【作者】 刘同宝；

【导师】 闵航； 林福呈；

【作者基本信息】 浙江大学 ， 微生物学， 2008， 博士

【摘要】 细胞自噬是真核生物中高度保守的过程,在此过程中,包括过剩或异常的细胞器在内的细胞物质被包裹进双层膜的自噬泡中并被运输到溶酶体或液泡中进行降解,从而使得生物大分子得以重新利用。细胞自噬在诸如对变化环境的适应、发育与分化过程中细胞形态的建成以及细胞寿命的决定等一系列生物事件中起着重要作用。细胞自噬相关基因最早在酵母中被鉴定,如今在所有的高等生物中已发现其同源基因。细胞自噬可由饥饿诱导产生并维持一个氨基酸库以便合成一些必需的蛋白质。稻瘟病是一种由稻瘟病菌引起的毁灭性病害,给全世界的水稻生产带来巨大威胁。稻瘟病菌(Magnaporthe oryzae)具有典型的生活史和侵染循环,且被认为是丝状真菌分子生物学和病原菌与寄主互作研究的模式生物。稻瘟病菌能够形成专门的侵染结构—附着胞,其能够产生巨大的胞内膨压(约8.0Mpa),这个压力使稻瘟病菌穿透植物叶片表皮并侵染寄主。本研究主要目的是阐明细胞自噬相关基因MgATG4的表达模式,细胞自噬与稻瘟病菌生长、发育和致病性之间的关系以及细胞自噬相关蛋白MgATG4与MgATG8之间的相互作用。获得的主要研究结果如下:从稻瘟病菌成熟附着胞的差减文库中克隆到了细胞自噬相关基因MgATG4,该基因编码一个53.93kDa的半胱氨酸蛋白酶。将稻瘟病菌MgATG4基因,互补到酵母ATG4基因缺失突变体中,能够恢复酵母的细胞自噬过程;对MgATG4进行时间表达模式分析,发现该基因在孢子、附着胞及菌丝中均有表达;细胞定位分析发现MgATG4蛋白在细胞质中均匀表达。通过构建MgATG4基因置换载体,得到MgATG4基因缺失突变体△mgatg4。△mgatg4突变体在CM培养基上的菌落形态发生变化,菌丝稀疏;产孢量显著下降,约为野生型Guy11的1/90;分生孢子的萌发及附着胞形成延迟;附着胞膨压显著下降,在完整的水稻或者大麦叶片上不能形成可见病斑,致病性丧失;在有磨损的大麦叶片上也未能产生病斑;突变子在OMA培养基上与2539菌株交配,能产生子囊壳,但延迟且量极少;基因MgATG4的缺失,使细胞自噬过程中断,液泡和细胞质中无自噬泡的积累。从稻瘟病菌成熟附着胞的差减文库中克隆得到了另一个细胞自噬相关基因MgATG8,该基因编码一个14.4kDa的ATG8蛋白。对MgATG8进行时间表达模式分析,发现该基因在孢子、附着胞及菌丝中均有表达。通过双分子荧光互补技术检测MgATG4与MgATG8在稻瘟病菌细胞发育过程中的相互作用,分别构建BiFC载体ATG4-YFPN、ATG4△-YFPN和YFPC-ATG8共转化稻瘟病菌Guy11菌株并用潮霉素和草胺磷双抗筛选。转化子BY-7经共聚焦显微镜检测结果发现在孢子、营养菌丝、附着胞形成过程及穿透过程中均没有检测到YFP荧光信号,但在饥饿诱导4h后的菌丝中检测到了MgATG4与MgATG8的相互作用。MgATG4蛋白序列中含有6个半胱氨酸残基。通过序列比对分析,发现MgATG4蛋白序列中的Cys206为保守半胱氨酸;分别构建MgATG4、MgATG8蛋白表达载体pPICZα-ATG4和pPICZα-ATG8,并采用定点突变技术将MgATG4第206位的半胱氨酸残基Cysteine突变为丝氨酸Serline。采用毕赤酵母表达系统表达野生型和突变体MgATG4-His融合蛋白以及MgATG8-His融合蛋白并纯化。体外蛋白酶切实验表明Cys206是MgATG4的活性位点。更多还原

