Dissection Of Wheat N9134 Network Response To Fungal Stress Via Transcription-proteome Association And Analysis Of Core Gene Family Functions

Crop production process will be threatened by various biological and abiotic stresses,which will have a huge impact on growth,development and yield.Wheat yield reduction caused by pathogen infection is a thorny problem in food safety production.Although different technologies have been used to resist,the use of resistant varieties is the safest,most costeffective and environmentally friendly method.It is of great significance to study the wheat pathogen interaction network and mechanism,identify genes related to pathogenic stress response,and provide more information for modern breeding.The resistance of winter wheat introgression line N9134 to stripe rust and powdery mildew comes from the short arm of chromosome 1B and the long arm of chromosome 5B,respectively.In the early stage,our research team used high-throughput sequencing and isobaric tags for relative and absolute quantitation(i TRAQ)technology to generate the transcriptome and proteome profiles of N9134 under the stress of Blumeria graminis f.p.tritici,(Bgt)E09 and Puccinia striiformis f.sp.tritici(Pst)CYR31 respectively.On this basis,the changes of transcription-protein expression profile in leaves of N9134 seedlings inoculated with stripe rust were studied,and the range of potential core resistance genes was narrowed by creating networks based on merging data generated by RNA-Seq following weighted gene co-expression network analysis(WGCNA)with the results of transcriptome-proteomeassociated analysis.The identification and expression analysis of Hsps protein,the key node of the response network of stripe rust and powdery mildew,under the stress of stripe rust and powdery mildew were carried out,and the SUN protein with the highest connectivity among the 32 induced proteins identified on 1BS chromosome was selected for functional research.The main results are as follows:1.Transcriptome and proteome-based network analysis reveals a model of gene activation in wheat resistance to stripe rustWe used the high-throughput sequencing and isobaric tags for relative and absolute quantification(i TRAQ)technologies to generate transcriptomic and proteomic profiles of seedling leaves at different stages under conditions of pathogen stress.By conducting comparative proteomic analysis using i TRAQ,we identified 2050,2190,and 2258 differentially accumulated protein species at 24,48,and 72 h post-inoculation(hpi).Using pairwise comparisons and weighted gene co-expression network analysis(WGCNA)of the transcriptome,we identified a stress stage-specific module enriching in transcription regulator genes.The homologs of several regulators,including splicing and transcription factors,were similarly identified as hub genes operating in the Pst-induced response network.Moreover,the Hsp70 protein were predicted as a key point in protein–protein interaction(PPI)networks from STRING database.Taking the genetics resistance gene locus into consideration,we identified 32 induced proteins in chromosome 1BS as potential candidates involved in Pst resistance.This study indicated that the transcriptional regulation model plays an important role in activating resistance-related genes in wheat responding to Pst stress.2.Identification of heat-shock proteins in wheat and analysis of functions about fungal responseThe heat shock proteins corresponding to fungal stress in wheat were classified as 119 Dna J(Hsp40)proteins(Ta Dna Js;encoded by 313 genes)and 41 Hsp70 proteins(Ta Hsp70s;encoded by 95 genes)into six and four groups,respectively,via a phylogenetic analysis.An examination of protein sequence alignment revealed diversity in the Ta Dna J structural organization,but a highly conserved J-domain,which was usually characterized by an HPD motif followed by DRD or DED motifs.The expression profiles of Hsp-encoding homologous genes varied in response to Bgt and Pst stress.A quantitative real-time polymerase chain reaction(RT-q PCR)analysis indicated a lack of similarity in the expression of Dna J70 b,Hsp70-30 b,and Hsp90-4b in Bgt-infected resistant and susceptible wheat.Furthermore,a direct interaction between Dna J70 and Ta Hsp70-30 was not detected in a yeast two-hybrid(Y2H)assay,but screening c DNA library and Y2 H evidence supported that Ta Hsp70-30 not only interacts directly with heat-shock transcription factor(Hsf)A9-like protein but also interacts with Ta Hsp90-4 by Hsp organizing protein.Instantaneous expression of N.benthamiana shows that the subcellular localization of Hsps protein is diverse.This study revealed the structure and expression profiles of the Hsp-encoding genes in wheat,which may be useful for future functional elucidation of wheat Hsps responses to fungal infections.3.Identification of SUN-interacting nuclear envelope proteins and its associated components in Wheat fungal stress responsesHere,SUN and KASH related proteins were identified from DAPs under fungi infection by using their conserved motifs,and finally four SUN proteins,one WIP protein,four WIT proteins,two WPP proteins and one Ran GAP protein were obtained.The transient expression in Nicotiana benthamiana showed that Ta SUN2,Ta Ran GAP2,Ta WIT1 and Ta WIP1 were nuclear membrane proteins,while Ta WPP1 and Ta WPP2 could be expressed on both nucleus and cell membrane.RT-q PCR analysis demonstrated that the transcription of Ta SUN2,Ta Ran GAP2 and Ta WPP1 were strongly responded to fungal infection.Furthermore,using the nuclear and split-ubiquitin-based membrane yeast two-hybrid system and Bimolecular Fluorescence Complementation syste(Bi FC),we substantiated the interaction between Ta SUN2 and Ta WIP1,as well as Ta WIP1/WIT1 and Ta WPP1/WPP2.Silencing of Ta SUN2,Ta Ran GAP2 and Ta WPP1 in wheat leaves promoted powdery mildew infection and hyphal growth,and reduced the expression of Ta BRI1,Ta BAK1 and Ta14-3-3,indicating that these nuclear membrane proteins play a positive role in resistance to fungal stress.Our study uncovered the characteristics of nuclear membrane proteins and drawn preliminary construction of SUN-WIP-WPP-Ran GAP complex in wheat,which laid a foundation for dissecting their detailed function in wheat in future.This study clarified that transcriptional regulation modules play an important role in activating genes related to wheat resistance to stripe rust stress,the functions of Hsps and SUN,the key nodes in fungal stress response network were analyzed.This will provide more information for the network and mechanism of wheat pathogen interaction and identification of genes related to pathogenic stress response.