Screening And Functional Characterization Of Genes Involved In Growth And Pathogenicity In Fusarium Oxysporum

Fusarium spp.is widely found in soils and plants and is an opportunistic pathogenic fungus that infects humans and animals and infects plants.Clinically,this fungal is the most common pathogen causing fungal keratitis.In recent years,with the increase of immunosuppressive population,the incidence of deep filamentous fungal infections has gradually increased,second only to Aspergillus spp.infection,which seriously threatens human health.At present,the research on the pathogenic mechanism of Fusarium mainly includes cell wall degrading enzymes,MAPK pathway and Fusarium’s own metabolic toxins,but there are still many unknown genes and mechanisms waiting for development and research.The main purpose of this study was to find genes related to the growth and development of Fusarium oxysporum and to provide new targets or strategies for the treatment of Fusarium.The previous laboratory has successfully established and optimized the ATMT transformation system of Fusarium oxysporum.In this study,802 strains of mutants were obtained by transforming Fusarium oxysporum by ATMT.The T-DNA insertion was confirmed by PCR and subculture to be stable and inherited.The construction and screening of mutants provides sufficient resources for studying the growth and development,pathogenesis and other mechanisms of Fusarium oxysporum.By screening 802 mutant phenotypes,5 strains of colony morphology and microscopic morphology were screened,which were FOM3,FOM13,FOM93,FOM344 and FOM716.The T-DNA insertion position was successfully located by TAIL-PCR method,and the affected genes were identified as dynein 5 and four putative proteins,respectively.Four classes of clinically used fungi were selected from itraconazole,voriconazole,amphotericin B and caspofungin.The susceptibilitytest was carried out on 5 mutants,and the pathogenicity was determined by C.elegans.The MIC value of FOM93 amphotericin B was 0.5 μg/mL,and the lethality was68.9%.There were no significant changes in other strains.The FOM93 strain disruption gene FOXG-03063 was analyzed as a putative protein.To verify whether the random insertion of T-DNA was correct,a gene-directed knockout strain Δ03063 and a restorer strain Δ03063-C were constructed.The strain Δ03063 was consistent with the colony and microscopic morphology of the mutant,all of which were hyphal growth retardation,mycelial refinement,and the surface of the colony was thick,and the strain Δ03063-C could return to normal form.The pathogenicity test proved that the pathogenicity of the knockout strain and the mutant strain were basically the same,and the restorer strain could restore the pathogenicity.This indicates that the loss of FOXG-03063 gene leads to growth and development of the strain and weakened pathogenicity.The qPCR method was used to verify the expression changes of mRNA-related genes.The results showed that the two subtypes of the key enzyme hexokinase(HK)in the glycolytic pathway and the pentose phosphate pathway,6-phosphofrutokinase(PFK),pyruvate kinase(PK),6-phosphate glucose dehydrogenase(6PGD),6-phosphogluconolactonease(PGL)and 6-phosphogluconate dehydrogenase(6PGDH)expression levels were significantly down-regulated due to the above enzymes Both are rate-limiting enzymes,indicating that the rate of carbohydrate metabolism is significantly slower,which may affect the supply of sugar to the growth of the strain.Summarizing the above results,deletion of the FOXG-03063 gene leads to the development of the strain and leads to a decrease in pathogenicity.At the same time,FOXG-03063 causes a decrease in the expression level of key enzymes in the glucose metabolism pathway,which may affect the growth and pathogenicity of the strain.