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, 9 (1), 10813

The Ustilago Maydis Null Mutant Strains of the RNA-binding Protein UmRrm75 Accumulate Hydrogen Peroxide and Melanin

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The Ustilago Maydis Null Mutant Strains of the RNA-binding Protein UmRrm75 Accumulate Hydrogen Peroxide and Melanin

Alma Laura Rodríguez-Piña et al. Sci Rep.

Abstract

Ustilago maydis is a dimorphic fungus that has emerged as a model organism for the study of fungal phytopathogenicity and RNA biology. In a previous study, we isolated the U. maydis UmRrm75 gene. The deletion of the UmRrm75 gene affected morphogenesis and pathogenicity. UmRrm75 gene encodes a protein containing three RNA recognition motifs. Here we determined that UmRrm75 has chaperone activity in Escherichia coli using the transcription anti-termination assay. Subsequently, we analyzed the growth of ΔUmRrm75 mutants at 15 °C and 37 °C, observing that mutant strains had reduced growth in comparison to parental strains. UmRrm75 gene expression was induced under these non-optimal temperatures. ΔUmRrm75 mutant colonies displayed a dark-brown color at 28 °C, which was confirmed to be melanin based on spectroscopic analysis and spectrometric data. Furthermore, ΔUmRrm75 mutant strains showed the presence of peroxisomes, and increased H2O2 levels, even at 28 °C. The ΔUmRrm75 mutant strains displayed a higher expression of redox-sensor UmYap1 gene and increased catalase activity than the parental strains. Our data show that deletion of the UmRrm75 gene results in higher levels of H2O2, increased melanin content, and abiotic stress sensitivity.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Transcription anti-terminator test of UmRrm75. Anti-terminator assay of UmRrm75 in the RL211 E. coli strain. Cultures of RL211 (negative control), RL211-pINIII (negative control), RL211-CspA (positive control) and RL211-UmRrm75 bacterial strains were adjusted to OD600 1.0, and they were spotted in serial dilutions on LB plates with 8 or 10 µg/mL chloramphenicol. Plates were photographed after 48 h of incubation at 37 °C. Data shown are representative of three independent experiments.
Figure 2
Figure 2
Growth assays of U. maydis parental and ΔUmRrm75 mutant strains. Serial dilutions of cultures of U. maydis were spotted on CM media and incubated at 37°C, 15 °C, and 28 °C (control). Plates were photographed after 3 and 6 days. Data shown are representative of three independent experiments.
Figure 3
Figure 3
UmRrm75 gene expression analysis in FB2 parental strain under thermal, osmotic, and oxidative conditions. (A) FB2 parental strain grown for 24 h in liquid MM at 15 °C, 28 °C and 37 °C. (B) FB2 strain grown for 24 h in liquid MM with 1 M sorbitol or 1 mM H2O2. Transcript levels of UmRrm75 were calculated by the qRT-PCR approach. Normalized fold change was calculated comparing the UmRrm75 gene expression (under stress condition) with control conditions, after normalization to the UmGAPDH using the (2−ΔΔCt) method. Analyses were performed in triplicate. Data represent the mean ± SEM. One-Way Analysis of Variance (ANOVA) and Tukey’s post-test analyses were performed. Significant differences are marked with an asterisk (P < 0.05).
Figure 4
Figure 4
IR and 1H NMR spectra analysis of pigments from ΔUmRrm75 null mutant and parental strains. (A) Infrared spectra of melanin extracted from 1/46 and 1/53 null mutant strains at 28 °C (B) Infrared spectra of melanin extracted from FB2 and SG200 parental strains at 37 °C. (C) 1H NMR spectra of melanin extracted from 1/46 and 1/53 null mutant strains at 28 °C. (D) 1H NMR spectra of melanin extracted from FB2 and SG200 parental strains at 37 °C. All spectra were compared with synthetic melanin as a reference.
Figure 5
Figure 5
Hydrogen peroxide detection in ∆UmRrm75 mutant and parental strains. (A) FB2, 1/2 and SG200 parental strains, and their respective mutant strains (1/46, 1/40 and 1/53) were grown in liquid MM with DCFH2-DA for 4 h at 15 °C, 28 °C and 37 °C. Images were taken with a 40x oil-objective. Data shown are representative of results of three biological replicates. (B) H2O2 quantification was performed using KI in U. maydis cells. FB2 parental and 1/46 mutant strains were grown in liquid MM for 10, 12, and 24 h. Data are reported as μmol/gCFW. Data are means ± SEM from three biological replicates (n = 3). Different letters indicate a significant difference according to One-Way Analysis of Variance (ANOVA) and Tukey’s post-test analysis.
Figure 6
Figure 6
Sensitivity of ∆UmRrm75 mutant and parental strains to H2O2. (A) Sensitivity assays was assessed in an agar diffusion test with a filter soaked with 1uL H2O2 (30% v/v) was placed on agar plate. (B) Halo size was quantified as the diameter of each ∆UmRrm75 mutant and parental strains grown for 6 days. Data represent the mean ± SEM (n = 4) and differences at P < 0.05 were considered significant. The different letters indicate a significant difference according to One-Way Analysis of Variance (ANOVA) and Tukey’s post-test analysis.
Figure 7
Figure 7
Detection of peroxisomes by the DAB method, and quantification of UmPex3 gene expression in ΔUmRrm75 mutant and parental strains. (A) TEM images of FB2, 1/2 and SG200 parental strains and 1/46, 1/40 and 1/53 mutant strains grown in liquid MM supplemented with 0 or 1 mM H2O2. Peroxisomes containing DAB reaction product are marked by the letter P. Bar correspond to 500 nm. (B) Transcript expression analysis of UmPex3 gene in FB2 parental and 1/46 mutant strains. Normalized fold change was calculated comparing the UmPex3 gene expression in 1/46 mutant with FB2 parental, after normalization to the UmGAPDH using the (2−ΔΔCt) method. Analyses were performed in triplicate. An unpaired t-test was performed. Data represent the mean ± SEM. Significant differences are marked with an asterisk (P < 0.0001).
Figure 8
Figure 8
Catalase treatment and enzyme activity, and UmYap1 expression in the ΔUmRrm75 mutant and parental strains. (A) Determination of catalase (CAT) activity in FB2 parental and 1/46 mutant strains at 10, 12 and 24 h. Data are means ± SEM from three biological replicates (n = 3). The different letters indicate a significant difference according to One-Way Analysis of Variance (ANOVA) and Tukey’s post-test analysis (P < 0.05). (B) Exogenous application of CAT in ΔUmRrm75 mutant strains. Cultures grown in liquid MM with DCFH2-DA at 28 °C; Left panel (without CAT), and right panel (250 U/mL CAT). Images were acquired on a fluorescence microscope at 40× (oil-objective) magnification. One representative microscopy image of DCFH2-DA fluorescence is shown from three biological replicates. (C) Transcript level analysis of UmYap1 gene in FB2 parental and 1/46 mutant strains. Normalized fold change was calculated by comparing the UmYap1 gene expression in 1/46 mutant with the FB2 parental, after normalization to UmGAPDH using the (2−ΔΔCt) method. Analyses were performed in triplicate. Unpaired t tests were performed and data represent the mean ± SEM. Significant differences are marked with an asterisk (P < 0.0001).

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