Salicylic Acid Regulates The Pathogenicity Of Fusarium Oxysporum By Inhibiting The FoTOR Signaling Pathway

As an important potato producer in the world,China has always ranked first in terms of potato planting area and total output in the world.However,potato fusarium wilt and dry rot are important fungal diseases in the production and storage of potato,which can lead to large-scale reduction of potato production.The main pathogen of potato wilt and dry rot is F.oxysporum,a worldwide distributed soil-borne pathogenic fungus with a wide range of hosts that can cause more than 100 plants wilt.Therefore,the control of F.oxysporum has important economic and practical significance for the potato industry.Salicylic acid(SA)is a phenolic compound that is ubiquitous in plants and regulates various developmental processes and stress responses in plants.It can also be secreted to affect the distribution of rhizosphere microorganisms in vitro.Target of rapamycin(TOR)is a key regulator of eukaryotic cell growth,metabolism,hormonal response,and stress response,and also plays an important role in regulating the pathogenicity of pathogenic fungi.The effect of the SA derivative aspirin on the TOR signaling pathway has been studied in animal cells.However,it is not clear whether SA produced by plants can target the TOR signaling pathway of pathogenic fungi and thus affect the pathogenicity of pathogenic fungi.In this study,in order to explore the pathogenic mechanism of phytohormone SA regulation of F.oxysporum and apply SA to inhibit the pathogenicity of phytopathogenic fungi,F.oxysporum was used as the pathogen material to analyze the FoTOR signaling pathway.Through genetics,biochemistry and molecular biology,this study reveals the mechanism by which phytohormone SA regulates the pathogenicity of F.oxysporum by inhibiting the FoTOR signaling pathway.The specific research results are as follows:(1)Fusarium oxysporum strains were isolated from potato tuber of dry rot.Alignment with the yeast TOR protein revealed two TOR gene homologs in the F.oxysporum genome.Phylogenetic analysis and kinase domain sequence alignments indicate that FoTOR is evolutionarily conserved across species.Moreover,the conserved TOR signaling pathway was present in F.oxysporum by using yeast TOR signaling pathway components as reference.We found that the deletion of the FoTOR1 gene may be lethal in F.oxysporum,but FoTOR2 is dispensable in mycelial growth by deleting the gene.TOR specific inhibitor rapamycin(RAP)inhibits the mycelial growth of F.oxysporum and necrotic areas caused by F.oxysporum.In addition,FoFKBP12 can mediate the inhibition of FoTOR protein activity by RAP.These results indicate that there is a conserved TOR signaling pathway in F.oxysporum.(2)In order to investigate whether SA can directly inhibit the growth of F.oxysporum,the mycelial growth of F.oxysporum experiment was carried out in vitro.The spores of F.oxysporum treated with different concentrations of SA,the results showed that SA inhibited the mycelial growth of F.oxysporum in a dose-dependent manner.The scanning electron microscope(SEM)results showed distorted and swollen hypha,twisted and shorter hyphal tips of F.oxysporum that were caused by SA treatment.The spores germination of F.oxysporum and the necrotic areas caused by F.oxysporum also decreased significantly with incre asing SA concentration.Western blot results showed that SA can induce the phosphorylation of FoSNF1.Gene knockout experiment indicated that the FoSNF1 knockout mutant(Δsnf1)is not sensitive to SA compared to wild-type F.oxysporum.Further,FoSNF1 interacted with FoRAPTOR through yeast two-hybrid assay,and the phosphorylation level of FoRAPTOR was significantly increased in the hyphae treated with SA,while the phosphorylation level of FoRPS6,the downstream component of FoTORC1,was significantly decreased.These results indicated that SA activates FoSNF1 and phosphorylates FoRAPTOR,a key component of FoTORC1,resulting in inhibition of the FoTORC1 signaling pathway.(3)A cDNA library of FoTOR1 was constructed,and a new FoTOR1 interacting protein(named as FoTIP4)was found by the c DNA library of FoTOR1.The FoTIP4 deletion mutant(ΔFotip4)experiment showed that the hyphal growth of ΔFotip4deletion mutant was retarded on PDA medium.In addition,the spores production ofΔFotip4 strain was significantly reduced,suggesting that FoTIP4 regulates the hyphal growth and spore development of F.oxysporum.The inoculation experiment of potato leaves and tubers showed that the lesion symptoms of ΔFotip4 mutant strains was significantly reduced compared with that of WT strain.Additionally,cellophane penetration assay was performed to verify invasive growth of ΔFotip4 mutant strains.Efficient penetration was detected in WT strain,but not in ΔFotip4 mutant strains,indicating that FoTIP4 may be involved in the regulation of CWDEs.The expression levels of CWED-related genes were significantly down-regulated in ΔFotip4 mutant strain.EMSA assay showed that FoTIP4 could bind to the promoter of CWDE-related genes.These results indicated that FoTIP4 can regulate the expression of CWDE-related genes in F.oxysporum.In conclusion,exogenous SA treatment can significantly inhibit the vegetative growth,spore germination and pathogenicity of F.oxysporum.There is a conserved TOR signaling pathway in F.oxysporum,and FoTORC1 plays an important regulatory role in F.oxysporum.The molecular mechanism of plant hormone SA regulating pathogenicity of F.oxysporum through FoTORC1 signal pathway was elucidated.SA inhibits FoTORC1 signal pathway through phosphorylation of FoRAPTOR,and FoTIP4,a key regulator of CWDEs gene expression downstream of FoSNF1-FoTORC1 signal pathway,mediates the inhibition of SA on mycelial growth and pathogenicity of F.oxysporum.