Characterization And Function Analysis Of The Genes Involved In Melanin Biosynthesis Pathway In The Phytopathogenic Fungus Setosphaeria Turcica

DHN melanin is common in ascomycetous and imperfect fungi, which can protect microorganisms from UV light and the action of microbial lytic enzymes. It is considered as one of the important virulence factors for certain plant and animal pathogenic fungi. The roles of melanin have been studied extensively in regard to the melanization of the appressorial cell wall and the penetration process in phytopathogenic fungi. In this research, four genes of Setosphaeria turcica, involved in DHN melanin biosythnthesis pathway, were cloned and named as St3HNR, StLAC1, StLAC2 and StMR respectively. St3HNR encoded naphthalene reductase, StLAC1 and StLAC2 genes belonged to laccase gene, StMR encoded a transcription factor. All of them shared high amino acid homology with the corresponding proteins in other pathogenic fungi. Functional analysis of St3HNR, StLAC1 and StMR genes were explored by creating the gene-knockout mutants and RNAi mutants. Main results in this paper were as follows:1. One 1,3,8-Trihydroxynaphthalene(1,3,8-THN) reductase gene (St3HNR), two laccase genes (StLAC1 and StLAC2) and one regulation gene (StMR) were cloned with the candidate gene cloning strategy. Among them, we had cloned the full length DNA and cDNA of St3HNR, StLAC1 and StMR and also obtained partial coding region of StLAC2. The ORF and amino acid sequence of St3HNR had been deposited in the GenBank database and the accession numbers were No.EU681832 and No.ACD47140.2. Gene structures of St3HNR, StLAC1 and StLAC2 were analysised. St3HNR included 918 bp DNA sequence with 804 bp coding region and consisted of 3 exons and 2 introns. Its predicted protein contained 267 aa with a molecular weight of 28.28 kDa. StLAC1 gene included 1855 bp and interrupted by one intron and its ORF of 1806 bp encoded 601 amino acid residues. StMR gene with 3276 bp encoded a protein of 1011 amino acid residues and was interrupted by 3 introns. All introns were accordance with GT-AG rules.3. Homology analysis showed that the deduced amino acid sequence from the St3HNR gene shared 95% similarity to fungal 1,3,8-THN reductase and a complete reductase domain was found, whereas 1,3,8-THN reductase had 45.9% sequence similarity to 1,3,6,8-THN(St4hnr) of S. turcica. It was probable that two genes belonged to two groups of reductase and catalyse different substrate. StLAC1 exhibited the high identities to known laccases in fungi with over 60%. The low identity for the StLAC2 on nucleic acid level was found for the laccase from Hortaea acidophila, Fusarium proliferatum, Gaeumannomyces graminis with 33% ~ 46%. StLAC1 and StLAC2 genes exhibited much less homology identity (26.34%). Stlac1 protein has three conserved copper binding- regions, especially conserved regions with high identity. The deduced amino acid sequence of the StMR shared 88% similarity with the transcription factor of Bipolaris oryzae and Cochliobolus heterostrophus. The predicted S. turcica Stmr protein has two ZnF_C2H2 domains and one GAL4 domain as Zn(II)2Cys6 binuclear cluster close to its N terminus, similar to Magnaporthe grisea Pig1p and Colletotrichum lagenarium Cmr1p. Interestingly, all three domains proteins are highly similar.4. Southern hybridization results showed that genes of St3HNR and StLAC1 had single copy in the genome of S. turcica, whereas there are multicopy of StLAC2.5. Eukaryotic gene expression vector of St3HNR, pPIC9K-St3HNR, was constructed and expressed in host yeast strain GS115 through electroporation. The St3hnr protein expression was confirmed by SDS-PAGE.6. The St3HNR gene-disruption vector was constructed based on the gene double-cross homologous combination theory and PEG-mediated gene transformation system. Transformants were screened by hygromycin B and PCR with specific primers corresponding to hygromycin phosphotransferase gene and St3HNR gene. Six St3HNR gene-disruption mutants, named as△3hnr1,△3hnr2,△3hnr3,△3hnr4,△3hnr5 and△3hnr6, were obtained by Southern blot analysis performed with the DIG-labeled HPH gene and St3HNR gene as probes respectively. Although wild type strain and mutants were similar in form of colony and hypha, sporulation, HT-toxin activity, rates of spore germination and appressorium formation, all mutants exhibited brownish-red colony, colourless hypha and conidium, weakend turgor pressure and penetration of appressorium, slow vegetative growth rate and low pathogenicity on health host. The pathogenicity of mutants was restored while they were inoculated on wound corn leaves, but the area of lesion was decreased compared with wild type.7. The laccase activity of S. turcica was tested by spectrophotometer under 420 nm with ABTS as the substrate, and the influence of the test conditions on the enzyme activity were studied. The results showed that optimal reaction pH value, concentration of substrate and reaction time were 2.6, 0.6 mmol/L and 5 min respectively. In addition, the best laccase activity of S. turcica was screened when it grew on the medium containing 250μmol/L CuSO4.8. The StLAC1 gene replacement vector was also constructed based on the same strategy as St3HNR. StLAC1 transformants named StLAC1-M2, StLAC1-M3 and StLAC1-M4, which exhibited same grey colony as wide type. Extracellular oxidase activity of laccase in transformants StLAC1-M3 and StLAC1-M4 were reduced.9. The silencing vector encoding hairpin RNA of the StMR fragment was constructed in a two-step PCR based cloning, and introduced into the S. turcica genomic DNA. Six transformants with different color colony from less-pigmented transformants to the wild-type phenotype were obtained after screening by hygromycin B. Most transformants exhibited low aerobic mycelia, dense mycelia, transparent and curl hypha, without conidial production.The above results can summarize the function of these genes: 1) St3HNR gene was involved in melanin biosynthesis and related to penetration of appressorium, pathogenicity, respectively. 2) This study presents evidence that S. turcica possesses functional laccase and at least two laccases genes, but StLAC1 gene is not essential for melanin biosynthesis. 3) Accumulation of melanin in S. turcica was affected by StMR gene and it is necessary to clarify the regulation mechanism in future. This work is helpful for us to study and understand the other conserved gene function in DHN melanin biosynthesis pathway of S. turcica.