Studies On The Function Of FonTup1 In Pathogenicity Of Fusarium Oxysporum F. Sp. Niveum,The Causal Agent Of Watermelon Fusarium Wilt Disease

Watermelon wilt disease,caused by Fusarium oxysporum f.sp.niveum?Fon?,is a destructive soil-borne fungal disease worldwide,and occurs at all stages of watermelon development.In recent years,owing to the continuous expansion of planting area and difficulties in crop roration,watermelon wilt disease has become as one of the most serious limiting factors in watermelon industry.However,the molecular mechanism of pathogenicity in Fon is largely unknwont.Tup1-Cyc8 complex is a highly conserved transcriptional repressor in eukaryotes,participating in the regulation of multiple biological processes.In this study,we focused on the functions of FonTup1 in Fon pathogenicity and their possible mechanisms.FonTup1 was identified by cloning and bioinformatics analyzing.Targeted deletion mutant?FonTup1 and its coresponding complement transformant?FonTup1-C were constructed using PEG-mediated protoplast transformation technology.The biological function of the FonTup1 was systematically explored through analyzing and comparing mycelial growth and sporulation phenotype,pathogenicity,environmental stress response and transcriptome profiling.Main results are as follows:?1?FonTup1 is involved in fungal development,conidiogenesis and pathogenicity in Fon.As comparied to wile type,the?FonTup1 strain displayed reduced mycelia growth and defects in conidiogenesis.Pathogenicity assay indicated that the deletion mutant?FonTup1 strain decreased significantly in its virulence on watermelon,and downregulated the expression levels of four infection-related genes.Expression levels of fusaric acid biosynthesis genes reduced significantly in the mutant?FonTup1,indicating FonTup1 is involved in regulating the biosynthesis of FA.?2?FonTup1 is involved in the cell wall integrity of Fon.Cell wall-damaging sensitivity analysis showed the sensitivity of?FonTup1 mutant strain to cell wall stress factor Congo Red is significantly reduced,and the expression levels of four cell wall biosynthesis-related genes were significantly lower than those of wild type.These results indicated that FonTup1 participated in regulating the cell wall integrity in Fon.?3?FonTup1 is required for response to environmental stress.The sensitivity of?FonTup1 mutant to oxidative stress factor Paraquat,Osmotic stress factor Sorbitol,NaCl increased,while its sensitivity to CaCl₂ decreased significantly.These results indicated FonTup1 is involved in the regulation of stress response in Fon.?4?The whole gene expression profilings of wild type and the?FonTup1 mutant were analyzed and compared through RNA-seq.The results indicated that,as compaed with wild type,there were 2999 differentially expressed genes?DEGs?in the?FonTup1mutant,of which 1670 genes were up-regulated and 1329 genes were down-regulated.The GO and KEGG enrichment analysis revealed DEGs in the?FonTup1 mutant were significantly enriched in biological processes and molecular function categories,mainly involving the pathways of carbon metabolism,amino acid metabolism,citric acid cycle,oxidative phosphorylation,secondary metabolite synthesis,etc.It is thus speculated that the deletion of FonTup1 disrupts the normal cell cycle and basic metabolic pathways,resulting in abnormal growth and reduced pathogenicity in Fon.?5?As revealed by RNA-seq analysis,the expression levels of malate dehydrogenase?MDH?family genes?FonMDH1,FonMDH2,FonMDH3?in the?FonTup1 mutant were significantly down-regulated.The biological function of these three MDH genes was further studied.The results indicate that the FonMDH3 is an essential gene,as knockout of FonMDH3 was lethal;the?FonMDH1 strain showed serious growth defects,and failed to grow in the liquid medium.However,the mycelial growth and conidiation was significantly reduced,and the morphology of conidia was abnormal in?FonMDH2 strain.Expression levels of 3 morphogenesis-related genes?FGA1,FGB1,SGE1?in the?FonMDH2 mutant were significantly lower than the wild type,and the pathogenicity of the?FonMDH2 strain was reduced.These results indicated that deletion of FonTup1 gene leads to the suppression of FonMDH genes expression,resulting in abnormal cell metabolism and failure to maintain normal growth state and morphological differentiation,which was one of the reasons for the reduced pathogenicity of Fon.