Molecular Mechanism Analysis Of The Salicylic Acid Signaling Pathway Involved In The Resistance Mediated By The Wheat Powdery Mildew Resistance Gene Pm21

Wheat powdery mildew is an epidemic fungal disease caused by Blumeria graminis f.sp.tritici,which seriously threatens the yield and quality of wheat.Develo Pment of new wheat varieties with high yield and disease resistance is the most economical and effective measure for prevention of wheat powdery mildew.Plant hormones play important regulatory roles in wheat disease resistance,among which salicylic acid plays a critical role in regulating wheat disease resistance.The wheat material 92R137,a wheat-Haynaldia villosa tranlocation line T6VS·6AL developed previously in our laboratory,confers broad spectrum resistance to wheat powdery mildew,and the broad-spectrum powdery mildew resistance gene Pm21 was located on 6VS of Haynaldia villosa.Since Pm21 has been widely used in production and is a broad-spectrum resistance gene from wild species,it is of great value to analyze its resistance mechanism for the sustainable utilization of Pm21 gene and the engineering breeding of resistance gene.In the early stage of this study,the Pm21-interacted proteins were screened by yeast two-hybrid technique,and three proteins involved in salicylic acid pathway were identified.In this study,the molecular mechanism of salicylic acid signaling pathway in Pm21-mediated powdery mildew resistance was explored by studying the effect of external application of salicylic acid on wheat powdery mildew resistance and the effect of endogenous salicylic acid content on the expression of signaling pathway response genes.At the same time,two PAL and one TGA genes interacting with Pm21 were cloned,and their functions and mechanisms were studied.In addition,based on the published genomic information of wheat and other cereal plants,PAL and TGA gene families were genome-widely explored and evolutionarily studied.1.Study on the effects of salicylic acid on Pm21 mediated broad-spectrum resistance of wheat powdery mildewAfter SA,SA inhibitor（ABT）and dd H₂O were sprayed on Nannong 9918 and mutant NM14,spores development,reactive oxygen species production and cell necrosis in different samples were observed at different time points after Bgt inoculation.The results showed that external application of SA could increase the accumulation of reactive oxygen species,produce stronger HR response,and enhance the disease resistance of plants at the early stage of Bgt infection,however,external application of ABT could inhibit the accumulation of reactive oxygen species,reduce the number of allergic cell necrosis,and reduce the disease resistance of plants in the late stage of Bgt infection.At the same time,the endogenous salicylic acid content and phenylalanine ammonia-lyase activity were evaluated in Nannong9918 and mutant NM14 using the samples with Bgt inoculated for different time.The results showed that the endogenous salicylic acid content of Nannong9918 and NM14 increased after Bgt inoculation.However,the endogenous salicylic acid content of Nannong 9918 was significantly higher than that of NM14 at 12 hpi and 24 hpi,suggesting that salicylic acid perhaps participated in Pm21-mediated resistance to powdery mildew at the early stage of pathogen infection.2.Cloning of TaPAL-2A/2D and TaTGA-2DThree proteins involved in salicylic acid pathway,PI190,PI207 and PI309,were isolated from 92R137 by yeast two-hybrid technique,which was corresponding to the TRAESCS2A02G380900,TRAESCS2B02G113200 and TRAESCS2D02G377200 in Ensemble Plants database website（http://plants.ensembl.org/index.html）.These three proteins were named as TaPAL-2A,TaTGA-2B and TaPAL-2D,respectively.Full-length c DNA of TaPAL-2A and TaPAL-2D genes related to salicylic acid synthesis and TaTGA-2B gene downstream of salicylic acid signal transduction were cloned from Nannong9918.3.Bioinformatics analysis of PAL and TGA gene familiesThe PAL and TGA family genes in wheat,barley,Arabidopsis,rice and maize were genome-widely scanned,and chromosomal localization,cluster analysis and evolutionary analysis were carried out on both gene families in wheat.The PAL family is divided into Clade1-Clade6.The members of Clade2 branch are all from Arabidopsis,and the other branches are all from gramamines.Most of the genes in the same species were clustered in the same clade,indicating that the PAL gene family diverged dramatically.At the same time,the number of PAL gene family members in gramineae species is relatively conservative,and there is no amplification phenomenon during the formation of tetraploidy and hexaploidy in wheat.The TGA family is divided into Clade1-Clade6.Except for Clade2,each branch has members from Arabidopsis,indicating that the TGA gene family is relatively conservative in different species.Members of the TGA gene family did not show gene amplification in the process of polyploidy formation and domestication of wheat.The expression patterns of PAL gene family and TGA gene family in wheat under different tissue,biological and abiotic stresses were analyzed,and the possible biological processes involved in PAL gene family and TGA gene families were speculated.The results showed that TaPAL-2A and TaPAL-2D expressed at high level but were not induced by drought and heat stress.The expression of TaTGA-2B in tissues is not specific,and the expression of TaTGA-2B is consistently high before and after induction by stripe rust and powdery mildew fungus.4.Functional analysis of TaPAL-2A/2D and TaTGA-2D genesIn order to investigate whether the cloned salicylic acid pathway related genes are involved in the specific resistance process of Nannong9918,the interaction ability between TaPAL-2A,TaPAL-2D,TaTGA-2B and Pm21 were investigated,and the cellular region where they interacted were preliminarily explored by using yeast biheterozygous,bimolecular fluorescence complementation and subcellular co-localization.The results showed that both TaPAL-2A and TaTGA-2B could interact with Pm21.RT-PCR analysis showed that TaPAL-2A and TaPAL-2D were up-regulated by Bgt in Nannong9918.TaPAL-2D gene was silenced in Nannong9918 by using VIGS technique,and silencing of TaPAL-2D in Nannong9918 could not significantly reduce the plant resistance to the disease.