| Nitrogen is an essential mineral nutrient and required for the regular growth and development in plants. Under limiting nitrogen stress, plant had developed a fine tune regulation mechanism to dispel the damage caused by nitrogen deficiency stress. A previous study have shown that OsSPL11, (LOC_Os12g38210), an ubiquitin E3 ligase, and a negative regulator for plant defense signaling and cell death lesion-mimic phenotype, was regulated under nitrogen starvation stress in rice. In this study, OsSpl11-mutated and over-expressed rice plants grown in hydroponic system were tested for the roles of OsSPL11 in plant growth and development under different nitrogen conditions. OsSPL11 expression was induced throughout the plant and highly induced in roots of OsSPL11-OX plants under N-limited condition compare to that under N-optimum condition. spil11 mutant plants showed reduced root density under low nitrogen condition, and reduced shoot and root biomass accumulation compared to that of wild type ones under both N-optimum and N-limited conditions.OsSPL11 was also demonstrated to regulate nitrogen limitation-induced leaf senescence. Compared to wild type plants, the spil11 mutant showed early visible senescence under N-limited condition. Moreover, early senescence affected the root development and nitrogen uptake in spi11 mutant under N-limited condition. Consistently, spil11 mutants exhibited lower expression of OsNRT1.1,OsNRT1.2, OsNRT2.1 and OsNAR2.1 in roots, while OsSPL11-OX plants exhibited higher expression of those genes under N-limited condition. Typical senescence associated genes OsSGR, OsNAP and OsNYC1 were obviously up-regulated in spil11 mutants, suggesting that OsSPL11 delayed senescence development in rice. Lesion mimic phenotype in spill mutant was also suppressed under N-limited condition, which is consistent with reduced expression of defense and cell death-associated genes. Thus, these results indicate that OsSPL11 contributes greatly to the adaption to N deficiency stress as a positive regulator in rice.Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastating bacterial diseases worldwide. Down-regulation of a resistance protein gene OsRP-1 (LOC_Os08g42700) in response to Xoo infection was previously observed. In the current study, it was found that OsRP-1 encoded a putative CC-NB-LRR domain-containing protein, and had a duplication of the kinase 1a,2 and 3a motif of the NBS region in the N-terminal half of the protein. The observed phylogenetic tree suggested that OsRP-1 was a homologous of Pib (LOC_Os08g47010) protein by showing within one group which is a resistance protein to Magnaporthe oryzae and also showed closely related to RPM1 from Triticum urartu. Temporal and spatial expression analysis revealed that OsRP-1 had relatively higher expression levels in leaf and stem than in root, with the highest expression levels occurring in the leaf. Expression of OsRP-1 was found to be down-regulated in both mock treatment and Xoo inoculation.To identify the OsRP-1 interactome and downstream signaling components, yeast two-hybrid (Y2H) screening was carried out. Total of 137 proteins were selected as potential protein-protein interactors. In the classification of GO annotation analysis,137 putative proteins were well distributed into 32 biological process,19 categories of molecular functions, and 20 categories of cellular components. To determine the positive interactors, transformation analysis for elimination of false positive was carried out for selection of the strength of the interactions. Among 11 protein selected, four proteins OsAHP2 (LOC_Os09g39400), OsPsaD (LOC_Os08g44680), OsMYB (LOC_Os09g12750), and OsAAA-ATPase (LOC_Os11g47970) were found to be positively interacted with OsRP-1 in the Y2H system.To verify the interaction between OsRP-1 and selected proteins, GST pull-down approach was used, by which OsAHP2, a putative histidine phosphotranfer protein was shown to interact with OsRP-1. Firefly luciferase complementation assay was performed as well to confirm the interaction of OsRP-1 and OsAHP2 in planta, and these two protein interaction was weak and not specific. Further GST pull-down analysis demonstrated that N-terminal domain of OsRP-1 was enough to interact with OsAHP2. To understand the functions of OsAHPs in response to Xoo infection, OsAHPs-RNAi rice lines were inoculated with Xoo at 4-week-old seedling stages. OsAHPs-RNAi plants showed significantly shorter lesion length than that of wild type plants, suggesting that OsAHPs negatively regulates the disease resistance. Expression of defense genes OsPR1a, OsPR1b, and OsPBZ1 was highly induced at 6 h after Xoo inoculation. These observations suggest that OsRP-1 and OsAHP2 interaction mediates a signaling pathway involved in bacterial disease resistance responses of rice. |
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