RESUMEN
The perennial gramineous grass Miscanthus condensatus functions as a major pioneer plant in colonizing acidic volcanic deposits on Miyake-jima, Japan, despite a lack of nitrogen nutrients. The nitrogen cycle in the rhizosphere is important for the vigorous growth of M. condensatus in this unfavorable environment. In the present study, we identified the nitrogen-cycling bacterial community in the M. condensatus rhizosphere on these volcanic deposits using a combination of metagenomics and culture-based analyses. Our results showed a large number of functional genes related to denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in the rhizosphere, indicating that nitrate-transforming bacteria dominated the rhizosphere biome. Furthermore, nitrite reductase genes (i.e., nirK and nirS) related to the denitrification and those genes related to DNRA (i.e., nirB and nrfA) were mainly annotated to the classes Alpha-proteobacteria, Beta-proteobacteria, and Gamma-proteobacteria. A total of 304 nitrate-succinate-stimulated isolates were obtained from the M. condensatus rhizosphere and were classified into 34 operational taxonomic units according to amplified 16S rRNA gene restriction fragment pattern analysis. Additionally, two strains belonging to the genus Cupriavidus in the class Beta-proteobacteria showed a high in vitro denitrifying activity; however, metagenomic results indicated that the DNRA-related rhizobacteria appeared to take a major role in the nitrogen cycle of the M. condensatus rhizosphere in recent Miyake-jima volcanic deposits. This study elucidates the association between the Miscanthus rhizosphere and the nitrate-reducing bacterial community on newly placed volcanic deposits, which furthers our understanding of the transformation of nitrogen nutrition involved in the early development of vegetation.
RESUMEN
Osteopontin (OPN) is elevated during the progression of experimental autoimmune uveoretinitis (EAU) in C57BL/6 (B6) mice. Furthermore, EAU symptoms are ameliorated in OPN knockout mice or in B6 mice treated with anti-OPN antibody (M5). Recently, OPN has been shown to promote the Th1 response not only in the extracellular space as a secretory protein but also in cytosol as a signaling component. Thus, we attempted to reduce OPN in both compartments by using a small interfering RNA (siRNA) targeting the OPN coding sequence (OPN-siRNA). EAU was induced in B6 mice by immunization with human interphotoreceptor retinoid-binding protein (hIRBP) peptide sequence 1-20. The OPN- or control-siRNA was administered with hydrodynamic methods 24 h before and simultaneously with immunization (prevention regimen). When plasma OPN levels were quantified following siRNA administration with the prevention regimen, the level in the OPN-siRNA-treated group was significantly lower than that in the control-siRNA-treated group. Accordingly, the clinical and histopathological scores of EAU were significantly reduced in B6 mice when siRNA caused OPN blockade. Furthermore, TNF-alpha, IFN-gamma, IL-2, GM-CSF and IL-17 levels in the culture supernatants were markedly suppressed in the OPN-siRNA-treated group, whereas the proliferative responses of T lymphocytes from regional lymph nodes against immunogenic peptides was not significantly reduced. On the other hand, the protection was not significant if the mice received the OPN-siRNA treatment on day 7 and day 8 after immunization when the clinical symptoms appeared overt (reversal regimen). Our results suggest that OPN blockade with OPN-siRNA can be an alternative choice for the usage of anti-OPN antibody and controlling uveoretinitis in the preventive regimen.