Molecular Regulation Mechanism Of A Susceptibility Gene TaSWEET14 From Wheat In Response To Stripe Rust Fungus

Wheat stripe rust seriously affects the safe production of wheat in China and threatens the national economy and people’s livelihood.As a kind of obligate biotrophic parasitic fungus,Puccinia striiformis f.sp.tritici absorbs nutrients from host cells for growth and development through infecting structure,haustorium.Carbohydrates are one of the most important forms of nutrition that pathogens obtain from host cells,and sugar transfer is dependent on sugar transporters.In our previous study,we identified a sugar transporter gene TaSWEET14 that was up-regulated by Pst.After silencing the gene by BSMV-VIGS(virus induced gene silencing),it was found that the expansion of hyphae of Pst was restricted and the amout of sporulation was significantly reduced,indicating that it plays an important role in the pathogenic process.In order to further analyze the molecular regulation mechanism of TaSWEET14,in this study we used yeast one-hybrid technique to screen and obtain the transcription factor of regulating TaSWEET14,and then the interaction target of the transcription factor was screened by yeast two-hybrid technique,and performed mutual verification and functional analysis.The results of this study laid the foundation for revealing the molecular mechanism by which the Pst promote sugar transport by hijacking the host sugar transporter TaSWEET14 to ensure its sugar supply.The main research of this study are as follows:1.Cloning and analysis of the promoter of TaSWEET14 geneIn the URGI database,BLAST alignments revealed that the SWEET14 gene,and the sequence of the initiation codon ATG upstream of 2000 bp containing the promoter transcriptional regulatory region was selected as a promoter candidate sequence.It was named A14 NP.A14NP was cloned by PCR using Su11 wheat genomic DNA as a template.Analysis of the cis-acting element on its sequence revealed that it contains the core elements of a typical eukaryotic gene promoter,regulatory elements involved in plant hormone induction,cis-acting elements involved in light regulation,and MYB transcription factor regulatory elements.2.Yeast one-hybrid screening for transcription factors regulating TaSWEET14 geneUsing the yeast one-hybrid system,the promoter of TaSWEET14 gene was used as bait to capture 7 candidate transcription factors in the c DNA library of wheat-stripe rust interaction.After verification,it was confirmed that only the wheat transcription factor TaMYB50 was able to grow at selection plate contained an Ab A concentration of 300ng/ml and was therefore identified as the final candidate transcription factor.3.Verification of interaction between TaSWEET14 promoter and transcription factor TaMYB50The transcription factor TaMYB50 and TaSWEET14 promoter were co-expressed in tobacco leaves by means of a tobacco transient expression system.The results showed that the GUS activity of the tobacco leaves expressing the TaSWEET14-promoter:GUS fusion plasmid alone was 1 time higher than that of the tobacco leaves co-expressing the TaSWEET14-promoter:GUS and transcription factor TaMYB50 fusion plasmids.This indicates that the transcription factor TaMYB50 binds to the TaSWEET14 promoter and inhibits transcription.4.TaMYB50 gene function analysisThe TaMYB50 gene is 231 bp in length and belongs to the MYB-related transcription factor family and contains one conserved MYB domain.TaMYB50 was located in the cell nucleus by transient expression of tobacco.Transcriptional activation verification assay confirmed that TaMYB50 has a repressor.Real-time quantitative PCR analysis confirmed that TaMYB50 was up-regulated in the compatible combination of wheat and Pst interaction and the up-regulation time piont was consistent with TaSWEET14.5.Screening and identification of TaMYB50 interaction targetsA Pst effector protein Ps15882 interacted with TaMYB50 was obtained from the c DNA library of wheat-stripe rust by using DUALhunter system.The realities of their interactions are clarified by Bi FC and Co-IP assays.The Ps15882 gene is 684 bp in length and has a protein isoelectric point(p I)of 7.55 and a molecular weight of 24.65 k Da.Pfam and Inter Pro Scan software analyzed this protein sequence without finding a special functional structure.The signal peptide prediction software Signal P 4.1 showed that the1-22 amino acids of Ps15882 was a signal peptide.The yeast signal peptide screening system and color reaction demonstrated that the signal peptide of Ps15882 has a secretory function.