| Wheat powdery mildew(Pm)is a fungal disease caused by the biotrophic pathogen Blumeria graminis f.sp.tritici(Bgt).The utilization of single race-specific Pm resistance gene usually become non-effective in a short period,mainly due to the emergence of new virulent Bgt isolates.Therefore,the exploration and utilization of genetic resources with durable and broad-spectrum resistance is of great significance.Haynaldia villosa(2n=14,VV)is a wheat wild relative,which was proved to have broad-spectrum resistance to wheat powdery mildew.T.aestivum-H.villosa translocation line T6VS·6AL conferring the Pm21 gene was developed and widely used in wheat breeding.Wheat varieties with improved Pm resistance have been commercially released.In previous research,R genes or Pm resistance related genes located on 6VS or other chromosomes have been cloned and functional characterized in H.villosa.However,the regulatory network of Pm resistance by Pm21 is elusive.In this study,a near-isogenetic line of T6VS·6AL using Yangmai 158 as recurrentparent,BP 102 and its receptor were inoculated with two Bgt isolates E26 and E31,respectively.Differentially expressed genes(DEGs)at five time points were investigated.The pathways and regulatory networks involved in wheat-Bgt interaction were studied.The main results obtained were as follows:1.Analysis of the RNA-seq dataYoung leaves from the BP102 and Yangmai 158 were inoculated with Bgt isolates E26 and E31,and sampled at 1,3,8,18 and 24 hours after inoculation(hai).The non-inoculated leaves at the corresponding timepoints were used as mock control.The RNA of a total of 30 samples were extracted and used for RNA-seq.Sequence evaluation showed that the Q20 and Q30 of all the analyzed samples were above 93%and 85%.The Clean reads were mapped to the reference genome sequences,and the mapped ratio was 68.11～79.21%.After filtering,a total of 54418 genes were considered as unigenes and used for further analysis.2.Comparison of DEGs in BP102 induced by Bgt isolates E26 and E31The Functional annotation based on Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)of DEGs showed that,BP 102 activated similar signal pathways when infected by the two different isolates,E26 and E31.Uponthe Bgt infection at early stage(1-3 hai),genes related to cell wall thickening were activated.The cell surface receptors recognized the pathogen attack and activated protein phosphorylation signaling.The ion transport and plant hormone signaling pathways were up-regulated.We proposed that during the wheat-Bgt interaction,the primary signaling could be enlarged and transduced to downstream by the MAPK cascade,ions transport and plant hormone signaling pathways.From 3hai to 8 hai,the ion transport and jasmonate signalingpathway were kept activated,and the plant cell wall thickening and secondary metabolites accumulation reached the peak level.At the later infection stage(18-24hai),the pathways related to vesicle transport and transmembrane transport of carbohydrates and amino acids were significantly enriched.We proposed that in response to Bgt infection,wheat transports secondary metabolites to the extracellular for resisting the Bgt invasion.In addition,we also observed minor differences for BP102 in response to Bgt infection at 18hai.Firstly,cell wall metabolism was specifically activated after E31 inoculation;secondly,for all the associated pathways,the E31 inoculated samples enriched more DGEs than E26 inoculated.To conclud,Both physical defense(cell wall thickening)and chemical defense(synthesis of flavonoids and terpenoids and visicle transport)played important role in powdery mildew resistance.The incompatible interaction pathways between BP 102 and E26/E31 were:the perception of Bgt by the cell surface-localized receptors,followed by activation of phosphorylation kinases,transducted by MAPK cascade,then further up-regulated the calcium signaling and salicylic acid signaling.3.Comparison of DEGs in Yangmai 158 induced by Bgt isolates E26 and E31GO and KEGG of DEGs showed that,Yangmai158 also activated similar signal pathways when infected by the two different isolates.Plant-pathogen interaction and plant hormone signaling pathways were enriched at early infection stage,while genes related to cell wall thickening and secondary metabolites accumulation were enriched at the later stage.At 1-3hai,the DEGs were mainly associated with plant pathogen interaction,plant hormone signal transduction,and MAPK cascade signaling pathway;At 3-8hai,the jasmonic acid signaling was kept activated,cell wall thickening and biosynthesis of flavonoids and terpenoids were initiated.At 18-24hai,cell wall thickening continuously enriched,and secondary metabolites accumulated.Some differences were also observed for Yangmai 158 in response to the infection of two different isolates.There were specific enrichment of the JA pathway when inoculated by E26 at 3 hai.At 18 hai of E31,we observed specific enrichment