY transcription factors have been suggested to play important roles in SAR.WRKY transcription factors play various roles in plant responses to both biotic and abiotic stresses,as well as physiological processes.However,different from SAR responses observed in Arahidopsis,three types of induced resistance have been demonstrated in wheat and barley,including acquired resistance(AR),systemic,immunity(SI)and BTH-induced resistance(BIR).However,roles of WRKY genes during SAR in these Triticeae crops are largely unknown.To investigate the transcriptional regulatory network of SAR and function of the NPR1 protein in these Triticeae species,In our previous study,we performed RNA-seq analysis on samples collected from either the region adjacent to Pseudomonus syringae DC3000 infiltration or BTH sprayed area in barley transgenic lines wNPR1-OE and HvNPR1-Kd,as well as wild-type plants.We then checked the expression profiles of all the WRKY genes in these two transcriptome databases,and found that HvWRKY6 and HvWRKY70 were higher induced in wNPRI-OE than in the wild-type plants upon both P.syringae DC3000 inf-ection and BTH treatment.Based on these results,we speculated that these two WRKY genes might serve as key transcriptional regulators shared by BTH-induced resistance and the NPR1-mediated AR.To investigate the subcellular localization of HvWRKY6 and HvWRKY70 proteins in plant cells,a green fluorescent protein(GFP)tag was fused to each end of the HvWRKY6 and HvWRKY70,and the recombinant constructs were transiently expressed in Nicotiana benthamiana using Agrobacterium.Clear fluorescence for HvWRKY6 and HvWRKY70 were observed in the nucleus.We have generated wheat transgenic lines overexpressing the HvWRKY6 or HvWRKY70 gene under the maize Ubiquitin promoter.Seedlings of transgenic materials and wild-type plants were infiltrated with P.syringae DC3000 or sterile water as a control,and Magnaporthe oryzae(Mo)isolate P131 was inoculated as the secondary pathogen in the region adjacent to the P.syringae DC3000 infiltration area.Compared with the wild-type plants,we observed more pronounced resistance to Mo isolate P131 in the HvWRKY6-OE.However,we did not observe any elevated resistance to Mo in HvWRKY70-OE.To investigate the possible regulatory downstream genes of the HvWRKY6 during the NPR1-meidated acquired resistance,a series of qRT-PCR assays were performed.Gene expression results indicated that HvWRKY6 might regulate TaPR1a,TaPR2 and TaPR4b genes during AR response induced by P.syringae DC3000.On the other hand,upon BTH treatment,the expression levels of all the tested PR and BCI genes were significantly higher induced in the HvWRKY70-OE than those in the wild-type plants,which indicated that the HvWRKY70 gene might be key regulator of the BTH-induced resistance.The generated wheat transgenic lines were then subjected to inoculate with wheat stripe rust(Puccia striiformis f.sp.tritici,Pst).The sporulation of the virulent Psi pathotype CYR32 was significantly diminished or delayed in the wheat transgenic lines HvWRKY6-OE and HvWRKY70-OE compared with the wild-type plants.In conclusion,we found HvWRKY6 and HvWRKY70 as key transcriptional regulators of SAR in Triticeae crops.The roles of these two WRKY genes in wheat SAR were initially clarified.The generated wheat transgenic lines over-expressing these identified WRKY genes may be potential resources for the improvement of wheat broad-spectrum disease resistance to various fungal diseases.