Cloning And Functional Analysis Of An NAC Transcription Factor Related To Powdery Mildew Resistance In Wheat

Powdery mildew is one of the three major diseases of wheat in China.The rapid evolution of the powdery mildew pathogen races has become a big challenge for the wheat breeding.Resistance genes cloning as well as mechanism studying of broad spectrum resistance will provide important gene resouces and theoretical evidence for the wheat powdery mildew resistance breeding.In our previous studies,a serine/threonine kinase gene(Stpk-V),the key member of broad spectrum resistance to powdery mildew resistance gene Pm21 from Haynaldia villosa,has been cloned,and the resistant transgenic plants transformed with Stpk-V has been developed.A powdery mildew susceptible mutant NM14 of resistant varity Nannong9918 has also been obtained by EMS treatment To study the mechanism of broad spectrum resistance,the digital gene expression data was obtained from the Stpk-V transgenic plants and its receptors,and from the resistant Nannong9918 and its mutant NM14.A NAC transcription was screened from the differencially expressed data,which showed different expression pattern between the two resistant lines and the suceptble lines.The NAC transcription genes was induced in the four materials,but in the resistant lines the high expression level was sustained for longer time.This result suggested that TaNACs was involved in the powdery mildew resistance pathway.NAC(NAM,ATAF1/2,CUC1/2)transcrption factor,a kind of transcription factor specific to plants,were found in Arabidopsis,rice,wheat,soybeans and other species.Previous studies on plant NAC transcription factors mainly focused on their roles in the plant growth,development and abiotic stress regulation.In recent years,NAC transcription factors were found to be induced by fungi,bacteria and virus,and participated in a wide range of plant defense responses.The gene cloning,gene expression pattern,structure characterization,transcrption activity testing,subcellular localization and function evaluation were conducted in this study on the basis of the above result.The function of this gene involed in the nonhost resistance were also studied.The main results are as follows:1.Screening and cloning of TaNACs geneAccording to the digital gene expression profile,TaNACs gene was cloned.Protein structure analysis indicated that the gene contained the five conserved domains of NAC family,including A,B,C,D and E.Three homologous genes were searched on the NCBI database using Blastn using the TaNACs sequence as query.Primers specific to the three genes were designed based on the multiple sequence alignment.And then the three homologous genes fromdifferent genomes were cloned from cDNA of Bgt icolulated Stpk-V transgenic plants,namedas TaNACs-A,TaNACs-B,TaNACs-D.2.Expression pattern of TaNACsgeneTo explore the expression pattern of TaNACs and its homologous genes,the expression was analysed in the resistant and susceptible materials before and after Bgt inoculated firstly.In the resistant materials,TaNACs and its homologous were induced by Bgt and reched the highest level,at 6 hai and then decreased slightly;while in the susceptible materials,the expression levelof TaNAof and its homologous genes rised again at 72 hai.Secondly,we examined the response of TaNACs to hormones as well as other pathogens in wheat.The results show that the responses of TaNACs and its homologous genes to Pst was similar to their responses to Bgt.the expression level of TaNACs and TaNACs-B were up-regulated in resistant materials at 24 hai,and then decreased;while in the susceptible materials,TaNACs were induced again at 72 hai.It should note that TaNACs-D is not affected by Pst and Bgt.Finally,the gene expression levels were tested in the powdery mildew resistant material treated with different hormones,and it was found that TaNACs and its homologous sequences were affected by jasmonic acid(JA)and Salicylic acid(S A).It was proposed that the TaNACs was particpated in different signaling pathways mediated by SA and JA because TaNACs responded differently to JA and SA.It was also suggested that the deiffernce of the promoter regions of the three TaNACs leading to the different expression pattern under the same treatment.3.Transcriptional activation activity assay of TaNACsTaNACs and its homologous sequences were inserted to BD vector and then transformed into yeast.The reporter gene GUS was activated in the transformed cells,showing that the TaNACs gene has a transcriptional activation activity and localization in the nucleus.4.Function analysis of TaNACs by using the transient expression assay and VIGS technologyUsing transient expressionassay,the three genes were transformed into the epidermal cells of susceptible Yangmai 158 to make the genes overexpressed.The result showed that the hautorium index was significantly decreased in the NAC gene overexpressed cells,suggesting that overexpressing TaNACs can improve wheat resistance to powdery mildew.On the other hand,it was found that the development of Bgt was promoted in the TaNACs silenced leaves of Nannong 9918 and Stpk-V high-generation stable transgenic plants.This study demonstrated that TaNACs was involved in the resistance response of wheat to powdery mildew from two aspects.5.Genetic transformation of TaNA Cs VgeneFor the further study of TaNACs gene function,transgenic plants were developed using Yangmai 158 as receptor by gene gun mediated transformatioa The resistance to powdery mildew was evaluated using the To generation lines and the Ti generation lines.The resistance of some TO generation lines showed increased resistance to powdery mildew.6.Study on the non host resistance of TaNACsThe homologous gene of TaNACs in barley was involed in the basal resistance to powdery mildew.In this study,we found that the TaNACs was also participated in the resistance progress to the powdery mildew in wheat.To find whether theTaNACs participated in the nonhost resistance,the wheat-Bghand barley-Bgt interaction system was used for the gene function study.