Interaction Mechanism Analysis Between Cotton And Verticillium Dahliae Based On Mμlti-omics

The lack of disease-resistant cotton germplasm and the frequent variation of the pathogen has led to urgent problems in cotton breeding and comprehensive control of Verticillium wilt.Although the pathogenicity of the fungus and plant immunity has been extensively studied,little is known about the interaction mechanism between V.dahliae and cotton.The analysis of the interaction mechanism between cotton and V.dahliae by multi-omics can provide a theoretical reference for solving the problem of cotton resistance to Verticillium wilt.At the same time,identifying of elicitors that can induce cotton immune activation can be used to innovate cotton germplasm resistant to Verticillium wilt.In this project,transcriptome analysis revealed the changes in the molecular regulatory network during the whole process of interaction between cotton and pathogen,and found the possible changes of interaction mode between cotton and pathogen at different stages.Combined with the comparative transcriptome and secretome of the early interaction between V.dahliae and cotton roots,the elicitor VP2 in V991 that can induce cotton immune activation was identified.Heterologous overexpression of VP2 in cotton can activate SA and JA signal pathways,enhance lignin metabolism and enhance cotton resistance to V.dahliae,thus realizing germplasm innovation of resistance to Verticillium wilt.The main results are as follows:1.Bidirectional transcriptome analysis of the interaction between cotton and V.dahliaeIn this study,the cotton hypocotyls infected by V991 and 1cd3-2 were sequenced by transcriptome,and the reads from the sequencing were matched to the cotton and V.dahliae genomes,respectively.Through GO enrichment analysis of the differentially expressed genes(DEGs)that specifically respond to V.dahliae in cotton at different time points,it was found that the genes involved in the cell wall synthesis pathway were significantly enriched in cotton after 3-9 days post-inoculation(dpi).During the 12-21 dpi,the genes involved in immune response and other related genes in cotton were significantly enriched,and there was a significant outbreak of reactive oxygen species(ROS)and an increase in antioxidant enzyme activity compared with the early stage.Further studies showed that a high concentration of hydrogen peroxide(5 m M)could significantly kill cotton protoplast cells.Therefore,this study suggested that the symptoms of leaf chlorosis and necrosis of cotton Verticillium wilt may be related to the excessive accumulation of ROS.The comparative transcriptome analysis of V991 and 1cd3-2 revealed that the differential expression of six types of genes involved in the virulence of V.dahliae at the transcriptional level may be one of the reasons for the virulence differentiation of V.dahliae.Especially 12-21 dpi,the genes involved in virulence in V991 were significantly induced.In addition,strain-specific genes of V.dahliae may also be an important cause of virulence differentiation.Combined with the V.dahliae comparative genome,V991-specific secretory proteins SP3 and SP6 were identified.The results of q RT-PCR showed that their expression in cotton increased from 12 to 21 dpi.Through homologous recombination,the SP3 and SP6 in the V991 genome were knocked out,and the result shows that the SP3 and SP6 knock-out transformants did not change the hypha growth rate.However,the knockout mutants of SP3 were significantly less virulence to cotton,suggesting that SP3 might be one of the key virulence factors of cotton diseases caused by the defoliating V.dahliae.Comprehensive bidirectional transcriptome analysis revealed that cotton and V.dahliae could be divided into two different stages during the long-term intercropping process: in which 3-9 dpi was the early interaction stage,in this stage,plants mainly rely on structural resistance such as cell walls to resist the invasion of the pathogen,and accordingly,genes related to the involvement of V.dahliae in metabolism and inhibition of plant immunity are significantly induced during this stage;12-21 dpi is the late interaction stage,during which the plant undergoes excessive activation of immune responses,which may be one of the main causes of cotton diseases;Accordingly,during the late interaction stage,V.dahliae secretes a large number of effectors,activating the immune response in plants,and accelerating plant disease.2.Using V.dahliae elicitor VP2 to activate plant immunity and improve cotton resistance to V.dahliaeComparative transcriptome analysis of V991 and 1cd3-2 interaction with the diseaseresistant cotton line Hai7124 and the disease-susceptible cotton line YZ1 for 3 days showed that V991 had the more up-regulated expression of DEGs.In combination with the secretome of V991,10 V991-specific induced secretory proteins(VPs)were screened.q RT-PCR of VPs injected into Nicotiana benthamiana(N.benthamiana)leaves for 48 h showed that VP2 significantly activated the expression of HR and PTIrelated genes.VP2 was highly up-regulated in the early stage of mycelial invasion and induced necrosis in a variety of plant leaves.The results of subcellular localization showed that VP2 was localized on the N.benthamiana cell membrane.VIGS experiments confirmed that the necrosis of N.benthamiana leaves induced by VP2 was dependent on Nb BAK1 but not on Nb SOBIR1.The amino acid sequence of VP2 in V991 and 1cd3-2 is very conservative,only two amino acids are mutated in the signal peptide region.However,during the invasion of cotton,ND-type VP2 was almost not induced in non-defoliating pathotype V.dahliae.The VP2 gene in V991 genome was knocked out and complemented by homologous recombination.The results showed that VP2 knockout transformants did not affect the hyphal growth of V991,but the pathogenicity to cotton was significantly reduced.This indicates that VP2 plays an important role in the virulence of V991.In addition,the VP2 is heterologous overexpressed into the cotton by utilizing a plant-transgenic technology,the VP2 transgenic cotton exhibited enhanced resistance to V.dahliae without affecting its growth and development.The results of RNA-seq showed that the genes differentially expressed in VP2 transgenic cotton compared to wild-type control were significantly enriched in cell wall modification,response to fungus,lignin metabolism,hormone metabolism,etc.Among them,the expression level of genes involved in plant immune response-related genes such as chitinase,OSM34,and peroxidase was higher in VP2-overexpressing cotton lines compared with the wild type,regardless of normal condition or inoculation with pathogen.The results of q RT-PCR showed that genes involved in JA and SA signaling-related genes were significantly activated in OEVP2 transgenic cotton,and the hormone contents of JA and SA were also significantly higher than that of the wild-type control.In addition,Gh PALs,genes involved in lignin synthesis-related genes in OEVP2 transgenic cotton,were significantly activated,and lignin content was also significantly increased.In summary,this study found that there was a phased immune regulatory transition in the interaction between cotton and V.dahliae using bidirectional transcriptome analysis of cotton and V.dahliae,which gave us new insights into the plant-fungal interactions.At the same time,the key elicitor VP2 of V991 was identified by analyzing the transcriptome and secretome of the early interaction between the pathogen and cotton roots.By using cotton transgenic technology,the heterologous overexpression of the effector protein VP2 from V.dahliae into cotton can increase the resistance of cotton to V.dahliae,which enriched the means of creating cotton germplasm resistant to Verticillium wilt.