| Rice blast disease caused by Magnaporthe oryzae is the mainly fungal disease in rice,threatening global rice production seriously.The most destructive form of rice blast,neck blast,often reaches epidemics without severe foliar symptoms and macroconidium production,suggesting that macroconidia may be not the only inoculum in the field.Wheat blast caused by another forma specialis of M.oryzae is referred as a wheat head disease and epidemics of wheat blast have destroyed many wheat fields that have no foliar or stem symptoms(only the heads became blighted).Therefore,a certain inoculum other than macroconidia must be responsible for the spreading and outbreak of wheat blast.The rice blast fungus M.oryzae,a model for studying fungal-plant interactions,produces both microconidia and macroconidia(i.e.pyriform conidia).However,most of studies focus on macroconidia.Microconidia germination has not been reported and its roles in plant infection are not clear.This project mainly focuses on microconidiation in liquid,germination,infectivity and gene expression of microconidia in M.oryzae.At first,we created a method producing abundent and pure microconidia.Microconidia harvested from the culture solution by filtration through Miracloth and Whatman filter paper were not contaminated with macroconidia or hyphal fragments.By examining microconidiation in a number of mutants,we found the Δpmk1 and Δmst12 mutants produced over 100-fold fewer microconidia than the wild type while Δmip11 had 4.5-fold up-regulated.Thus,microconidium production was related with the Pmk1 MAPK pathway.About 4.9% of microconidia produced germ tubes after incubation on 1% water agar for 72 h.Microconidia can’t form appressoria no matter on plant surfaces or hydrophobic slides.Most of germinated microconidia could not form colonies,which maybe caused by the lack of mitosis in germ tubes.Microconidia were regarded as spermatia in sexual reproduction in previous publications.No attraction or obvious changes were observed in the germination and germ tube growth of strain 70-15(MAT1-1-1)microconidia in the presence of Guy11(MAT1-1-2)on 1% water agar and oatmeal agar.Thus,microconidia had no functions as spermatia in our test conditionsa and they may have other functions in M.oryzae.Microconidia failed to infect intact host leaves,which was consistent with the fact that microconidia had no appressoria.Microconidia had limited carbon storage in comparison with macroconidia and lacked enough lipid and glycogen to generate sufficient turgor pressure for penetration.However,microconidia could colonize and cause necrotic lesions on wounded plant leaves,stems,and flowering heads.Fungal colonies derived from germinated microconidia or recovered from lesions caused by microconidia were normal in growth,conidiation,and pathogenesis.The condition of producing microconidia in liquid submerged culture is similar to growth conitions in planta rather than sexual reproduction,so it is likely that microconidia could be produced in diseased plants,especially in vascular system.We detected microconidia in asymptomatic tissues of diseased rice plants under laboratory and field conditions,and found microconidia or microconidia mimic.In addition,we identified two genes specifically expressed in microconidia,six genes expressed over 20-folds up-regulated than hyphae and macroconidia by comparing the transcriptome sequencing data.Most of these genes’ functions were unknown,which may be related with undefined functions of microconidia.Thus,it is important for exploring the roles of microconidia to focus on genes specifically and up-regulated expressed.Our results demonstrate that microconidia are able to germinate,infect hosts and can be produced in planta.Due to their smaller size and abundance,microconidia produced in planta may allow the pathogen to spread readily via the vascular system and result in systemic colonization,which may be an important factor in the outbreaks of rice blast.Results from this study provide the important theory reference for controlling rice blast,wheat blast,and other important diseases caused by Magnaporthe species.Another work was carried out about pathegonicity mechanism of macroconidia.Deletion of the MAP kinase MoMPS1,cause the pathogen no penetration into plant cuticle and non-pathogenic.Programmed cell death of macroconidia was delayed.It is known that autophagic cell death in macroconidia is necessary for successful infection by the rice blast fungus.Thus we speculate that deletion of MoMPS1 affect the autophagy process,leading to low ability of transferring energy storage to the appressoria.This project works on the relationship of MoMps1 kinase and autophagy-associated phenotype in ?Momps1.Ten candidate genes related concurrently with the Mps1 pathway and autophagy mechanism were screened by yeast two-hybrid assays.The interaction of MoAtg13,MoAtg26,MoPex1 and MoMps1 were observed.In addition,MoAtg13 interacted with MoPex1.Atg13 had conserved functions in various eukaryotic cells,which was regarded as the adaptor or accessory protein in the Atg1 kinase complex participating in the Tor-mediated initiation of autophagy.The ortholog of MoAtg26 in Pichia pastoris took part in pexophagy.MoPex1 was a peroxininvolved in peroxisomal biogenesis.The ?Mopex1 mutant signifcantly impaired the mobilization and degradation of lipid bodies during appressorium formation.Thus,MoMps1 may participate in mobilization and degradation of lipid droplets via interacting with MoAtg13 and MoPex1 during appressorium development.The absence of MoMPS1 leads to the defect of Ⅱ type programmed cell death in macroconidia.Results of this study lay the foundation for exploring the molecular mechanism of MoMPS1 affecting penetration peg formation during appressorium development,contributing to reveal the pathogenesis of macroconidia. |
