Involvement of lysosomal integral membrane protein-2 in the activation of autophagy.

Lysosomal integral membrane protein-2 (LIMP-2) is a type III transmembrane protein that is highly glycosylated and mainly localized to the lysosomal membrane. The diverse functions of LIMP-2 are currently being uncovered; however, its participation in macroautophagy, usually described as autophagy, has not yet been well-investigated. To determine the possible involvement of LIMP-2 in autophagic activity, we examined the intracellular amount of microtubule-associated protein 1 light chain 3 (LC3)-II, which is well-correlated with autophagosome levels, in exogenous rat LIMP-2-expressing COS7 and HEK293 cells. Transient or stable expression of LIMP-2-myc significantly increased the levels of LC3-II. Conversely, knockdown of LIMP-2 decreased the LC3-II levels in NIH3T3 cells. Furthermore, approaches using lysosomal protease inhibitors and mCherry-GFP-LC3 fluorescence suggested that exogenous expression of LIMP-2 increased the biogenesis of autophagosomes rather than decreased the lysosomal turnover of LC3-II. Considering the results of the biochemical assay and the quantitative fluorescence assay together, it is suggested that LIMP-2 has a possible involvement in autophagic activity, especially autophagosome biogenesis.

Broad and systemic immune-modulating capacity of plant-derived dsRNA.

Double-stranded RNA (dsRNA) is well characterized as an inducer of anti-viral interferon responses. We previously reported that dsRNA extracted from a specific edible plant possesses an immune-modulating capacity to confer, in mice, resistance against respiratory viruses, including the H1N1 strain of the influenza A virus (IAV). We report here that the systemic immune-activating capacity of the plant-derived dsRNA protected mice from infection by a highly virulent H5N1 strain of the IAV. In addition, subcutaneous inoculation of the dsRNA together with the inactivated virion of the H5N1 strain of the IAV suppressed the lethality of the viral infection as compared with individual inoculation of either dsRNA or HA protein, suggesting its potential usage as a vaccination adjuvant. Moreover, intra-peritoneal inoculation of the dsRNA limited the growth of B16-F10 melanoma cells through the activation of NK cells in murine models. Taken together, this study demonstrated the systemic immune-modulating capacity of a plant-derived dsRNA and its potential for nucleic acid-based clinical applications.

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific, toxin-producing fungus Alternaria alternata†Japanese pear pathotype.

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant-pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.