Mechanisms Of NADPH Oxidase(NOX) Genes On Sexual Reproduction And Pathogenicity In Curvularia Lunata

Curvularia leaf spot,caused by [Curvularia lunata(Walk.)Boed.] is an important maize leaf disease around the world.It widely occurs in maize planting areas in America,Europe,Asia and Africa and seriously threats to the safe production of maize.C.lunata penetrates maize leaf epidermal cells through its infective structure appressoria,meanwhile,secretes cell wall degrading enzymes and toxins to destroy maize stratum corneum,cell wall,cell membrane and defense enzyme system,and then sucessfully infect and cause disease.Appressoria of hemibiotrophic plant pathogenic fungi are used as important weapons in their biotrophic stage.However,the regulatory mechanism of appressorial formation is not yet fully understood.NADPH oxidase(NOX),as a key enzyme that produces extracellular reactive oxygen species(ROS)in eukaryotic organisms,participates in infection structure formation of various plant pathogenic fungi.In this paper,the functions of five subunits of NADPH oxidase in C.lunata were studied,and their regulatory mechanisms on growth,development and pathogenicity were explored.The main research results obtained are as follows:1.Reactive oxygen species and NADPH oxidase are necessary for the growth and development,appressorial formation and melanin biosynthesis in C.lunata.ROS were produced within conidia,germ tubes,and appressoria during conidial germination and appressorial formation.When C.lunata was treated with ROS scavenger ascorbic acid and NADPH oxidase inhibitor DPI,hyphal development and appressorial formation were inhibited.The effects were in an AA/DPI concentration-dependent.2.The constituent subunits and regulatory subunits of the NADPH oxidase in C.lunata were identified.Phylogenetic analysis,protein functional domain,and physical and chemical properties analysis revealed that C.lunata had three constituent subunits Cl NOX1,Cl NOX2 and Cl NOX3 and two regulatory subunits Cl NOXR and Cl RAC1.Then deletion and complementation mutants of the five subunit genes were obtained.3.Cl NOX3 and Cl RAC1 are not essential for C.lunata development and pathogenicity.Mycelial growth,conidial production,appressorial formation,pseudothecium and ascus formation,stress resistance,and disease index of Cl NOX3 and Cl RAC1 deletion and complementation mutants were examined.The results show that deletion of Cl NOX3 and Cl RAC1 did not affect C.lunata mycelial growth and development,infection structure formation and pathogenicity.4.Cl NOX1 and Cl NOXR regulate C.lunata sexual reproduction.Cl NOX1 localized on the cell plasma membrane.Deletion of Cl NOX1 and Cl NOXR caused reduced pseudothecium and ascus number,smaller pseudothecia,down-regulated expression level of MAT1-2-1 and Cl NPS2 genes.Meanwhile,ROS production and pathogenicity of the mutants were reduced.Deletion of redox transcription factor gene Cl AP1 in C.lunata resulted in reduced pseudothecium and ascus number,and the expression level of MAT1-2-1 and Cl NPS2 genes in the mutants was down-regulated.Knockout of superoxide dismutase gene Cl SOD1 weakened pseudothecium and ascus formation,while deletion of catalase gene Cl CAT4 promoted pseudothecium and ascus formation.Removing of catalase genes Cl CAT1 and Cl CAT3 did not affect pseudothecium and ascus formation.Deletion of Cl K1 which is homologous to Fus3/Kss1,resulted in smaller pseudothecia and reduced ascus number,and the expression level of MAT1-2-1 and Cl NPS2 genes in the mutants was down-regulated.Deletion of serine/threonine protein kinase gene Cl SCHA in C.lunata resulted in reduced pseudothecium number,and the expression level of MAT1-2-1 gene in the mutants was down-regulated.Removing of small G protein gene Cl G2 did not affect pseudothecium and ascus formation.Cl NOX1 and Cl NOXR transcriptionally regulated the expression of Cl SCHA and Cl K1 genes.The above data show that ROS related genes Cl NOX1,Cl NOXR,Cl AP1,Cl SOD1 and Cl CAT4,MAPK signalling pathway gene Cl K1,and c AMP-PKA signalling pathway gene Cl SCHA are involved in C.lunata sexual reproduction.Cl NOX1 and Cl NOXR produced ROS signaling pathway,Cl SCHA mediated c AMP-PKA signaling pathway,and Cl K1 mediated MAPK signaling pathway are cross-linked in regulating C.lunata sexual reproduction.5.Cl NOX2 affects C.lunata development and pathogenicity by regulating melanin biosynthesis.Cl NOX2 localized on the plasma membrane and generated superoxide anion and intracellular ROS.Deletion of Cl NOX2 and cell wall integrity signaling pathway MAPK gene Cl M1 resulted in lighter colony color.The biological and pathogenic phenotypes of both mutants are similar to those of DHN melanin biosynthesis gene Cl BRN1 deletion mutants.Cl NOX2 and Cl M1 respectively regulates the expression of DHN melanin biosynthesis gene Cl SCD1 and Cl BRN1,and Cl M1 negatively regulates Cl NOX2.The above data show that Cl NOX2 related ROS signaling pathway and Cl M1 mediated cell wall integrity signaling pathway are cross-linked in regulating intracellular DHN melanin biosynthesis,which in turn affects C.lunata development and pathogenicity.