Study On The Pathogenic Effects Of ROS Degrading Genes And Pectate Lyase Gene Of Fusarium Graminearum

The ascomycete Fusarium graminearum (teleomorph Gibberella zeae) is a plant pathogen which can infect the spike, stalk, crown and root of wheat, barley, rice, oats and other cereal crops, causing head blight, stalk rot, crown rot and root rot. It brings huge losses to agricultural production. Over the years, researchers have been trying to find an effective and environmentally friendly control method, but with little success. Learning the key virulence genes of pathogen will help to understand the process of interaction between plants and fungi, and finally it will help to find new disease target sites.By analyzing the gene expression profiling of F.graminearum infecting wheat coleoptile, a predicted Cu-Zn SOD (SOD1) which preferentially expressed during invasive growth in plant was detected. Constitutive expression of a SOD1-RFP fusion protein in F.graminearum showed SOD1-RFP was localized inside the cell. In this study, SOD1was expressed in Escherichia coli, and purified protein was used for enzyme activity assay. Result confirmed that SOD1encodes a Cu-Zn SOD. The single-gene knockout mutants was constructed and a number of its phenotypes were analysed. Results demonstated that this protein is able to protect F.graminearum from extracelluar superoxide stress, and mutants showed reduced virulence on hosts.In this study, SOD1mutants showed reduced germination percentage when H2O2was added to culture. DAB staining assay of F. graminearuminfectiag coleoptiles showed obvious staining spot. All these results indicate that F.graminearum needs weapon to defend itself against extracellular H2O2.By analyzing the gene expression profiling of F.graminearum infecting wheat coleoptile, a predicted Catalase (CAT1) which preferentially expressed during the early stage of invasive growth in plant was detected. It has a signal peptide and a conserved catalase domain. Constitutive expression of a CAT1-RFP fusion protein in F.graminearum showed CAT1-RFP was localized on the fungal cell wall. In this study, CAT1was expressed in Escherichia coli, and purified protein was used for enzyme activity assay. Result confirmed that CAT1encodes a catalase. The single-gene knockout mutants were constructed and a number of its phenotypes were also analysed. Results demonstated that this protein is able to protect F.graminearum from extracelluar H2O2stress, and mutants showed reduced virulence on hosts.The double mutants of these two genes were constructed, and infection assay also showed reduced virulence comparing to wild type strain and single-gene mutants.Pectate lyase can eliminate cleavage of (1â†'4)-a-D-galacturonan by β-elimination as a member of pectinases. In this study, PelA was expressed in Escherichia coli and knock-out mutants were constructed. Biochemical analysis showed that PelA has pectate lyase activity in vitro. External environment such as temperature, calcium ion concentration and pH affected the enzyme activity, and enzyme activity reached the highest at50℃,0.5mmol·L-1CaCl2and pH8.5. PelA knock-out strains didn’t show significant difference in virulence from wild-type strains during infection of wheat coleoptiles.In conclusion, the functions of a Cu-Zn SOD, Catalase and PelA were verified by gene knockout, enzyme activity assay and infection assay. Results showed that Cu-Zn SOD and Catalase is virulence related of F. graminearum defending against ROS during infection. This study also identified a pectate lyase, PelA, in Fusarium graminearum for the first time, and its biochemical characteristics was verified in vitro.