Expression Pattern And Functional Analysis Of An Autophagy-related Gene MgATG4 In Magnapor The Oryzae

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.