Pre-mRNA Regulation Mediated By Puccinia Striiformis Effector Pst_A23 Suppresses Plant Immunity

Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),an obligate biotrophic fungi,causes significant yield loss in main wheat-growing areas in China,even in the whole world.The most effective approach to control the disease epidemic is worthy of breeding excellent resistant cultivars.However,the main reason for the disease outbreak is the "loss" of resistance caused by the frequent pathogenicity variation of Pst.Therefore,on the basis of revealing the pathogenicity variation and pathogenic mechanism of Pst,it is of great significance to develop the strategy of sustainable and effective control of the disease by the organic combination of transgenic technology and genetic breeding.In this study,based on the genome sequencing of Pst CYR32,From the genome and secretome of Pst CYR32,Pst?A23 was identified to suppress cell death caused by mouse BAX protein in Nicotiana benthamiana via PVX virus(potato virus X)mediated by Agrobacterium tumefaciens.Well-established system of Pst-wheat was used to explore the functions of the important pathogenic factor Pst?A23 and its host targets in the Pst?A23-regulated pathways in the infection process.This study first reveals Pst effector as a splicing regulator to suppress host plant immunity and enriches our understanding of pathogenic mechanism for the biotrophic parasitic fungi,which provides an important theoretical basis for the establishment of the sustainable strategy for effective disease control.The main findings in this study are listed as follows:1.The sequence analysis of Pst?A23Protein Pst?A23 is rich in serine,lysine and arginine,accounted for 11.6%,6.2% and 13.0%,respectively.BLASTX analysis indicated that Pst?A23 was a Pst-specific effector protein,but also a highly conserved effector without change in nucleotide sites in selected races of CYR32,PST-130,PST-21,PST-43,PST-08/21 and PST-87/7.2.The function analysis of effector Pst?A23The functional signal peptide of Pst?A23 was confirmed by the yeast secretion system and the subcellular localization.Transiently expressing Pst?A23 suppressed Flg22 and bacteria-induced callose deposits,and reduced the expression levels of the defense related gene PR1,PR2 and WRKY12 in N.benthamiana.Furthermore,Pst?A23 was highly induced at 24 and 48 hpi(hour post inoculation).When Pst?A23 was transiently silenced by HIGS(host induced gene silencing),fungal biomass was significantly reduced with fewer uredia and increased host cell death at infected sites.Histological observation showed that the length of hyphae and infection area were reduced compared with that in the control plants.The transgenic plants of silencing Pst?A23(L2 and L10)were created to confirm that the pathogenicity of Pst was reduced with reduction of uredia,compromise of the development of Pst and increasing of host cell death.In addition,the overexpressing Pst?A23 transgenic plants showed more uredia with increasing fungi biomass.These results indicated that Pst?A23 was a significant pathogenic factor of Pst.3.The understanding molecular mechnism of Pst?A23 in suppressing plant immunityInterestingly,Pst?A23 was specifically accumulated in plant nucleus with irregular spots.The merged fluorescence showed that Pst?A23:GFP and At SR45:RFP were co-localized into nuclear speckles.However,R2A:GFP with the mutant arginine residues(R)at the C-terminal lost the ability to localize in nuclear speckles,making clear that R2-rich domain was indispensable for nucleus localization.To gain further insight into the role of Pst?A23 in plant cell,we used IP/MS to screen plant protein interactors and obtained two candidate targets Ta SR34 and Ta U1.Pst?A23-Ta SR34 and Pst?A23-Ta U1 interactions were confirmed by bimolecular fluorescence complementation(BIFC),microscale thermophoresis(MST),co-immunoprecipitation(Co-IP)and firefly luciferase complementation imaging assays.To accurately evaluate the regulation role of Pst?A23 in wheat,we sequenced the transcriptome of transgenic plant of overexpressing Pst?A23.Interestingly,1278 significantly different genes(911 up-regulated and 367 down-regulated)were identified and gathered to the pathway of plant-pathogen interaction,plant hormone signal transduction and biosynthesis of secondary metabolites.Splicing event analysis from the RNA-seq data indicated that 792 events were significantly spliced(P<0.05 and |?psi|>0.1).Among five major types of significantly different splicing,alternative 5' splice site(267 events)was the most abundant differential alternative splicing type,followed by intron retention(205 events),alternative 3' splice site(177 events),exon skipping(142 events)and mutually exclusive exons(1 events).semi-quantitative RT-PCR was used to identify that the splicing ratio of Traes CS6A02G254500 and Traes CS2A02G510800 were significantly reduced.On the contrary,the splicing ratio of Traes CS1D02G241000,Traes CS6D02G182300 and Traes CS7D02G272500 were increased.The splicing ratio of two selected defense-related genes,Ta Xa21 and Ta WRKY53 genes were also accurately confirmed by q RT-PCR and reduced in the overexpressing Pst?A23 transgenic plants.When Ta