【作者】 王芬；

【导师】 薛春生；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2021， 博士

【摘要】 由新月弯孢菌[Curvularia lunata(Walk.)Boed.]侵染引起的玉米弯孢叶斑病是一种重要的世界性病害,广泛发生于美洲、欧洲、亚洲及非洲的玉米产区,严重威胁着玉米安全生产。C.lunata通过附着胞侵入玉米叶片表皮细胞,同时产生细胞壁降解酶和毒素破坏角质层、细胞壁、细胞膜和防御酶系统,进而完成侵染和致病。附着胞是半活体营养类型植物病原真菌活体营养阶段的重要致病手段,其调控机制目前还不十分清楚,NADPH氧化酶(NOX)是真核生物产生胞外活性氧(ROS)的关键酶,参与多种植物病原真菌侵染结构的形成。因此,本文研究了新月弯孢菌NADPH氧化酶五个亚基调控其生长发育和致病力的作用与机制。取得的主要研究结果如下:1.明确了活性氧及NADPH氧化酶是玉米弯孢叶斑病菌生长发育、附着胞形成和黑色素生物合成所必需的。玉米弯孢叶斑病菌在孢子萌发和附着胞形成过程中分生孢子、芽管和初始附着胞都能产生活性氧。活性氧清除剂抗坏血酸和NADPH氧化酶抑制剂DPI处理玉米弯孢叶斑病菌后,菌丝生长发育和附着胞形成均受抑制,且随着抗坏血酸和DPI浓度的增加,抑制效果越显著。2.鉴定了玉米弯孢叶斑病菌NADPH氧化酶组成亚基和调控亚基。通过系统发育分析、蛋白质功能结构域和理化性质分析,确定了玉米弯孢叶斑病菌NADPH氧化酶由3个组成亚基Cl NOX1、Cl NOX2、Cl NOX3和2个调控亚基Cl NOXR和Cl RAC1组成,并且获得了这五个亚基基因敲除和互补突变体。3.Cl NOX3和Cl RAC1不是维持玉米弯孢叶斑病菌生长发育和致病力的必需组份。通过分析Cl NOX3和Cl RAC1基因敲除与互补突变体的菌丝生长、分生孢子产量、附着胞形成、假囊壳与子囊形成、抗逆和病情指数等指标,发现Cl NOX3和Cl RAC1不影响玉米弯孢叶斑病菌的生长发育、侵染结构和致病力。4.明确了Cl NOX1和Cl NOXR调控玉米弯孢叶斑病菌有性生殖机制。Cl NOX1定位在细胞质膜上,Cl NOX1和Cl NOXR敲除突变体假囊壳和子囊数量减少,假囊壳较小,交配型基因MAT1-2-1和非核糖肽合成酶基因Cl NPS2表达下调,同时活性氧生成量和致病力也显著下降。敲除调控活性氧平衡的氧化还原转录因子基因Cl AP1,突变体假囊壳和子囊生成量减少,MAT1-2-1和Cl NPS2基因表达下调;敲除超氧化物歧化酶基因Cl SOD1假囊壳及子囊生成量减少;敲除过氧化氢酶基因Cl CAT4假囊壳及子囊生成量增加,敲除Cl CAT1和Cl CAT3不影响假囊壳和子囊形成。敲除Fus3/Kss1同源基因Cl K1假囊壳较小,子囊生成量减少,MAT1-2-1和Cl NPS2基因表达下调;敲除丝氨酸/苏氨酸蛋白激酶基因Cl SCHA假囊壳数量减少,MAT1-2-1基因表达下调;敲除小G蛋白基因Cl G2不影响假囊壳和子囊形成。Cl NOX1和Cl NOXR转录水平调控Cl SCHA和Cl K1基因表达。以上结果表明,活性氧平衡相关基因Cl NOX1、Cl NOXR、Cl AP1、Cl SOD1和Cl CAT4,MAPK信号通路基因Cl K1,c AMP-PKA信号通路基因Cl SCHA参与调控玉米弯孢叶斑病菌有性生殖,Cl NOX1/Cl NOXR相关的ROS与c AMP-PKA和MAPK信号途径共同调控玉米弯孢叶斑病菌有性生殖。5.明确了Cl NOX2调控黑色素生物合成影响C.lunata生长发育和致病力机制。Cl NOX2定位在质膜上,参与超氧根阴离子和胞内活性氧生成。Cl NOX2和细胞壁完整性信号途径MAPK基因Cl M1敲除突变体菌落颜色变浅,生物学和致病力表型与DHN黑色素生物合成基因Cl BRN1敲除突变体相似。Cl NOX2和Cl M1在转录水平上分别调控DHN黑色素生物合成基因Cl SCD1和Cl BRN1表达,且Cl M1负调控Cl NOX2基因表达。以上结果表明,Cl NOX2介导的活性氧信号途径与Cl M1介导的细胞壁完整性信号途径共同调控胞内DHN黑色素生物合成,进而影响玉米弯孢叶斑病菌致病力。更多还原

【Abstract】 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.更多还原