Functional Study Of A FAD Binding Protein Encoding Gene VdFAD In Verticillium Dahliae

Verticillium dahliae is a soil-borne plant pathogenic fungus.Verticillium wilt caused by this pathogen poses a huge threat to global cotton production and seriously affects the development of agricultural economy.Therefore,the prevention and control of plant diseases caused by V.dahliae is a problem that needs to be solved urgently.During the growth and reproduction,V.dahliae will produce a special structure,namely microsclerotia which plays an important role in the process of reproduction and pathogenicity.As the dormant structure of V.dahliae,microsclerotia has strong resistance to environmental stresses and plays a key role in the disease cycle.The microsclerotia will germinate and generate hyphae under the induction of plant root secretion.Subsequently,hyphaes wrap around the roots,and a few hyphaes adhere closely to the surface of plant roots to form adherent spore with infectivity.Adhesive spores further differentiate to form penetrating spike which helps pathogen toinvade host plant cells and colonize vascular tissues.After colonization,a large number of hyphaes will block the vascular bundles and interfere with the transportation of water and nutrients,resulting in severe wilting and even death of plants.So far,the pathogenic mechanism of V.dahliae has not been fully elucidated.Therefore,there is an urgent need to reveal the underlying mechanism involved in the pathogenic process to provide guidance for the prevention and control of cotton verticillium wilt.Based on the previous transcriptome data,we found a FAD binding protein coding gene VdFAD which shows a high expression during the infecting process of V.dahliae to tomato plant,implying that VdFAD plays an important role in the process of infecting plants.However,little is known about its function in V.dahliae.In this study,we made a comprehensive functional analysis of VdFAD and the results as followed:1)VdFAD is an unknown protein containing FAD binding domain.Bioinformatics and expression pattern analysis revealed that the VdFAD gene is 1819bp in length and encodes 534 amino acids residues.Through sequence analysis,the protein with highhomology to VdFAD is a FAD binding protein in Purpureocillium lilacinum,shared with 69.7%homology.Bioinformatics analysis found that VdFAD has an N-terminal signal peptide of 20 amino acids residues in length,and contains a FAD binding domain,whose function has not been annotated.Analyzing the expression pattern of VdFAD,found that VdFAD expression is low during the vegetative growth process,but highly expressed during the infection of cotton by V.dahliae.These results indicate that VdFAD may be involved in the pathogenic process of V.dahliae infection.2)VdFAD gene is involved in the response of V.dahliae to stress.The wild-type strain,knockout strainΔVdFAD and overexpression strain VdFAD^OEwere cultured on CZA and PDA medium supplemented with various stress conditions（Sorbitol,Congo red,Na Cl,MND and H₂O₂）,It was found that the knockout strainΔVdFAD was more sensitive to Congo red than the wild type,and the addition of Congo red to the PDA medium found that the knockout strainΔVdFAD was not significantly different from the wild-type,because Congo red is a fungal cell wall inhibitor,this suggests that VDFAD affects fungal cell wall integrity only under barren conditions.Further analysis of conidia cell wall epitope carbon sources between the mutant strain and the wild-type strain showed no significant difference in the fluorescence signals of Con A and WGA on the conidia wall surface of the mutant strain compared with that of the wild-type strain,indicating that VDFAD knockout did not affect the conidia cell wall epitope carbon sources.In addition,compared with the wild-type strain,the VdFAD overexpression strain VdFAD^OEunder H₂O₂stress have a smaller colony diameter and lower expression levels in oxidative stress-related genes such as gamma-glutamylcysteine synthetase,glutathione reductase,thioredoxin etc.These results indicate that VdFAD is involved in the redox reaction of response to stress in V.dahliae.3)VdFAD gene affects the conidia yield of V.dahliae.Conidial yield analysis of wild-type strain,knockout strainΔVdFAD and overexpression strain VdFAD^OEin CZA solid medium.The results showed that,there was not significantly difference between overexpression strain VdFAD^OEand wild-type strain in the conidia yield while the conidia yield of the knockout strainΔVdFAD was about 3.5 times higher than that of the wild-type strain.These results indicate that VdFAD affectes the conidial yield of V.dahliae.4)VdFAD gene affects the synthesis of microsclerotia and melanin of V.dahliae.To determine whether VdFAD functions in the formation of microsclerotia,conidia from the wild-type strain,knockout strainΔVdFAD strain and overexpression strain VdFAD^OEwere coated and cultured in CZA medium.The results showed that there were no microsclerotia produced by wild-type and VdFAD^OEstrain,while a large number of microsclerotia were observed in theΔVdFAD strain plates.Furthermore,expression analysis of the melanin-related genes showed that melanin synthesis-related genes,such as Vayg1,Vafl M,VDSCD,Vd LAC,and VT4HR,had a higher expression level inΔVdFAD strain than that in wild type and VdFAD^OEstrain.These results indicate that VdFAD regulates the microsclerotia and melanin synthesis of V.dahliae.5)VdFAD gene influences the virulence of V.dahliae to cotton.To analyze the effect of VdFAD on the virulence of the strain,wild-type,ΔVdFAD and VdFAD^OEstrain were used to infect cotton seedlings.The results showed that,compared with the wild-type strain,the knockout strainΔVdFAD caused larger disease spots on cotton plants with higher disease index.The grade 3 and 4 of disease index on the cotton seedlings infected by the knockout strainsΔVdFAD-1 andΔVdFAD-2 was 54.28%and60.6%,respectively.The cotton seedlings infected by VdFAD^OE-1 and VdFAD^OE-2accounted for 35.29%and 34.15%in term of grade 3 and 4 of disease index,and this index was 29.73%caused by wild-type strain.These results suggest knockout of VdFAD increases the pathogenicity of V.dahliae and VdFAD negatively influences the pathogenicity.6)Exogenous VdFAD protein has no significant effect on microsclerotia formation of the knockout strainΔVdFAD.VdFAD is an extracellular protein containing a signal peptide,implying it functions outside the cell.To explore its extracellular function.VdFAD protein were expressed and purified through the Pichia pastoris system and the protein was added to the knockout strainΔVdFAD plates.The observation showed that the exogenous VdFAD had no effect on the microsclerotia formation ofΔVdFAD strain,suggesting that VdFAD in vitro could not restore the function of VdFAD inΔVdFAD strain.7)ΔVdFAD vs WT transcriptome analysis.After 4 days growth culturing the in CZB liquid medium,RNA from wild type andΔVdFAD strain was extracted for transcriptome analysis.The data showed that,compared with the wild type strain,the number of differentially expressed genes（DEGs）in theΔVdFAD strain was 706,of which there were 315 up-regulated genes and 391 down-regulated genes.The melanin-related genes such as Vayg1,Vafl M,VDSCD,Vd LAC and VT4HR were up-regulated in theΔVdFAD strain,which was consistent with the previous q RT-PCR analysis results.The enrichment analysis of GO classification showed that a total of 49GO functions were significantly enriched（P<0.05）.Moreover,in the top 20 functional gene enrichment levels,there were 4 GO terms involved redox processes and 4 GO terms related to cell membranes and cell walls.Additionallly,KEGG enrichment analysis showed that a total of 20 metabolic pathways were significantly enriched（P<0.05）and 8 pathway related to material metabolism.These results further proved evidence that VdFAD is involved in microsclerotia and redox in V.dahliae.In summary,VdFAD is highly expressed during the host infection process of V.dahliae.This gene affects the cell wall integrity of V.dahliae,and participates in microsclerotium melanin synthesis and involves in redox reaction pathway and pathogenicity.