A Rice E3 Ubiquitin Ligase OsBBI1 Contributes To Broad-spectrum Resistance Against Magnaporthe Oryzae

Ubiquitination is one of the most important post-translational protein modifications and plays key roles in plant growth, development and hormone responses through degradation of targeted proteins via the ubiquitin-proteasome system. Recent studies have demonstrated that ubiquitination-related components have critical functions in regulation of defense response against biotic and abiotic stress. During my Ph.D thesis study, I identified a rice gene, OsBBIs (for blast and BTH-induced 1), which encodes an E3 ubiquitin ligase, and I performed detailed functional analysis of OsBBIl using a Tos17 interstion line and overexpression transgenic lines in rice disease resistance against Magnaporthe oryzae, the causal agent of blast disease.During the research of gene expression profile of rice induced by Magnaporthe oryzae, we found that the expression of a RING protein rice gene upregulated in response to Magnaporthe oryzae infection or BTH treatment, and speculated that the OsBBIl may involve in rice disease resistance against Magnaporthe oryzae. The OsBBIl gene encodes a C3HC4-type RING zinc finger protein and contains a highly conserved RING domain at its C-terminus. OsBBIl recombinant protein was purified from prokaryotically expressed E. coli cells and E3 ubiquitin ligase activity was analyzed. The results showed that the OsBBIl recombinant protein had E3 uiquitin ligase activity in vitro. However, a truncated version of OsBBIl, in which the RING domain was deleted, did not show any E3 uiquitin ligase activity. Thus, OsBBIl is a novel RING protein that has E3 ubiquitin ligase activity and that the RING domain in OsBBIl protein is critical to its E3 ligase activity. Semi-quantitative analysis showed that the OsBBIl gene was constitutively expressed in rice root, leaf, stem and sheath segments. However, expression of OsBBIl was induced by treatment with benzothiadiazole and by infection with M oryzae, indicating that OsBBIl might have a function in disease resistance in rice against M. oryzae.A Tos17-inserted mutant line, designated as OsBBIl, in the Nipponbare background was identified that displays no significant morphological phenotype. RT-PCR analyses with a pair of primers amplifying the coding region of the OsBBIl gene showed no detectable transcript of OsBBIl in osbbil plants. With seedling inoculation, the OsBBIl plants showed increased susceptibility to races ZD1 (strain 99-188), ZD3 (strain 98-271) and ZG1 (strain 94-10), resulting in an average of 1 grade higher in disease index than the wild type Nipponbare plants. Similar results were also obtained in detached leaf assays. Disease lesions on the OsBBIl leaves were significantly larger than on the wild type leaves after inoculated with two races of M. oryzae. These results indicate that OsBBIl is required for immunity in rice against M. oryzae.To further explore the function of OsBBIl in rice blast disease resistance, independent OsBBIl-overexpressing transgenic lines (OsBBIl-OE) were generated and disease phenotype of these transgenic lines against M. oryzae was analyzed. RT-PCR analysis showed that expression level of OsBBIl in representative stable transgenic lines (OE-255, OE-256 and OE-259, T3-T5 generations) were higher than that in the wild type TP309 plants. The overall disease scores in the three OsBBIl-OE lines were lower than those in the wild type plants, disease severities on the OsBBIl-OE plants showed average 1.1-2.5 grades lower than those on the wild type TP309 plants. Similar results were also obtained in detached leaf assays. The lesion sizes on the OsBBIl-OE plants were significantly reduced as compared with those on the wild type plants, resulting in 50%-80% and 45%-50% reduction when infected by ZB1and ZB15, respectively. These results confirm that OsBBIl-mediated resistance is efficient against multiple races of the blast fungus.Cellular defense responses were analyzed and compared between mutant/overexpression lines and wild types after inoculation with M. oryzae. The results suggested that the ratios of accumulation of reactive oxygen species (ROS), autoflurescence and wall associated cross-linked proteins in sheath epidermic cells of the OsBBIl mutant line were less than that of wild type plants, and the ratios of accumulation of ROS, autoflurescence and wall associated cross-linked proteins in sheath epidermic cells of the OsBBIl-overexpressing lines were much higher than that in the wild type. Transmission electron microscopy analysis showed that, when compared with the wild types, the average thickness of epidermal cell wall of leaf sheath in the OsBBIl mutant plants was significantly reduced, while that of overexpression plants was significantly enhanced. Further studies showed that expression levels of ROS-associated genes (OsRbohAl, OsRbohBl, OsRbohA2 and OsRbohB2) and cell wall associated genes (OsGSL2-1, OsCAD2-1 and osCAD3-1) were lower in OsBBIl mutant plants and were higher in the OsBBIl-OE plants, as compared with those in wild type plants, after inoculation with M. oryzae.Semi-quantitative RT-PCR analysis showed that expressioin of PRla, a well-known PR gene in rice, was up-regulated in OsBBIl-OE plants after inoculation with M. oryzae. Taken togather, these results indicate that OsBBIl might function in disease resistance against M. oryzae through modification of cell wall defense response and that OsBBIl may also participate in common defense signaling pathways.