Two Hybrid Histidine Kinases Involved in the Ethylene Regulation of the Mycelial Growth and Postharvest Fruiting Body Maturation and Senescence of Agaricus bisporus.

Ethylene regulates mycelial growth, primordium formation, and postharvest mushroom maturation and senescence in the white button mushroom, Agaricus bisporus. However, it remains unknown how ethylene is detected by the mushroom. In this study, we found that two hybrid histidine kinases in the mushroom, designated AbETR1 and AbETR2, showed domain structures similar to those of plant ethylene receptors. The transmembrane helices of AbETR1 and AbETR2 were expressed in yeast cells and showed ethylene-binding activities. Mushroom strains with downregulated expressions of AbETR1 and AbETR2 showed reduced sensitivity to the ethylene inhibition of mycelial growth, ethylene regulation of their own synthesis, postharvest mushroom maturation, and senescence and expression of maturation- and senescence-related genes. Therefore, AbETR1 and AbETR2 are expected to be biologically functional ethylene receptors and exhibit a different mode of action from that of the receptors of plants. Here, we fill gaps in the knowledge pertaining to higher fungus ethylene receptors, discover a novel mode of action of ethylene receptors, confirm ethylene as a novel fungal hormone, and provide a facilitated approach for preventing the maturation and senescence of postharvest button mushrooms. IMPORTANCE Ethylene regulates diverse physiological activities in bacteria, cyanobacteria, fungi, and plants, but how to perceive ethylene by fungi only remains unknown. In this study, we identify two biologically functional ethylene receptors in the basidiomycete fungus Agaricus bisporus, which fills the gaps of deficient fungal ethylene receptors. Furthermore, we found that decreased expression of the ethylene receptors facilitates preventing the maturation and senescence of postharvest button mushrooms, indicating that the two fungal ethylene receptors positively regulate the ethylene response, in contrast to that in plants.

Enhancing the 1-Aminocyclopropane-1-Carboxylate Metabolic Rate of Pseudomonas sp. UW4 Intensifies Chemotactic Rhizocompetence.

1-aminocyclopropane-1-carboxylic acid (ACC) is a strong metabolism-dependent chemoattractant for the plant beneficial rhizobacterium Pseudomonas sp. UW4. It is unknown whether enhancing the metabolic rate of ACC can intensify the chemotaxis activity towards ACC and rhizocompetence. In this study, we selected four promoters to transcribe the UW4 ACC deaminase (AcdS) gene in the UW4 ΔAcdS mutant. PA is the UW4 AcdS gene promoter, PB20, PB10 and PB1 are synthetic promoters. The order of the AcdS gene expression level and AcdS activity of the four strains harboring the promoters were PB20 > PA > PB10 > PB1. Interestingly, the AcdS activity of the four strains and their parent strain UW4 was significantly positively correlated with their chemotactic activity towards ACC, rhizosphere colonization, roots elongation and dry weight promotion. The results released that enhancing the AcdS activity of PGPRenable them to achieve strong chemotactic responses to ACC, rhizocompetence and plant growth promotion.

Heterologous Expression of Rhizopus Oryzae CYP509C12 Gene in Rhizopus Nigricans Enhances Reactive Oxygen Species Production and 11α-Hydroxylation Rate of 16α, 17-Epoxyprogesterone.

The 11α-hydroxylation of 16α, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The 11α-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the 11α-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The H2O2 levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the 11α-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.

Promotion of the growth and plant biomass degrading enzymes production in solid-state cultures of Lentinula edodes expressing Vitreoscilla hemoglobin gene.

Vitreoscilla hemoglobin (VHb), encoded by the Vitreoscilla hemoglobin gene (vgb), is highly effective at binding oxygen and delivering it to both prokaryotes and eukaryotes under hypoxic conditions. In this study, we introduced the vgb gene into shiitake mushrooms, and the mycelia of the transformatants grew faster. In particular, they spread into the solid substrate located in the lower part of the test tubes and bags where the oxygen was hypoxic and produced more ß-glucan and plant biomass degrading enzymes compared to the original strain. The maximum growth rate of the transformants was 8.5%-15.9% higher than that of the original strain on sawdust-based cultures in plastic bags. The laccase and amylase activities were 17.7%-40.3% and 16.7%-37.9% higher than that of the original strain, respectively. In addition, the ß-glucan contents of the transformant mycelia from the submerged fermentation were 12.9%-24.0% higher than that of the original strain. These results reveal that the expression of VHb in mushroom fungi promots the mycelial growth in solid-state cultures under the hypoxic condition as well as enhances ß-glucan and plant biomass degrading enzymes production.

[Expression and significance of Tim-3 on peripheral blood monocytes in patients with chronic hepatitis B].

OBJECTIVE: To detect T cell immunoglobulin and mucin domain-containing molecule 3(Tim-3) expression on peripheral blood monocyts (PBMs) and explore its effect on inflammatory cytokines production of PBMs in patients with chronic hepatitis B (CHB). METHODS: The expression of Tim-3 protein and mRNA on PBM subsets was respectively determined using flow cytometry and real-time quantitative RT-PCR (qRT-PCR) in CHB patients at different phases. After PBMs were stimulated with both recombinant human galectin-9 and lipopolysaccharide (LPS) in active CHB patients, the supernatants were collected to measure the levels of proinflammatory factors including TNF-α, IL-1ß and IL-6 by ELISA. RESULTS: Compared with healthy controls and CHB patients at immune active (IA) phase, the expression of Tim-3 was significantly up-regulated in PBMs of CHB patients at immune tolerance (IT) phase (P<0.01). The level of Tim-3 expression on both CD14(+);CD16-; and CD14(+);CD16(+); monocyte subsets of CHB patients at IT phase was significantly higher than that of the CHB patients at IA phase and of healthy controls (P<0.05). qRT-PCR further demonstrated that the level of Tim-3 mRNA on monocytes of CHB patients at IA phase was also higher than that of healthy controls (P=0.040). There was a positive correlation between the expression of Tim-3 on CD14(+); monocytes and serum ALT level in IA CHB patients (r=0.362, P=0.028). The levels of proinflammatory factors in supernatants, such as TNF-α, IL-1ß and IL-6 increased significantly in monocytes stimulated by LPS and galectin-9, compared with ones by LPS alone. CONCLUSION: Tim-3 expression, which is up-regulated on PBMs of CHB patients, is negatively correlated to the level of ALT in sera of IA CHB patients, and Tim-3/galectin-9 pathway might promote proinflammatory factors production of monocytes stimulated with LPS.