Differential Regulation Of Laccase StLAC1 And StLAC6 Pathogenicity Mechanism In Setosphaeria Turcica

Laccases are multicopper oxidases,which play important roles during important biological processes including morphogenesis,host-pathogen interaction,pigments synthesis,and stress response.In order to clarify the biological role of different laccase isozymes in Setosphaeria turcica,on the basis of previous obtained laccase StLACl and StLAC6 gene knockout mutants of S.turcica,the recovery and overexp-ression strains of StLAC1 and StLAC6 were further created,and their functions in growth,development and infection were analyzed.At the same time,in order to deeply analyze the way it works,the effects of StLAC1 and StLAC6 knockout on gene expression and metabolite synthesis of S.turcica were compared by transcriptome and metabonomics,so as to provide a basis for analyzing and comparing the action mechanism of different laccase isozymes.The main results were as follows:1.The recovery and overexpression vectors were constructed by seamless cloning method,and the recovery and overexpression strains of key laccase genes StLAC1 and StLAC6 were created by protoplast transformation technology.2.Phenotypic analysis of wild-type strain,knockout mutant ΔStLAC1,revertant strain C.ΔStLAC1 and overexpression strain OE.StLAC1 showed that StLAC1 affected melanin synthesis and fungal cell wall structure.It also affected the pathogenicity of the pathogen by affecting the germination and infection ability of the appressorium.Solid-state NMR detection showed that the deletion of StLAC1 did not affect the structure of melanin,but only affected its polymerization in the cell wall.3.Phenotypic analysis of wild-type strain,knockout mutant ΔStLAC6,revertant strain C.ΔStLAC6 and overexpression strain OE.StLAC6 showed that StLAC6 gene did not affect the growth of the pathogen,but negatively regulated the melanin synthesis and pathogenicity of the pathogen.Crude toxin of knockout mutant made leaves more active oxygen,but increased the sensitivity to chemical fungicides.4.Metabonomics was used to analyze the intracellular and extracellular differential metabolites of wild-type strain,ΔStLAC1 and ΔStLAC6 of S.turcica.The results showed that the deletion of StLAC1 gene had a greater impact on the metabolites,and the differences between StLAC1 and StLAC6 affected the melanin synthesis precursor metabolites,and StLAC1 gene mainly affected the lipid metabolites.5.In order to clarify the morphological and pathogenic mechanism of StLAC1 on S.turcica,transcriptome was used to analyze the effect of StLAC1 deletion on gene transcription level,and it was found that StLAC1 deletion affected the expression of genes related to membrane components,cell metabolic activities and mitochondrial function,causing the disorder of membrane structure and sphingolipid signal,and the change of energy utilization mode of the pathogen.