Screening And Functional Analysis Of Regulatory Factors Of Wheat Rhythm Gene TaLHY

Wheat stripe rust caused by Puccinia striiformis f.sp.tritici is one of the main diseases that seriously damages wheat production.Plants can use their biological clock to help them adapt to changes in the external environment.The endogenous clock core components mainly regulate the expression of other genes through transcription-translation feedback loops,helping plants gain a growth advantage under biotic and abiotic stress conditions.WRKY transcription factors and the clock gene LHY(LATE ELONGATED HYPOCOTYL)both play important roles in plant defense responses against pathogens.TaLHY is a wheat core rhythmic gene cloned in previous laboratory work,and five splicing isoforms are formed during its transcription process.RNAi silencing and overexpression of TaLHY transgenic plants have proved that TaLHY is involved in the resistance to stripe rust disease,but its molecular regulatory mechanisms in disease resistance are still unclear.Based on this,this study used yeast one-hybrid(Y1H)screening to identify the upstream regulatory factors of the TaLHY gene,and preliminary investigations using fluorescence quantitative PCR,subcellular localization,LUC analysis,DNA Pull-down,and VIGS were used to explore the functions of the regulatory factors and their regulatory mechanisms in TaLHY,aiming to provide basic information for the regulatory network of TaLHY involved in the resistance to stripe rust disease in wheat.The main research results are as follows:1.A total of 164 clones were obtained from the yeast one-hybrid screening library,and150 different genes were identified by BLAST comparison to the Chinese Spring reference genome.Forty-three genes were found to interact with the TaLHY promoter after one-to-one yeast one-hybrid verification,including six proteins containing HMG-box,AP2/ERF domain,AT-hook motif,TGA domain,PHD domain,and one protein containing a WRKY domain,which are likely to be the transcriptional regulatory factors of TaLHY.2.Transcriptional activation verification experiments were conducted to validate the transcriptional activation function of the WRKY domain protein.It was found that the protein contains a transcriptional activation domain and is localized in the nucleus of wheat protoplasts.Through online database analysis and homologous sequence alignment,it was determined that the protein is TaWRKY20,a member of the class I WRKY family transcription factors with two WRKY domains.3.Using qPCR method,the tissue-specific expression of TaWRKY20 was analyzed.The results showed that TaWRKY20 was mainly expressed in leaves of the resistant stripe rust wheat variety CN19,followed by spikes,stems and roots,with the lowest expression level in grains.In the susceptible stripe rust wheat variety MY11,the highest expression levels were in leaves and stems,followed by spikes,and the lowest in roots and grains,indicating that TaWRKY20 is expressed in both resistant and susceptible wheat leaves.TaWRKY20 responds quickly to stripe rust fungus,salicylic acid,and chitin induction,and its response speed is slightly faster than TaLHY.4.Yeast one-hybrid,LUC analysis,and DNA Pull-down confirmed that TaWRKY20 can positively regulate its expression by binding to the W-box element in the TaLHY promoter region.This indicates that TaWRKY20 functions as an upstream regulatory factor of TaLHY and regulates its transcriptional expression in response to stimuli such as stripe rust fungus,chitin,and salicylic acid,participating in the defense response to stripe rust disease.5.The relative expression level of TaWRKY20 was detected using RNAi silencing and overexpression of TaLHY transgenic plants.It was found that the expression level of TaWRKY20 decreased or increased correspondingly with the decrease or overexpression of TaLHY,suggesting that TaLHY has a reverse regulatory effect on TaWRKY20.Using CUT&Tag,yeast one-hybrid,and LUC analysis,it was demonstrated that the splicing isoform protein TaLHY718 aa can bind to the MBS element on the TaWRKY20 promoter to positively regulate its expression.In addition,the different splicing forms of TaLHY can synergistically regulate the transcriptional activity of the TaWRKY20 promoter,indicating that TaLHY may maintain the basal level of TaWRKY20 under no external stimulation by synergy among its different splicing isoforms.6.Transient silencing of TaWRKY20 through VIGS weakened the resistance of wheat cultivar CN19 to stripe rust pathotype CYR32.The effector PSEC17 of rust fungus that inhibits wheat PTI response can transiently suppress the transcriptional activity of TaWRKY20 promoter in wheat protoplasts,indicating that TaWRKY20 may participate in regulating the wheat stripe rust defense response process through early PTI.In summary,this study identified a WRKY transcription factor TaWRKY20 that regulates TaLHY and has a feedback mechanism between them.TaWRKY20 is induced in the early stages of PTI pathway during rust fungus infection,positively regulating TaLHY to participate in the defense response against wheat stripe rust.The splicing bodies of TaLHY synergistically regulate the expression of TaWRKY20 to maintain its basal level in wheat in the absence of external stimuli.