Study On Pathogenic Mechanisms Of Fungal_trans Transcription Factor VdFTF1 In Verticillium Dahliae

Verticillium dahliae causes vascular wilt disease and infects more than 200 host species worldwide,including many economically important crops,such as lettuce,cotton and tomato,but no effective biologicalcontrol methods are available due in part to its soil-borne nature.In this research,we identified pathogenesis-related gene significantly reduced virulence toward cotton from T-DNA insertional mutant library by Agrobacterium tumefaciens-mediated transformation（ATMT）served as an efficient protocol genetic manipulation of Verticillium dahliae for insertional mutagenesis.To isolate the host sequences flanking the inserted T-DNA,we employed a modified thermalasymmetricinterlacedPCR（TAIL-PCR）technique.BLASTandthen analysis of bioinformation（SMART）showed that the protein encoded by VEDA₀1836 contains a fungal-specific TF（Fungal_trans）domain and encodes a fungal-specific TF domain-containing protein（named VdFTF1 in this study）.To investigate the subcellular location of VdFTF1-GFP fusion protein was clearly localized to cell nuclei.Based on the above data support the hypothesis that the VdFTF1acts as a transcription factor.Subsequently,further research on the effects of VdFTF1 preliminary function study in V.dahliae.Molecular cloning,expression pattern,and function of VdFTF1 during early infecting process.We cloned the full length sequence ORF of VdFTF1.Real-time PCR observation illustrates its expression high level in the infected cotton of the fifth day.To generate VdFTF1 deletion and complementation expressive vectors,Agrobacterium tumefaciens-mediated transformationof Verticillium dahliae was conducted and the transformants were selected on PDA supplemented with hygromycin or geneticin at 50μg/mL for gene deletion or complementation,respectively.The VdFTF1-deletion strains displayed normal vegetative growth,mycelial pigmentation,and conidial morphology,but exhibited significantly reduced virulence in cotton,suggesting that VdFTF1 disruption did not alter vegetative growth,mycelial pigmentation,or conidial morphology.These results indicated that VdFTF1 is not required for radial growth and production of conidia but plays an essential role in virulence on cotton.With the rapid development of the next generation sequencing,the RNA sequencing（RNA-Seq）is widely used in the analysis of transcriptomes,gene expression and regulation in the whole genome range,also enables a genome-wide investigation of genes and molecular mechanisms underlying the pathogenicity of Verticillium dahliae.Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes（494 genes positively and 308 genes negatively）,among which 233 genes were associated with catalytic processes.These genes included 69 potential secreted proteins,a number of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton.Previous research has also suggested that many plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process.Bioinformatics approaches make possible the largescale prediction and analysis of secretory proteins from the Verticillium dahliae transcriptome.Thus,genes encoding extracellular proteins have a higher probability of being involved in virulence.An examination of the pathogenesis-related secretome of Verticillium dahliae is important for understanding the molecular mechanism of pathogen-host interaction.Secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.Real-time PCR observation illustrates differential expression of VdFTF1-regulated genes under different conditions was assessed using six different treatments:1)basic C’zapek-Dox agar plates containing 30 g/L sucrose;2)C’zapek-Dox plates containing 10 g/L carboxymethylcellulose（CMC）sodium;3)C’zapek-Dox plates containing 10 g/L pectin;4)24 hours of growth on cotton roots after root-dipping;5)two or three days of growth on cotton roots after root-dipping;and 6)PDA.Four isolates were used in the experiments:the wild-type（WT）strain,a VdFTF1-deletion mutant（ΔFTF1-1）,and two complemented strains(EC^KO-1 and EC^KO-2).Strains were grown for two days on C’zapek-Dox treatments.19 genes that were significantly regulated by VdFTF1 encoded secreted proteins containing CAZyme modules,which generally function in plant cell wall degradation,including auxiliary（AA2,AA3 and AA9）,glycoside hydrolase（GH3,GH5 and GH28）,and polysaccharide lyase（PL1）activities.Among these genes,15 were positively regulated in the wild-type strain during infection of cotton,but negatively regulated after the deletion of Vd FTF1.The in vitro assay revealed several VdFTF1-regulated genes that were positively activated during growth on carboxymethylcellulose（CMC）sodium or pectin-containing medium that were significantly suppressed after deletion of VdFTF1.This finding suggested that the secreted CAZymes regulated by VdFTF1 play important roles in plant cell wall degradation during infection of cotton.To further confirm that secreted CAZyme genes regulated by VdFTF1 participated in pathogenesis,the expression patterns of these genes were surveyed in the conidial suspension treated with cotton roots for two days.The transcription pattern of putative cell wall degrading enzyme genes under VdFTF1regulation is consistent with a role in virulence.Targeted gene deletion of one VdFTF1-regulated gene significantly impaired fungal colonization,as measured by qPCR,as well as aggressiveness and symptom severity in cotton.Functional disruption of putative secreted CAZyme VEDA₀7232,VEDA₀5900,VEDA₀3732,VEDA₀5776,VEDA₀4217,VEDA₀9568,VEDA₀9651 regulated by VdFTF1,some resulted in decreased virulence on cotton,but the others displayed small changes with Vd991.These genes regulated by VdFTF1 participated in different levels of pathogenicityIn conclusion,VdFTF1,which encodes a TF containing a Fungal_trans domain,regulates the gene expression of plant cell wall-degradation enzymes in Verticillium dahliae,which are required for full virulence on cotton.