Regulatory Mechanism Of Stripe Rust Fungus Regulating TaSTP3 And TaSWEE14d To Transport Host Sucrose

Access to host-derived sugars is essential for pathogen survival and proliferation.However,few studies have focused on how sugar translocation is strategically manipulated during pathogen colonization.Puccinia striiformis f.sp.tritici(Pst),as a biotrophic obligate fungus,is completely dependent on host-derived sugars for growth and reproduction.It is really conducive to develop effective strategies of disease control by understanding of the molecular mechanism by which strip rust fungi take in sugar from host.In this study,we revealed two pathways of wheat sucrose transporters regulated by Pst to obtain host-derived sugar for growth and reproduction by molecular biology skills.Ⅰ: The Ta WRKY61-Ta WRKY82-Ta VQ5 module transcriptionally regulates the wheat sugar transporter Ta STP3 to mediate the translocation of host-derived sucrose to stripe rust fungi for growth and reproduction.A STP gene with high expression level induced by Pst was previously identified from 79 Ta STPs genes of wheat.RNAi-Ta STP3 transgenic wheat could inhibit the growth and development of Pst mycelia.While after inoculation with Pst in Ta STP3 overexpressing transgenic wheats,the spore quantity of leaves had a significant increase,the area of Pst mycelia increased and the susceptible phenotype was enhanced.These resultes indicate that Ta STP3 might participate in Pst infection of wheat.Five candidate transcription factor genes Ta WRKY17,Ta WRKY19,Ta WRKY61,Ta WRKY82 and Ta PHR1 were previously screened by yeast one hybrid assay.On this basis,this study further clarified the transcriptional regulation of Ta STP3 during Pst infection of wheat.The following main results are obtained:1.GC-MS analysis showed that sucrose content in Ta STP3 overexpressing plants had a significant increase but decreased in RNAi-Ta STP3 plants.The yeast mutant complementation assay showed that Ta STP3 is a dual-transporter to transport glucose and sucrose.In addition,transcriptome analysis of Ta STP3 overexpressing transgenic wheats showed a significant downregulation of several defense-related genes,particularly those whose promoter regions contain sugar responsive elements(SREs),suggesting transcriptional inhibition in these genes in response to enhanced sugar accumulation.2.Five candidate transcription factor genes Ta WRKY17,Ta WRKY19,Ta WRKY61,Ta WRKY82 and Ta PHR1 screened by yeast one hybrid assay were generated overexpressing transgenic wheats.Phenotype identification and functional analysis showed that Ta WRKY19,Ta WRKY61 and Ta WRKY82 TFs specifically activate Ta STP3 during Pst infection of wheat.3.The expression level of Ta WRKY19 was induced by Pst,but the Ta WRKY61 and Ta WRKY82 did not respond to the infection from Pst;and Ta WRKY61 and Ta WRKY82 could interact with each other but did not interact with Ta WRKY19.We hypothesized that Ta WRKY19 has a separate regulatory mode,while Ta WRKY61 and Ta WRKY82 co-regulate Ta STP3 expression,and transcriptional cofactors may be involved to form a transcriptional regulatory complex.By means of transcriptome data and Bi FC assay,we screened a VQ protein that was induced by Pst and interacted with Ta WRKY61 and Ta WRKY82.Then,dual luciferase reporter systems further demonstrated that the Ta WRKY61-Ta WRKY82-Ta VQ5 module enhanced Ta STP3 expression.Ⅱ: The second way is that wheat sugar transporter Ta SWEET14 d hijacked by Pst through Pst15882-Ta MYB50 module to obtain host-derived sucrose for growth and reproduction.The transcriptome data,q RT-PCR and GWAS were previously used in this study to screen and identify a SWEET gene Ta SWEET14 d,which is specifically induced by Pst in wheat.A transcription factor Ta MYB50 regulating Ta SWEET14 d was previously screened by yeast onehybrid assay.On this basis,this study further elucidated the function of Ta SWEET14 d during Pst infection of wheat and how stripe rust fungus hijacks it to acquire host-derived sucrose for growth and reproduction.The following main results are obtained:1.We generated Ta SWEET14 d overexpressing transgenic wheat and knockout wheat of Ta SWEET14 d using CRISPR/Cas9 based gene editing.After inoculation with Pst in Ta SWEET14 d overexpressing transgenic wheats,the number of spores in leaf increased,the area of Pst mycelia increased and the susceptible phenotype was enhanced.Knockout wheat of Ta SWEET14 d could inhibit the growth of Pst mycelia,suggesting that Ta SWEET14 d promotes wheat susceptibility to Pst.2.The yeast mutant complementation assay and GC-MS analysis showed that Ta SWEET14 d could transport sucrose,and the sucrose content in Ta SWEET14 d overexpressing transgenic wheat had a significant increase.Subcellular localization analysis showed that Ta SWEET14 d was localized on the Golgi apparatus in tobacco leave cells and wheat protoplast cells.3.We generated Ta MYB50 overexpressing transgenic wheat and transient silencing plants of Ta MYB50,a transcription factor gene regulating Ta SWEET14 d.Phenotype identification and functional analysis showed that Ta MYB50 is a positive regulator of resistance to Pst infection and could repress the expression of Ta SWEET14 d.EMSA and dual luciferase reporter system analysis showed that Ta MYB50 directly binds to TA-box of Ta SWEET14 d promoter to repress its expression.4.The Pst effector Pst15882 interacts with Ta MYB50 in cytoplasm and nucleus.Pst15882 could inhibit Bax induced programmed cell death in tobacco leaves and callose accumulation in wheat leaves transiently expressing Pst15882.Silencing Pst15882 could restrict the growth of Pst and repress the expression of Ta SWEE14 d.Dual luciferase reporter system analysis showed that Pst15882 may eliminate Ta MYB50-mediated transcriptional repression of Ta SWEET14 d.Host sugar transporters are the core targets in the two ways of wheat stripe rust fungus to transport host sucrose.Host sugar is enriched at the interface between Pst and host cells through transcriptional regulation,providing more available host-derived sucrose for stripe rust fungi.Here,our findings shed light on a novel view for biotrophic fungi uptake host nutrients,and also laid a molecular mechanism foundation for the generation of wheat germplasm resistant to Pst.