| Laccases are multicopper oxidases,which are generally in the form of gene families encoding isozymes with different activities.Laccases play important roles during important biological processes including morphogenesis,host-pathogen interaction,pigments synthesis,and stress response.Due to the large number of laccase gene family members and the wide range of substrates,the understanding of its function is mostly phenotypic analysis with less of the biochemical pathways.Setosphaeria turcica is a hemibiotrophic ascomycete fungus that infects crops such as corn and sorghum,forming gray-brown lesions to cause wilting and a reduction in photosynthetic potential reducing yield.It was found that the knock-out of laccase genes StLAC1 and StLAC2 affected the growth,development and pathogenicity of S.turcica.In this paper,the laccase gene family was studied systematically.The key laccase genes were identified at the transcriptional and protein levels and the function in the growth,development and pathogenesis was studied by genomics and metabolomics.The main results were as follows.1.LMCOs were found in the S.turcica genome using a Hidden Markov Model for three Pfam copper oxidase families.They had conserved amino acid residues in the Cu-binding sites,but shared a low homology of 19.79%-48.70%.They were classified into five LMCO superfamilies,of which Stlac1,Stlac2,Stlac4,Stlac6 are Ascomycetes multicopper oxidase.Certain cis-acting elements of transcription factors were found in the upstream of these genes including NIT2（the major positive-acting nitrogen regulatory gene）,ADR1（apositive regulator of peroxisomal protein genes）,XRE（xenobiotic-responsive element）,CRE-A（responsible for carbon catabolite repression）and so on.By transcriptomic analysis,it was found that the expression of StLAC2 and StLAC6 was the highest in S.turcica isolate 01-23mycelia,St LAC3 was higher and the expression of other genes was relatively low.Laccase activity and transcription levels were affected by culture time,carbon source,nitrogen source,copper ion and iron ion.The laccase genes were differentially expressed during different stages of infect of maize leaves.The relative expression of mostly genes increased except of StLAC6 and StLAC8.2.There were three active laccase isozymes:Stlac1,Stlac2,and Stlac6 in S.turcica 01-23,identified by Native-PAGE and ESI-MS/MS.Stlac2 was mainly expressed intracellularly,while Stlac1 and Stlac6 were detected both intracellularly and extracellularly.These laccase isozymes were heterologously expressed in prokaryotic expression systems using Escherichia coli as host.The IPTG concentration,induction temperature,initial bacterial concentration,rotational speed and the addition of copper ion were optimized to obtain the recombinant isozymes Stlac1,Stlac2 and Stlac6,with the activity of 1.058 U/mg,0.861U/mg and 0.028 U/mg using ABTS as the substrate.The enzymatic properties showed that the recombinant laccases had a lower optimum pH（3-4.5）and a higher optimum temperature（60-70℃）.The effect of metal ions on the enzyme activity was different,but Fe3+had a strong stimulating effect on the activities of these isozymes.The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive because of incorrect glycosylation at the position of Asn97.3.By protoplast transform,the mutants of key laccase gene StLAC6 were constructed and two mutants were got.The phenotype analysis showed that the deletion of StLAC6 gene had no effect on the growth,morphology,sporulation and infection ability,but the pathogenicity ofΔStLAC6 to susceptible maize was increased.The crude toxin ofΔStLAC6 caused browning of the wounds on maize leaf,and the melanin synthesis increased.The peroxisome inΔStLAC6 hypha was increased with abnormal morphology and the numbers of liposome and phenolic content increased.The deletion of StLAC6 gene resulted in a significant increase in the relative expression of StLAC1,StLAC4 and StLAC5,and also affected the expression of neighboring genes of StLAC6,including genes encoding N-acetyltransferase,cytochrome P450 monooxygenase and enzymes related to phospholipid metabolism.4.The metabolite extraction method of S.turcica hyphae was optimized and the mycelia of wild type strain,ΔStLAC1,ΔStLAC6 were analyzed by metabolomics by UPLC-Triple TOF.The results showed that,compared with wild type strain,the deletion of laccase gene caused significant changes in the metabolites of the pathogen.The differential metabolites were more with most of them unique inΔStLAC1,while less inΔStLAC6 and most of them simultaneous change inΔStLAC1 andΔStLAC6.Among the metabolites,a variety of secondary metabolites were first identified in S.turcica.The secondary metabolites such as phytosphingosine,prostaglandin E2 and mycotoxins-related metabolites such as zearalenol and citrinin were quantified by ELISA assay,and the result was identical with metabolomics.5.Metabolomics analysis showed that Stlac1 and Stlac6 were involved in the synthesis of lipids and aromatic compounds,especially polyketones.The deletion of StLAC6 resulted in the increase of mycotoxins such as fusarochromanone,citrinin and diplosporin,which affected the pathogenicity of fungi.The deletion of StLAC1 mainly affected the synthesis of phospholipids and steroids,which affecting the pathogenicity and morphology of fungi.The change of some prostaglandins inΔStLAC1 andΔStLAC6 was opposite.By molecular docking of protein-metabolite and fluorescence titration,it was found that prostaglandins E2and citrinin were natural substrates of Stlac6,riboflavin was natural substrates of Stlac1,so laccases were involved in these metabolic pathways directly.But laccase was not combined with phospholipids and fatty acids,but the change of them caused by deletion of laccase,which affected the morphology,melanin synthesis and pathogenicity of S.turcica through signal transduction pathways. |
