Transcriptomic And Gene Functional Analysis Of Microsclerotia In Verticillium Dahliae

Verticillium dahliae Kleb.is a notorious phytopathogenic fungus,which can infect more than 200 plant species and its hosts are still expanded.Recently,vascular wilt disease caused by V.dahliae was seriously happened on Cotinus coggygria in Fragrant Hill and other regions in Beijing,which has caused severe mortality of trees and numerous economic losses,with seriously detrimental effects on the red leaf scenery.In the late stages of the disease cycle,V.dahliae will form large numbers of resting structures,known as microsclerotia,which will enter into the soil with the plant disease residues,and can survive in the soil for many years.Once meeting suitable environmental conditions and host plants,they will germinate and infect the plants.Therefore,microsclerotia play a critical role during the disease cycle and act as a big challenge for controlling the vascular wilt disease.However,the molecular mechanism of the microsclerotia formation is still unclear.The available of the genomic sequences and improved genetic transformation systems greatly accelerate the processes of exploring and functional analyzing the microsclerotia formation and pathogenesis related genes,which provide a new platform to reveal the molecular mechanism of microsclerotia formation and pathogenesis.In this study,we performed the transcriptomic analysis of microsclerotia formation processes by using mRNA-Seq,and then characterized the functions of several transcription factor genes with gene knockout method including two bZIP,one C2H2 and one MADS-box genes,which might involve in microsclerotia formation.1.Clarification of the changes in genes expression pattern and involved processes during microsclerotia formation:According to the microscopic examination,the cytological changes had been revealed from conidia to matured microsclerotia,and four key nodes of microsclerotia developmental processes were identified and used for transcriptomic analysis,including prophase stage of microsclerotia?MS1-60h?,initial stage of microsclerotia?MS2-72h?,middle stage of microsclerotia?MS3-96h?and matured stage of microsclerotia?MS4-14d?.Transcriptional sequencing analysis revealed that the number of expressed genes continuously increased from 9412 to 9939 during CO to MS4.A total of 724 genes were differentially expressed in all microsclerotia developmental stages compared with CO,including 600 significantly up-regulated genes and 124 significantly down-regulated genes.Gene functional annotation and enrichment analysis of these differentially expressed genes showed that they were involved in many cellular processes,such as oxidoreductase activity,melanin biosynthesis,protein catabolism,carbohydrate metabolism and so on.In addition,the results showed that different V.dahliae strains contained various genomic sequences in Lineage-Specific regions,and many alternative splicing events were identified among all stages which were intron retain dominant.2.Several functionally important candidate C2H2 transcription factor genes were identified in V.dahliae:A total of 79 C2H2 transcription factor genes were identified in the genome of V dahliae,which revealed an expanded C2H2-Homeobox subfamily among them by using the bioinformatics methods.Subsequently,the expression patterns of the C2H2 transcription factor genes during microsclerotia formation,poor nutrition conditions?lacking glucose or lacking sodium nitrate?and in liquid simulated xylem fluid medium?SXM?were calculated with digital gene expression profiling.The results showed that 36 C2H2 transcription factor genes were significantly up-regulated during microsclerotia formation stages,22 C2H2 transcription factor genes were significantly up-regulated under poor nutrition conditions,and 11 C2H2 transcription factor genes were significantly up-regulated in SXM condition.Further analysis revealed that 5 genes?VDAG 03208,VDAG 03437,VDAG₀5960,VDAG₀7359,VDAG₀8766?were significantly up-regulated in all three conditions,indicated that these C2H2 transcription factor genes might play important roles in microsclerotia formation,nutrition metabolism,and pathogenicity.3.A C2H2 transcription factor gene VdCrzl?VDAG₀3208?was involved in microsclerotia formation and pathogenicity in V dahliae:A C2H2 transcription factor gene VDAG₀3208 was selected from transcriptional data.Further bioinformatics analysis revealed that VDAG₀3208 was the ortholog of Crzl in Saccharomyces cerevisiae,therefore named as VdCrzl.qRT-PCR and promoter fusion with green fluorescent protein?GFP?analyses revealed that VdCrzl was highly induced its expression in the initial stages of microsclerotia formation and SXM condition.Functional analysis of VdCrzl with the gene knockout method showed that deletion of VdCrzl significantly reduced the production of microsclerotia and the virulence on smoke tree seedlings.In addition,VdCrzl deletion mutant displayed significant reduction in resistance to high concentration of Ca2+stimulus,whereas deletion of VdCrzl did not affect the fungal growth and conidial production.Subcellular localization analysis showed that VdCrzl would transfer into the nucleus from cytoplasm with the Ca2+ stimulus.The results showed that VdCrzl play important roles in microsclerotia formation,pathogenicity and response to the Ca2+ stimulus.4.Two bZIP transcription factor genes VDAG 08640 and VDAG 08676 were not involved in microsclerotia formation,but co-regulated the response to oxidative stress:Two bZIP transcription factor genes VDAG 08640 and VDAG₀8676 were identified from the transcriptional data which were differentially expressed during microsclerotia formation.VDAG₀8640 was significantly induced during microsclerotia formation while VDAG₀8676 reduced its expression levels during microsclerotia formation.Bioinformatics analysis revealed that VDAG 08676 was the ortholog of Atfl that is ubiquitious in fungi,whereas the orthologs of VDAG₀8640 were identified only in few fungal species.Functional characterization of these two genes showed that deletion of VDAG 08640 or VDAG 08676 did not affect the microsclerotia formation,and the VDAG 08640 deletion mutant displayed no distinguishable phenotypes in fungal growth,conidial production,stresses response and pathogenicity compared with the wild-type strain.However,the VDAG₀8676 deletion mutant displayed significant reduction in conidial production and fungal pathogenicity.Double deletion of VDAG 08640 and VDAG 08676 showed that double deletion mutant could still delevop microsclerotia as wild-type strain but it exhibited higher sensibility to the hydrogen peroxide than single gene deletion mutants and wild-type strain.5.MADS-box transcription factor gene VdWcml was an important microsclerotia formation and pathogenicity related factor in V.dahliae:A MADS-box transcription factor gene VdMcml was identified from transcriptional data which was significantly induced during microsclerotia formation.Deletion of VdMcml revealed that deletion mutants displayed obvious defects in fungal growth,conidial production,conidial morphology,chitin content in cell wall and pathogenicity.Observation of the infection processes with WT-GFP and ?VdMcml-GFP strains revealed that the conidia of ?VdMcml-GFP strain could not attach to the root surface while the conidia of WT-GFP strain could attach and infect the root normally.Further analysis showed that deletion of VdMcml would reduce the adhesion ability of conidia.Remarkably,deletion of VdMcml significantly affected the microsclerotia formation.Digitial gene expression analysis of wild-type strain and VdMcml deletion mutant at 14d of microsclerotia formation showed that hundreds of genes were significantly down-regulated in VdMcml deletion mutant,which were involved in various processes.Among them,a secondary metabolism backbone gene VDAG 07928 and its whole gene cluster from VDAG 07920 to VDAG 07931 were significantly reduced their expression levels in VdMcml deletion mutant compared with wild-type strain,and most of the genes in this cluster were highly induced during microsclerotia formation.The results indicated that MADS-box transcription factor gene VdMcml was required for fungal growth,conidial production,cell wall integrity,pathogenicity,microsclerotia formation and so on in V.dahliae.