Preliminary Study On Molecular Mechanism Of TaNH2 Involved In Wheat Resistance To Puccinia Striiformis F. Sp. Tritici

Puccinia striiformis f.sp.tritici（Pst）is an obligate biotrophic fungus.Wheat stripe rust caused by Pst is a serious threat to food security in China.The most economical and effective measure to control wheat stripe rust is breeding and planting disease-resistant varieties.However,the rapid virulence variation of Pst leads to a high risk of loss of resistance in wheat cultivars.Therefore,there is an urgent need to identify disease resistance factors in wheat and reveal the molecular mechanisms mediated by the disease resistance factors,which can provide theoretical basis and genetic resources for stripe rust resistance breeding.The previous research has demonstrated that TaNH2（NPR1 homolog）,a key component of Salicylic acid（SA）signaling pathway,positively regulates resistance to Pst in wheat.However,TaNH2-mediated resistance mechanism needs to be further elucidated.This study conducted a series of studies on TaNH2-mediated disease resistance mechanism.The following results were obtained.1.TaNH2 interacts with the b ZIP transcription factor TaTGA2.1.To identify TaNH2-interacting proteins,we performed yeast two-hybrid screening.From the c DNA library constructed from Pst-infected wheat leaves,we found totally 11 proteins that potentially interact with TaNH2.The interaction between TaNH2 and TaTGA2.1 was confirmed by Y2H,Bi FC,LUC,and Co-IP assays.Sequence analysis revealed that TaTGA2.1includes 336 amino acids with a molecular weight of 37.45 k Da,and has a highly conserved b ZIP domain,which belongs to the TGA transcription factor family of b ZIP family group D.Subcellular localization assays revealed that TaTGA2.1 is localized to the nucleus.2.TaTGA2.1 negatively regulates wheat resistance to Pst.Firstly,the expression levels of TaTGA2.1 were significantly up-regulated during interaction between wheat and Pst.Secondly,Silencing of TaTGA2.1 by virus-induced gene silencing（VIGS）assay resulted in increased resistance to Pst significantly,with the more necrosis area,higher H₂O₂ production,smaller Pst colony area and higher expression levels of PR genes compared to control.Thirdly,transgenic wheat plants overexpressing TaTGA2.1were generated.Overexpression of TaTGA2.1 led to enhanced susceptibility to Pst significantly,with the smaller necrosis area,lower H₂O₂ production,higher Pst colony area and lower expression levels of PR genes compared to control.The above results showed that TaTGA2.1 negatively regulates wheat resistance to Pst.3.TaNH2 inhibits the transcriptional regulatory activity of TaTGA2.1.To explore the mechanism of how TaNH2 regulates TaTGA2.1,dual luciferase reporter gene assay was performed.The result revealed that TaTGA2.1 could activate the expression of Ta CNGC16,possibly through binding to the cis-acting element with TGACG motif in the promoter regions of Ta CNGC16,and TaNH2 inhibits the expression of Ta CNGC16 activated by TaTGA2.1.In a word,TaNH2 may positively regulate resistance to Pst in wheat by suppressing the transcriptional regulatory activity of TaTGA2.1.In summary,this study confirmed that TaNH2 interacts with the b ZIP transcription factor TaTGA2.1,TaTGA2.1 negatively regulates wheat resistance to Pst,and TaNH2 inhibits the transcriptional regulatory activity of TaTGA2.1.This study preliminary analyzed on molecular mechanism of TaNH2 involved in wheat resistance to Pst,and will provide theoretical basis and genetic resources for stripe rust resistance breeding.