Regulation of dye-decolorizing peroxidase gene expression in Pleurotus ostreatus grown on glycerol as the carbon source.

Dye-decolorizing peroxidases (DyPs) (E.C. 1.11.1.19) are heme peroxidases that catalyze oxygen transfer reactions similarly to oxygenases. DyPs utilize hydrogen peroxide (H2O2) both as an electron acceptor co-substrate and as an electron donor when oxidized to their respective radicals. The production of both DyPs and lignin-modifying enzymes (LMEs) is regulated by the carbon source, although less readily metabolizable carbon sources do improve LME production. The present study analyzed the effect of glycerol on Pleurotus ostreatus growth, total DyP activity, and the expression of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), via real-time RT-qPCR, monitoring the time course of P. ostreatus cultures supplemented with either glycerol or glucose and Acetyl Yellow G (AYG) dye. The results obtained indicate that glycerol negatively affects P. ostreatus growth, giving a biomass production of 5.31 and 5.62 g/L with respective growth rates (micra; m) of 0.027 and 0.023 h-1 for fermentations in the absence and presence of AYG dye. In contrast, respective biomass production levels of 7.09 and 7.20 g/L and growth rates (µ) of 0.033 and 0.047 h-1 were observed in equivalent control fermentations conducted with glucose in the absence and presence of AYG dye. Higher DyP activity levels, 4,043 and 4,902 IU/L, were obtained for fermentations conducted on glycerol, equivalent to 2.6-fold and 3.16-fold higher than the activity observed when glucose is used as the carbon source. The differential regulation of the DyP-encoding genes in P. ostreatus were explored, evaluating the carbon source, the growth phase, and the influence of the dye. The global analysis of the expression patterns throughout the fermentation showed the up- and down- regulation of the three Pleos-dyp genes evaluated. The highest induction observed for the control media was that found for the Pleos-dyp1 gene, which is equivalent to an 11.1-fold increase in relative expression (log2) during the stationary phase of the culture (360 h), and for the glucose/AYG media was Pleos-dyp-4 with 8.28-fold increase after 168 h. In addition, glycerol preferentially induced the Pleos-dyp1 and Pleos-dyp2 genes, leading to respective 11.61 and 4.28-fold increases after 144 h. After 360 and 504 h of culture, 12.86 and 4.02-fold increases were observed in the induction levels presented by Pleos-dyp1 and Pleos-dyp2, respectively, in the presence of AYG. When transcription levels were referred to those found in the control media, adding AYG led to up-regulation of the three dyp genes throughout the fermentation. Contrary to the fermentation with glycerol, where up- and down-regulation was observed. The present study is the first report describing the effect of a less-metabolizable carbon source, such as glycerol, on the differential expression of DyP-encoding genes and their corresponding activity.

Enhanced oxidative stress resistance in Ustilago maydis and its implications on the virulence.

The phytopathogenic fungus Ustilago maydis causes corn smut by suppressing host plant defenses, including the oxidative burst response. While many studies have investigated how U. maydis responds to oxidative stress during infection, the consequences of heightened resistance to oxidative stress on virulence remain understudied. This study aimed to identify the effects on virulence in U. maydis strains exhibiting enhanced resistance to hydrogen peroxide (H2O2).To achieve this, we exposed U. maydis SG200 to 20 escalating H2O2 shocks, resulting in an adapted strain resistant to concentrations as high as 60 mM of H2O2, a lethal dose for the initial strain. Genetic analysis of the adapted strain revealed five nucleotide substitutions, two minor copy number variants, and a large amplification event on chromosome nine (1-149 kb) encompassing the sole catalase gene. Overexpressing catalase increased resistance to H2O2; however, this resistance was lower than that observed in the adapted strain. Additionally, virulence was reduced in both strains with enhanced H2O2 resistance.In summary, enhanced H2O2 resistance, achieved through either continuous exposure to the oxidative agent or through catalase overexpression, decreased virulence. This suggests that the response to the oxidative stress burst in U. maydis is optimal and that increasing the resistance to H2O2 does not translate into increased virulence. These findings illuminate the intricate relationship between oxidative stress resistance and virulence in U. maydis, offering insights into its infection mechanisms.