Functional Characterization Of Thioredoxingenes In Magnaporthe Oryzae And Secreted Unique Genes In Fusarium Graminearum

Plants are constantly exposed to the adverse external environment in nature,including biological and non-biological stress.The former contains pathogenic bacteria,fungi,oomycetes,virus,nematodes,insects,and so on.In the course of co-evolution of plant and pathogens,they formed complex interaction.Plants possessed many kinds of recognition and resistance mechanisms to prevent or limit pathogen infection.At the same time,pathogen initiated diverse life strategies and pathogenicity mechanisms to avoid or overcome plant resistance mechanism.Rice and wheat,two main grain crops in the world,are constantly threatened by pathogenic fungi.Magnaporthe oryzae,a heterothallic ascomycete,is one of the most destructive rice pathogens.It is the causal agent of rice blast disease that occurs wherever rice is cultivated.M.oryzae is also a model system for studying fungal-plant interactions.The homothallism ascomycetous fungus Fusarium graminearum is an important plant pathogen causing Fusarium head blight(FHB)disease of wheat and barley as well as stalk and ear rot disease on maize.This pathogen not only causes severe crop yield and quality losses,but also produces harmful mycotoxins,such as deoxynivalenol(DON)and zearalenone,to contaminates grains.In this thesis,we mainly focus on the pathogenicity genes involved in signal pathway and that may act as effector during plant infection period in this two important filamentous fungi.The main results and conclusions are as follows:1.In the rice blast fungus Magnaporthe oryzae,the Mst11-Mst7-Pmk1 MAP kinase(MAPK)pathway is essential for appressorium formation and infectious growth.The redox status can affect the dimerization of MAPK kinases and activation of downstream MAP kinases in human and mammals.To determine their roles in MAPK signaling,we isolated and characterized the two thioredoxin genes,TRX1 and TRX2,in M.oryzae.TRX2 had a higher expression level and was found to be the predominant thioredoxin gene.Whereas the trx1 mutant had no detectable phenotypes,the trx2 mutant had severe defects in conidium formation and morphology and was down-regulated in the expression of COM1,HTF1,and CON7 transcription factor genes.Moreover,the trx2 mutant also displayed pleiotropic defects in growth,light sensing,responses to oxidative and cell wall stresses,and invasive growth.Deletion of TRX2 reduced the extracellular peroxidase/laccase activities and expression levels of MoSKN7,COM1,HTF1,and CON7.The trx1 trx2 double mutant had more severe defects than the trx2 mutant and was non-pathogenic in infection assays.The trx2 and trx1 trx2 mutants still formed appressoria by germ tubes but were defective in appressorium differentiation at hyphal tips.Furthermore,the trx1 trx2 mutant failed to successfully penetrate and develop branching invasive hyphae in plant cells.Pmk1 phosphorylation was significantly reduced or barely detectable in the trx2 and trx1 trx2 mutants.Interestingly,the expression of Mst7-3xFLAG or-GFP fusion proteins in the trx2 mutant was only detectable on western blots of SDS-PAGE but not native gels,indicating that deletion of TRX2 affected proper folding or intra-/inter-molecular interactions of Mst7 and detection of the epitope tag.Among the four conserved cysteine residues in Mst7,only Cys305 is important for its function and FLAG-epitope detection.We further showed that Trx2 directly interacts with Mst7 in co-IP assays and expression of the dominant active MST7 allele could partially rescue the defects of the trx1 trx2 mutant.Taken together,our data indicated that thioredoxins play important roles in intracellular ROS signaling,conidiogenesis,and pathogenesis in M.oryzae.TRX2 is the predominant thioredoxin gene and it may regulate penetration and invasive growth by affecting the activation of Mst7 and its downstream Pmk1 MAPK.2.To invade and colonize their hosts,fungal pathogens utilize a serious of effector proteins to suppress plant immunity.Many of these effectors are secreted,cysteine-rich proteins that may have a role in virulence.In the genome of Fusarium graminearum,the causal agent of the wheat and barley head blight disease,we identified 53 genes that encode secreted proteins and have no homologous sequences in GenBank or other sequenced fungal genomes.Deletion mutants of all these SU(secreted unique protein)genes were generated and assayed for defects in growth,conidiation,sexual reproduction,DON production,and pathogenicity.Three of them,SUP1,SUP2 and SUP4,were found to be important for plant infection.Whereas SUP1 is required for infection of both wheat heads and corn stalks,SUP2 and SUP4 are only involved in wheat infection.In transient expression assays with the PVX-based system,all of them could block Bax-induced programmed cell death induced in Nicotiana benthamiana and wheat.The expression of SUP2 and SUP4 was significantly up-regulated during early infection stages up to 72 h.Fluorescence observation and western blot analysis with GFP-fusion proteins contained the signal peptides or not confirmed that Sup2 and Sup4 were secreted proteins.Live-cell imaging revealed that Sup2 and Sup4 accumulated at the plant-fungal interface during the very short-term biotrophic phase of infection and were maybe translocated into wheat cells.Further analysis showed that Cys84 and Cys105 are important for avirulence activity and translocation of Sup2.Additional,five wheat proteins interacted with Sup2 were identified by yeast two-hybrid assays,including phosphoglycolate phosphatase,cysteine protease,membrane related protein-likepolyketide cyclase,eukaryotic translation initiation factor and expansin EXPA7.These results showed that Sup2 and Sup4 may act as novel effectors and were important for wheat infection.