Functional Analysis Of The Melanin Biosynthesis Genes St4HNR And St3HNR In Setosphaeria Turcica

Preliminary results in our laboratory showed that DHN melanin was accumulated in Setosphaeria turcica which was related to pathogenicity. Two naphthol reductases genes, St3HNR and St4HNR, in DHN melanin biosynthesis of S. turcica were cloned. In order to confirm the function and the relationship of the two genes in DHN melanin biosynthesis, Knock-out technique was used to construct St4HNR gene deleted mutants, St3HNR and St4HNR double genes deleted mutants. The function of the genes was identified by analyzing the mutants.The St4HNR gene-disruption vector was constructed using pBS plasmid and antibiotic G418. Gene-knockout vector was transformed into widetype isolate 01-23 and mutant△St3HNR by PEG-mediated transformation method. St4HNR gene-deletion and St3HNRSt4HNR double genes-deletion mutants were screened by G418 and PCR with gene-specific primers.By phenotypic analysis, we found that both the mutant strain△St4HNR and△St3HNR△St4HNR which reduced melanization showed white from a positive of view. But, on the other side, the mutant strain△St4HNR was a dark-brown colony, and the strain△St3HNR△St4HNR was lighter brown. The color of the strain△St4HNR was lighter than the strain wild-type and the strain△St3HNR△St4HNR was lighter than the strain△St4HNR. It was obtained△St3HNR strain whose color was heavier than the strain△St4HNR and△St3HNR△St4HNR in our former study. We initially speculated that when St4HNR was missing, St3HNR may play a role in the first step dehydrogenation of DHN melanin biosynthesis. Comparing with the wild-type strain, the mutant strains exhibited faster vegetative growth rate, transparent and irregular-shaped mycelium, without conidial production. Melanin biosynthesis of the strain△St4HNR is dropped to 16.5μg/g and△St3HNR△St4HNR is dropped to 4.7μg/g.△St3HNR△St4HNR strains after induction does not produce appressorium. The△St4HNR strain appressorium turgor is 4.32 MPa, significantly lower than the wild-type appressorium turgor pressure 5.45 MPa.The time of the△St4HNR strains appressorium formation and penetration capability were 12 h later than the wild-type strains and infection ratio were dropped to 42%. Those results indicated that deletion of the St3HNR and St4HNR reduced the pathogenicity of S. turcica by inhibiting DHN melanin biosynthesis and turgor pressure of appresoria.