Cell Biological Study On Bio-function Of Barley Mlo Gene

The recessive loss-of-function mutations of barley Mlo gene mediate durable, highly effective, and broad-spectrum resistance to all known races of Blumeria graminis f. sp. tritici. The mutants could produced more effective papilla to arrest the penetration of pathogen appressoria. It is also found that the mutant plants, however, became more hyper-susceptible to the rice blast fungus Magnaporthe grisea and the cereal hemi-biotrophic fungus Bipolaris sorokiniana. These suggest that the barley Mlo gene plays an ambivalent role in the host defensive mechanism on the infection of different pathogens.This research presents a detailed investigation of the cytological events in barley cells infected by Magnaporthe grisea during the disease development. The H₂O₂ accumulation, papilla response and hypersensitive response in barley genotypes cultivar Ingrid (Mlo), BC Ingrid mlo-3, BC Ingrid mlo-4, and BC Ingrid mlo-5, were examined here. Further, the ultrastructure of the interactions was also observed. The main results were as follows:1. The diaminobenzidine (DAB) assay is sensitive for the detection of H₂O₂ in barley leaf. It is revealed that H₂O₂ accumulated quickly at the fungal penetration sites and nearby on Ingrid at the 12h after inoculation. At the 24-48 h after inoculation, the staining of the invaded cell became more intense and extended into the whole host cell, in which the cell wall and the agglomerate of protoplasm that capsulizing the hyphae were brownness. In contrast with Ingrid, H₂O₂ also had been detected at the fungal penetration sites on mlo mutant lines, but with less degree than that on Ingrid.There was half of the invaded sites in which both the epidermal cell and the nearby mesophyll cells showed brown deposition. And the others showed local DAB staining, but the development of the hypha do not affected. The results suggested that the accumulation of H₂O₂ in host cell may be one of the major factors for the host defensive mechanism with M. grisea.2. Notably, highly localized cell wall appositions (papilla) and H₂O₂ accumulation appeared quickly at 12h after inoculation in Ingrid, and the formerbecame the prominent defense response after inoculation, which efficiently blocked fungal penetration. The frequency of effective papilla was up to 35% in Ingrid. In mlo genotypes, the papilla formation are successfully penetrated by the fungus, and the effective papilla in mlo-3, mlo-4 and mlo-5 were just 15%, 12%, and 15%, respectively.3. It was confirmed by ultrastructual analysis that the epidermal cell necrosis was of hypersensitive response (HR), which is the host initiative defensive mechanism. High frequency of HR in epidermal cells of Ingrid was examined after inoculation. Most of these incidences cells as an HR effectively restricted fungal development in a single epidermal cell, and the effective HR in Ingrid is 75%. However, in the attacked cells with HR in BC Ingrid mlo lines, the fungal development was undisturbed and the secondary hyphae spread into mesophyll cells. Only one third of the invaded cells showed whole cell brown deposition at 24-48 h after inoculation, the effective HR in mlo-3, mlo-4 and mlo-5 were 38%, 26%, and 35%, respectively.4. The ultrastructural analysis released that the death of mesophyll cells could not be strengthened in the mlo mutation genotype. The fungal penetrated the mesophyll cells directly, and then put them to death. The mesophyll cells in mlo mutants were morphologically normal at the early stage of penetration. There was no HR-like cell death to be detected in the mesophyll cells with electron microscope. Reveal that the H2O2 accumulation in these cells did not result in active cell death.5. The MLO-GFP expression vector for transient expression in single epidermal cell was constructed and coated on golden particles to bombarded leaves in BC Ingrid mlo-3. Bright fluorescence was observed in the cell wall and the nucleus in epidermal cells. The GFP was also detected in the nucleus of mesophyll cells. It is implied that the MLO is located on the cell wall and nucleus of the epidermal cells.