【Abstract】 Autophagy is a highly conserved process in eukaryotes in which the cytoplasm, including excess or aberrant organelles,is sequestered into double-membrane vesicles and delivered to the degradative organelle,the lysosome/vacuole,for breakdown and eventual recycling of the resulting macromolecules.This process has an important role in various biological events such as adaptation to changing environmental conditions, cellular remodeling during development and differentiation,and determination of lifespan.Autophagy-related genes were first identified in yeast,but homologs are found in all eukaryotes.Moreover,autophagy is dramatically induced under starvation conditions for maintaining an amino acid pool so that essential proteins may be synthesized.Rice blast,the most serious disease of cultivated rice throughout the world,was caused by Magnaporthe oryzae,a filamentous ascomycete fungus,and has threatened the rice production worldwide.Magnaporthe oryzae has a typical life history and infection cycle,and has been developed as a model organism for the investigation of fungus-host interaction.Plant infection by the rice blast fungus is brought about by the action of specialized infection cells called appressoria.These infection cells generate enormous turgor pressure（as much as 8.0 Mpa）,which is translated into an invasive force that allows a narrow penetration hypha to breach the plant cuticle.This work is aimed to investigate the expression pattern of MgATG4,the role of autophagy in the process of cell development,differentiation and the relationship between autophagy and pathogenicity.Moreover,the interaction between autophagic proteins MgATG4 and MgATG8 was also involved.An autophagy-related gene MgATG4,which encodes a cysteine proteinase with a putative molecular weight of 53.93kDa,was cloned from a previously constructed suppression subtractive cDNA library of mature appresoria in M.oryzae strain Guy-11. To determine whether MgATG4 in the rice blast fungus is functionally related to the Atg4 ofS.cerevisiae,a full-length MgATG4 cDNA fragment of M.oryzae was introduced into S.cerevisiae strainΔatg4 using pYES2.The results showed that MgATG4 restored the corresponding defects in the starvation-sensitive phenotype ofΔatg4 mutant of S. cerevisiae,indicating that MgATG4 of M.grisea is presumably homologous to Atg4 of yeast in structure and function.To detect the expression of MgATG4 in Magnaporthe,we analyzed the temporal control of MgATG4 gene expression by construction and expression of a MgATG4 promoter-green fluorescent protein（GFP） fusion in Magnaporthe.Expression of GFP in MgATG4（p）:eGFP transformants was detected in conidia and during appressorium development.Furthermore,expression of GFP was also observed in vegetative hyphae in axenic culture with CM and CM-N media.When grown in CM-N medium,the green fluorescence emitted by GFP protein in hyphae was brighter than those grown in complete medium,implying that the MgATG4 promoter was induced by starvation.To investigate the localization of MgATG4 in M.oryzae,we constructed a MgATG4-fluorescent green protein gene fusion expression cassette.Expression of MgATG4-GFP was uniformly detectable in the cytoplasm of conidia,mycelia,and appressoria.A targeted gene deletion strategy was adopted to determine the role of MgATG4 in fungal development and pathogenicity at the molecular level.Deletion mutants ofΔmgatg4 formed fewer sparse aerial hyphae and conidiogenesis was reduced significantly and autophagy was blocked,indicating that MgATG4 is essential for autophagy. Furthermore,Δmgatg4 mutants showed delayed conidial germination and appressorium formation,lower turgor pressure of the appressorium.As a result of decreased appressorium turgor,theΔmgatg4 mutant lost its ability to penetrate the two host plants tested,namely rice and barley.The developmental and pathogenic phenotypes were recovered following re-introduction of an intact copy of MgATG4 into the mutant, suggesting that autophagy is thus necessary in the development of M.oryzae and essential to pathogenicity of the fungus.In mating experiments,the wild-type strain and the MgATG4-rescued strain produced many perithecia with viable ascospores with the opposite mating-type strain,2539,whileΔmgatg4 mutants need about 10 more days of incubation and formed less perithecia.Another autophagy-related gene MgATG8,which encodes an Atg8 family protein with a putative molecular weight of 14.4 kDa,was cloned from the same mature appresoria cDNA library.Temporal expression analysis revealed that MgATG8 expressed during plant infection and vegetative growth by Magnaporthe.To directly examine the in vivo MgATG4-MgATG8 interaction by BiFC assay,we generate the ATG4-YFPN, ATG4Δ-YFPN and YFPC-ATG8 fusion constructs and transformed them in pairs into the wild type Guy 11 strain and selected with hygromycin and glufosinate ammonium.The resulting transformant,BY1-7,was analysed by Southern hybridization and examined for YFP signals.Conidia of BY1-7 were incubated on onion epidermal cells for 2,4,8,12, 24 and 48 h,and observed under fluorescence microscopy.Although YFP fluorescehce was not detectable in conidia,appressoria,and vegetative hyphae,fluorescence signals were observed in vegetative hyphae grown under nitrogen starvation.Meanwhile, infectious hyphae that developed inside onion epidermal cells by the transformant had very weak or no YFP signals.These data.indicate that the MgATG4-MgATG8 interaction was enhanced during nitrogen starvation.MgATG4 contains 6 cysteines,one of which is highly conserved among homologues of fungi and mammals.The only conserved residue is the putative active residue of the protease Cys206,which also in the yeast S.cerevisiae,AtATG4,mouse ATG4A and ATG4B,HsATG4A,HsATG4B.The open reading frame encoding MgATG4 was amplified by PCR and subcloned into a pPICZαA Pichia expression vector plasmid.The construct,designated pPICZαA-ATG4,was used for the in vitro studies.Another construct,pPICZαA-ATG8,was constructed with the same strategy.One mutant gene was constructed based on the WT construct:MgATG4^C206S,in which cysteine 206 was replaced with serline.Mutagenesis was carried out by the pfu Turbo DNA polymerase with the QuickChange Site-Directed Mutagenesis Kit according to the manufacture’s protocol.Recombinant MgATG4-His6（WT and the mutant） and MgATG8-His6 were expressed in Pichia GS-115 strain and purified with the ProBond^TM Purification System. Assay of cleavage of MgATG8 in vitro revealed that recombinant MgATG4 harboring a mutation of cysteine to serline at position 206(MgATG4^C206S-His6) did not cleave MgATG8,neither in the absence nor in the presence of DTT.Based on these results and the sequence homology described above,we conclude that Cys206 is part of the active sites of MgATG4.In summary,autophagy is necessary for the formation of conidia and appressoria and for normal development and pathogenicity of Magnaporthe.The MgATG4 null mutants show defects in autophagy,the ability to survive starvation,conidiation,conidial germination,and appressorium turgor generation.As a result,theΔmgatg4 mutant loses its penetration ability and pathogenicity to the two tested host plants,namely rice and barley.BiFC assay indicated that the MgATG4-MgATG8 interaction was enhanced during nitrogen starvation.Assay of cleavage of MgATG8 in vitro revealed that Cys206 is part of the active sites of MgATG4.更多还原