Identification Of Virulence Motif Of Important Stripe Rust Fungus Effector PstGSRE1 And Functional Characterization Of TaLOL2 In Wheat

Wheat（Triticum aestivum L.）is an important cereal crop for human beings.Stripe rust,caused by Puccinia striiformis f.sp.tritici（Pst）,is one of the common biotic diseases of wheat,which severely reduces the yield and quality of wheat.Pstis a biotrophic plant pathogen,secreting numerous effectors to regulate host immunity systems through haustoria.Study on the pathogenic mechanisms of Psteffectors and the host immune responses will contribute to the in-depth dissection of Pst-wheat interaction and subsequently provide theoretical basis for generating wheat cultivars with durable resistance to stripe rust.Our previous study identified a glycine-serine-rich effector PstGSRE1,which contains a glycine-serine-enriched conserved motif m9.PstGSRE1 and its m9 motif can block the nuclear accumulation of wheat transcription factor TaLOL2 to suppress wheat defense responses.On the basis of this study,we further explored the virulence motif and the interaction region with TaLOL2 of PstGSRE1-m9.Furthermore,the stable wheat transformants overexpressing TaLOL2 were generated for functional analysis.And the downstream candidate genes of TaLOL2 were preliminarily analyzed.The main results are as follows.1.We generated 21 mutants for PstGSRE1-m9 and transiently expressed the mutants in Nicotiana benthamiana by agroinfiltration.The results showed that glycine and serine of PstGSRE1-m9 are not necessary for its virulence.And the 11^thto 17^th aa region of PstGSRE1-m9（PstGSRE1-m9-M7）were determined to be the virulence motif.Interactions between the14 mutants of PstGSRE1-m9 and TaLOL2 were verified by luciferase complementation assay（LUC）.The results indicated that glycine and serine are necessary for the interaction between PstGSRE1-m9 and TaLOL2.Bimolecular fluorescence complementation（BiFC）and yeast two-hybridization（Y2H）were used to further verify that the M7 region of PstGSRE1-m9 is important to interact with TaLOL2.2.Wheat transformants overexpressing TaLOL2 were inoculated with Pstraces CYR22,CYR29,CYR31,CYR32,CYR33 and CYR34.Compared with the Fielder plants（Wild type,WT）,transgenic plants showed enhanced wheat resistance against Pstraces CYR29,CYR31,CYR32,CYR33 and CYR34.The histological observation and statistics analysis results showed that the accumulation area of H₂O₂ near the infection sites were significantly expanded and the growth of hypha were obviously suppressed in transgenic plants comparing with WT.The qRT-PCR revealed that pathogenesis-related genes（TaPR1,TaPR2）,and TaNOX,which is associated with reactive oxygen synthesis,were up-regulated.And the relative transcript levels of TaSOD and TaCAT3,genes related to eliminate reactive oxygen,were significantly suppressed in transgenic plants during pst-wheat interaction.These data further demonstated that TaLOL2 plays a positive role in the resistance to wheat stripe rust.Furthermore,the candidate regulatory motifs bound by TaLOL2 were identified via DAP-seq.And the results preliminarily indicated that TaLOL2 positively regulates the relative transcript levels of downstream disease-resistant genes during the infection of Pst.In short,PstGSRE1-m9-M7 is the key regulatory motif of PstGSRE1.TaLOL2 positively regulates the transcription of the downstream disease-resistance genes and the wheat resistance against stripe rust.The project provides a good candidate target for new rust fungicide,and theoretical basis for the sustainable and environment-friendly management of wheat stripe rust.