WRKY53 and Ta Xa21 were silenced by virus-induced genes silencing(VIGS),larger necrosis areas were appeared accompanied by the reduction of 70-80% in expression levels of Ta PR1 and Ta PR2,but the hyphal length and infected area were significantly increased.Taken together,Pst?A23 regulated the pre-m RNA to suppress the plant immunity during Pst-wheat interaction.We evaluated a series of RNA motifs from splicing sites(50 bp)of significantly splicing genes in overexpressing plant using MEME(Motif-based sequence analysis tools)and found that 194 conserved RNA motifs were selected,including the top three motifs(61745 GAAGA,52458 UCUGC and 41390 UUCUU),accounted for 17.7%,15.1% and 11.9%,respectively.Furthermore,Pst?A23-RNA motifs(GAAGA,UCUGC and UUCUU)interaction was confirmed by MST and RNA-EMSA assay,and the R-rich domain at C-terminal was contributed to their interactions.4.Ta SR34 splicing factor had positive role in wheat resistance to PstTa SR34 had two copies on the chromosome 2A and 2D,and one longer sequence(180 bp)in the front of the gene on the chromosome 2B(temporarily named Ta SR34L).We first detected their localization in tobacco and confirmed that Ta SR34 localized in nuclear speckles and nucleoplasm,which co-localized with Pst?A23 in nuclear speckles.However,the sub-localization of Ta SR34 L was not specific.Although,the expression level of Ta SR34 has no change by q RT-PCR in compatible reaction,Ta SR34 in the chromosome 2A did not express,and the transcript level of Ta SR34 L in the chromosome 2B had no change,but in the chromosome 2D,Ta SR34 was highly induced to 3-fold and 7-fold at 24 hpi and 48 hpi in incompatible reaction.In addition,there is no change in plant defense and the development of Pst when silencing Ta SR34 L and Ta SR34 in wheat in compatible reaction.But silencing Ta SR34 obviously compromised wheat resistance with a reduction of host cell death and the expression levels of Ta PR1 and Ta PR2 and increasing of uredia in incompatible reaction.Histological observation showed that hyphae length,infected areas and fungal biomass obviously increased when silencing Ta SR34.The results indicated that Ta SR34 in wheat genome 2D contributed to wheat resistance to Pst,but Ta SR34 L in genome 2B did not.Furthermore,The transgenic plant of silencing Ta SR34(L1 and L3)showed that more uredia were appeared,along with a reduction of the expression levels of Ta PR1 and Ta PR2 genes.5.Ta SR34 splicing factor active SA pathway and enhance Arabidopsis thaliana resistanceTransiently expressing Ta SR34 induced cell death in N.benthamiana,but Ta SR34 L did not.The trangenic Arabidopsis thaliana of overexpresssing Ta SR34 companied with dwarf and cell death phenotype.Interestingly,the number of callose deposits and the expression levels of plant defense-related genes At PR1,At WRKY33 and At WRKY53 was significantly increased in Ta SR34-overexpressed transgenic Arabidopsis inoculated with Flg22 compared with that inoculated with water,indicating that Ta SR34 activated the plant basal immune responses.The RNA-seq of Ta SR34-overexpressing transgenic plant was used to detect about 2389 up-regulated genes and 1839 down-regulated genes.Interestingly,most down-regulated genes mainly accumulated in the process of photosynthesis and metabolic pathways,but up-regulated genes focused on biosynthesis of secondary metabolites and hormone related process.Furthermore,about 65 SA-related genes were up-regulated and the transcript levels of three SA synthesis genes(PAL4,PAL1 and EDS16)were significantly elevated by q RT-PCR.The total SA levels were also confirmed by enzyme linked immunosorbent assay(ELISA).Taken together,these results indicated that Ta SR34 might affect on salicylic acid biosynthesis to regulate the transcript levels of some related genes and plant defense reponse.6.Ta SR34 splicing factor regulated the splicing events of defense-related genesRNAseq data indicated that a total of 65 events(including 61 genes)were significantly differentially spliced in Ta SR34-overexpressing transgenic lines(P<0.05).We randomly selected candidate gene to identify the splicing ratio and the splicing ratio of these significant genes were increased or reduced compared with that in wild type.The splicing ratio of At LSD1(lesion simulating disease resistance response 1)was reduced,but the splicing ratio of At TGA6 gene related to SA pathway was increased in the overexpressed Ta SR34 plant,resulting in the increasing of the transcript levels of functional At TGA6 and the second intron retention of At LSD1.Meanwhile,Ta SR34-Ta U1 interaction was confirmed by BIFC,MST and Co-IP assay.Furthermore,Pst?A23-Ta U1 interaction was stronger than Ta SR34-Ta U1 interaction.The sequence analysis indicated that the conserved RNA motifs at splicing sites regulated by Ta SR34 are highly similar to those regulated by Pst?A23.Ta SR34 also bound to RNA targets of Pst?A23 by MST and RNA-EMSA assay,indicating that Ta SR34 and Pst?A23 shared the same RNA motifs.Taken together,these results showed that Pst?A23 might competitively bind to Ta U1 and RNA motifs to kick the protein Ta SR34 to regulate pre-m RNA splicing,resulting in weakening of plant defense response.