of genes related to plant-pathogen interaction and the activation of phosphorylation.At late infection stage of E31,DEGs associated with cell wall thichening and secondary production metabolites biosynthesize were more that infected by E26.4.The basal resistance play role in powdery mildew resistance of wheatThe DGEs in BP 102 and Yangmai 158 were compared.The results showed that there were common enriched DGEs in the two genotypes,indicating the pathways related to basal resistance were activated.At 1 hai,cell wall metabolism,MAPK cascade,JA signaling and carbohydrate metabolism pathway were activated.The phenylaprapanoid synthesis were initiated;At 3 hai,ion transport,pentose phosphate and carbohydrate metabolism pathway were enriched;At 8 hai,there were JA pathway were kept activated,cytokinin signaling was initiated and phenylaprapanoid accumulated;At 18 hai,the ion transport was kept activated and the MAPK cascade was reactivated;At 24 hai,the cell wall metabolism,terpenoids metabolism,and pentose phosphate pathway were enriched.We proposed that cell wall metabolism,MAPK cascade,pathways related to JA signaling,ion transport and phenylaprapanoid metabolism may involved in the basal resistance during the interaction of BP102/Yangmai 158 with Bgt.5.The resistance pathway mediated by the T6VS·6AL translocationThe comparison of GO and KEGG enrichment between BP102-E26 vs Y158-E26 and BP102-E31 vs Y158-E31 also identified those genes specifically enriched in BP102.These DGEs were proposed to be related to the resistance pathway mediated by the translocated chromosome 6VS.At 1-3 hai,specific DEGs in BP102 were associated with cell surface receptor signaling and calcium signaling pathways,protein phosphorylation and SA metabolism.It is proposed,at the early infection stage of the resistant genotype,these pathways facilitated the perception of Bgt by the cell surface-localized receptors,followed by immune signaling transduction.At 3-8 hai,the expression of the genes were associated with plant cell wall thickening and secondary metabolites accumulation reached the peak level,which is earlier than that in Yangmai 158.At 18-24 hai,vesicle-mediated transport were specifically enriched in BP 102.The DEGs in BP 102 were mainly associated with transmembrane transport of ion,carbohydrates and amino acids,suggesting that the transport of the secondary metabolites to the extracellular may be associated with powdery mildew resistance.The genes related to the key resisance pathways were further analyzed.Except the rapid activation of genes related to cell wall thickening and secondary metabolites accumulation,RLP,CDPK,MAPKs,IRAKs and genes related to the SA pathway were up-regulated atl hai in BP102..The qRT-PCR of NPR1,CDPK etc confirmed the RNA-seq data.To conclude,powdery mildew resistance of BP102 may be attibuted to the rapid activation of the following signal transduction pathway:Bgt triggered the cell surface-localized receptors,further activated the expression of phosphorylation kinases,transducted the defense signal via MAPK cascade and calcium signaling and establish the resistance through the SA signaling.Earlier triggered physical and chemical defense played an important roles inT6VS·6AL-mediated powdery mildew resistance.6.The Weighted Gene Co-Expression Network(WGCNA)of translocation T6VS·6AL of T.aestivum-H.villosa and Bgt interationTo understand the regulatory network between wheat and Bgt,WGCNA were performed.The DEGs in BP 102 could be classified into 22 modules.Referring to the assembled genome sequences of 6VS,the DGEs on 6VS were mainly included in the modules 1,2,3,and 4.The fourth module in which the Stpk-V and NLR1-V were included was further analyzed.It was found that in this module,the enriched DGEs were closely related to plant-pathogen interaction,plant hormone signal transduction,regulation of autophagy,endocytosis,phosphatidylinositol signaling system,and flavone and flavonol biosynthesis.This module was also closely correlated with the two samples,BP102-E26-1h and BP102-E31-1h.In this module,there were an enrichment of RLP,CPK,WRKY,EREBP,STPK_IRAK,and EREBP,which has proved to play important role in disease resistance.Among them,the EREBP was mostly enriched and should be one of the key hub in wheat-Bgt interaction regulatory network.7.The proposed powdery mildew resistance network mediated by the T6VS·6ALCombined the above analysis,we proposed a powdery mildew resistance network mediated by the T6VS·6AL:The RLP rapidly recognized the infection of Bgt,amplified the defense signals by phosphorylation kinase IRAK and MAPK signal cascade,activted the transcription factors,and up-regulated the calcium signaling and salicylic acid signaling pathways,induced the expression of NPR1,PR1 and R genes such as NLR1-V.In the early infection stage,cell wall thickening,antibacterial flavonoids and terpenoids accumulation also contributed to powdery mildew resistance. |
