| Rice(Oryza sativa)is one of the major cereal crops in the world,and more than half of the population use rice as staple food.Rice blast disease caused by Magnaporthe oryzae has been long as a serious threat to the yield and quality of rice.Therefore,it is of great importance to discover broad-spectrum resistance genes and to uncover the molecular mechanism of rice blast resistance.It is equally important to unravel the pathogenicity of M.oryzae,in order to develop strategies to avoid the pathogen infection.In this study,transcriptome analysis on rice and invading M.oryzae showed that rice PRR(Pattern recognition receptor)gene expression was mainly up-regulated in early infection stage;howerer,the expression of genes involved in cell wall-related metabolic pathways was suppressed.In addition,the expression of M.oryzae cell wall-degrading enzyme-coding genes was up-regulated.These results suggest that rice or fungal cell wall-related genes and rice PRR genes play an important role in extracellular immunity.Notebly,two fungal genes Mo Ma84 and Mo Ma94 that encode mannosidases were up-regulated during infection.Genetic transformation and phenotypic analysis showed that Mo Ma84/94 mutation did not impair the growth and development of M.oryzae,but the pathogenicity of Moma84/94 double mutant was significantly reduced.Further,the resistance of Mo Ma84-ecotopic expression transgenic rice to rice blast infection was compromised.Enzymatic study showed that Moma84 could degrade the cell wall of M.oryzae,and the main production,manninotriose and mannotetrose,activated the immunity in rice.Further ROS(Reactive oxygen species)burst assays showed that several other mannan oligosaccharides could also activate the immunity in rice,and mannopentaose had the strongest activativity.In addition,we screened a rice gene Os DUF5,which encodes a secreteory protein containing two DUF26(Domain of unknown function 26)domains,and its expression was significantly induced during M.oryzae infection.Rice blast inoculation assays showed that Os DUF5-overexpression lines showed enhanced rice blast resistance.The split-luciferase complementation and co-immunoprecipitation assays demonstrated that Os DUF5 strongly interacted with the chitin receptor Os CEBi P(Chitin elicitor-binding protein),and proceeded to form a polymer Os DUF5-Os CEBi P-Os CERK1(Chitin elicitor receptor kinase 1).Microthermophoresis and ROS burst assays demonstrated that mannopentaose was involved in the Os CEBi P-Os CERK1-dependent PTI immune response via binding to Os DUF5.Further,we screened the rice complementary DNA library by yeast twohybrid assay and identified a rice protein Os LRR12,which interacted with Os DUF5.Os LRR12 encodes a protein containing several leucine-rich repeat(LRR)domains,and showed similar expression pattern as Os DUF5 during M.oryzae infection.The split-luciferase complementation,Pull-down and co-immunoprecipitation assays demonstrated that Os LRR12 directly interacted with Os DUF5,and the interaction between Os LRR12 and Os DUF5 was significantly enhanced in the presence of chitin and mannopentaose.Rice blast inoculation assays showed that the resistance of Os LRR12-overexpression lines to rice blast infection was enhanced.Further bioinformatic analysis showed that most of the DUF26 protein-coding genes existed as gene clusters in Nipponbare genome,and Os DUF5 was highly conserved in rice3 k varieties.Importantly,field experiments showed that Os DUF5-overexpression lines exhibited normal plant architecture and yield,implying the potential for rice breeding.Based on the above results,two novel M.oryzae pathogenicity-related genes,Mo Ma84 and Mo Ma94,were identified,and the role of mannopentaose served as a PAMP in PTI response was clarified.Morover,this study also found that a host mannopentaose-binding protein,Os DUF5,was involved in the rice mannopentaose and chitin-mediated immune response through the Os DUF5-Os CEBi P-Os CERK1 pathway.In summary,Os DUF5 plays an important role in plant immune response.More importantly,Os DUF5-overexpression plants not only exhibited an enhanced rice blast resistance,but also maintained the stable yield.Therefore,Os DUF5 is of great importance for balancing rice yield and disease resistance,laying a theoretical foundation for breeding disease-resistant and high-yielding rice varieties. |
