Introduction

Ustilago maydis is a biotrophic fungus that infects maize (Zea mays) and teosinte (Zea perennis) to produce a disease known as corn smut. U. maydis is an obligate parasite that can only complete its sexual and infectious cycle in the host plant1. This basidiomycete has been widely used for studying the mechanisms of fungal pathogenicity2,3. Furthermore, U. maydis is considered an excellent model for the study of DNA recombination and repair, vesicle trafficking, and RNA biology3,4.

RNA binding-proteins (RBPs) are key players in gene expression regulation in all organisms because they mediate rapid changes in expression profile in order to help organisms adapt or overcome environmental changes5. RBPs can regulate RNA activity and structure by participating in RNA maturation, nuclear export, stability, transport, and translation6. RBPs bind to RNAs with high affinity through RNA-binding domains, modulating the RNA structure. In fungi, the RBPs are involved in growth, development, morphology, pathogenicity, and stress response. In Aspergillus nidulans, RBPs play an important role in cell cycle regulation7. RBPs in Saccharomyces cerevisiae are involved in splicing and mating regulation5. In U. maydis, RBPs participate in filamentation and pathogenicity8,9,10.

In a previous study, we identified the UmRrm75 gene in U. maydis, which encodes a protein containing three RNA recognition motifs interspersed by glycine-rich regions10. Deletion of the UmRrm75 gene resulted in several alterations, such as a donut-like morphology, decreased mating and post-mating filamentous growth, and reduced virulence in maize10. In this study, we determined that expression of UmRrm75 gene was induced under thermal stress (15 °C and 37 °C), and showed evidence of RNA chaperone activity. We detected greater sensitivity to temperature stress in ΔUmRrm75 mutants relative to the parental strains. We noticed that ΔUmRrm75 mutant strains accumulate a brown-pigment, greater H2O2 content, and also showed the presence of peroxisomes under both optimal and stress temperatures. Finally, the catalase activity in the ΔUmRrm75 mutant strains was analyzed, highlighting that the mutants were activating the detoxification system.

Results

The UmRrm75 protein exhibits RNA binding activity in E. coli

In order to analyze the RNA chaperone activity of the UmRrm75 protein, we used the bacterial transcription anti-termination system11. The open reading frame (ORF) of the UmRrm75 gene was cloned into the pINIII expression vector, and transformed into the E. coli RL211 strain. The RL211 strain contains the chloramphenicol acetyltransferase (cat) gene preceded by a strong loop (ρ-independent trpL) terminator. The melting of this anti-terminator loop confers chloramphenicol (Cm) resistance in E. coli11,12, which makes the system efficient in detecting RNA binding activity. In this assay, we included the RL211 strain expressing cspA gene as a positive control of RNA binding activity (RL211-cspA), and the RL211 and RL211-pINIII (empty vector) strains as negative controls. Bacterial growth was evaluated by the drop dilution test in a medium containing 8 and 10 µg/mL Cm. As observed in Fig. 1, the RL211-UmRrm75 strain achieved growth until the fourth dilution at 8 µg/mL Cm, and up to the third dilution at 10 µg/mL Cm, similar to the observed for RL211-cspA strain. As expected, no growth was observed in the RL211 negative control, and for the RL211-pINIII strain a slight growth was obtained until the first dilution for both Cm concentrations. These results indicate that the UmRrm75 protein was capable of binding and melting RNA secondary structures in E. coli.

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.

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Deletion of the UmRrm75 gene affects fungal growth under temperature stress conditions

We evaluated the effect of non-optimal temperatures on the growth of ∆UmRrm75 mutant strains (1/46, 1/40 and 1/53), and their respective parental strains (FB2, 1/2, and SG200) (Supplementary Table 1). The ∆UmRrm75 mutants and parental strains were grown on a complete medium (CM) in serial dilutions (1 × 102–1 × 105) for 6 days at 15 °C and 37 °C (non-optimal temperatures), and as a control the temperature 28 °C was used. Although at 28 °C, the ∆UmRrm75 mutants growth was slower than the parental strains, the reduction in growth was more noticeable at 37 °C and 15 °C (Fig. 2). After 3 days of incubation at 37 °C and 15 °C, the ΔUmRrm75 strains did not show growth, whereas at 28 °C yeast growth was observed until the second dilution (Fig. 2). After 6 days of incubation, null mutants were able to grow until the second dilution at 37 °C or 15 °C, displaying a reduced colony size. These data showed that the ΔUmRrm75 mutant strains exhibited a slower growth at non-optimal temperatures.

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.

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The UmRrm75 gene expression is induced under abiotic stress conditions

We evaluated the expression of the UmRrm75 gene under several abiotic stresses in parental strain FB2. The FB2 strain was grown in liquid minimal medium (MM) at 15 °C, 28 °C, and 37 °C for 24 h. Expression levels were determined by qRT-PCR and normalized against the optimal condition (28 °C). UmRrm75 gene was expressed at very high levels under non-optimal temperatures, 13.2-fold at 37 °C, and 31-fold at 15 °C, in contrast to those levels observed in the control condition at 28 °C (Fig. 3A). We also analyzed the UmRrm75 expression levels in FB2 strain grown in liquid MM supplemented with 1 M sorbitol or 1 mM H2O2 for 24 h. We observed an increase in UmRrm75 expression levels of 1.7-fold with sorbitol and 6-fold with H2O2 treatments (Fig. 3B). These results revealed that UmRrm75 gene was mainly regulated by thermal stress conditions.

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).

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The ∆UmRrm75 mutant strains accumulate a dark brown pigment under optimal and non-optimal temperatures

We observed that UmRrm75 null mutant colonies grown under optimal conditions (28 °C) exhibited a dark brown pigmentation after 6 days of growth (Fig. 2). This pigment was also observed when the ΔUmRrm75 strains were grown at 37 °C. In the parental strains, the brown pigmentation was only observed under 37 °C stress treatment (Fig. 2). These data show that the ΔUmRrm75 mutant colonies exhibit an accumulation of a dark-brown pigment, even under optimal temperature conditions.

The ∆UmRrm75 mutant strains accumulate melanin

In fungi, the accumulation of melanin and other non-enzymatic metabolites are part of the mechanisms of protection against oxidizing agents13. We performed several chemical tests to determine if the dark-brown pigment accumulated in ∆UmRrm75 strains (1/46 and 1/53), and their respective parental strains (FB2 and SG200) corresponded to melanin compounds. Our first physicochemical data revealed that the pigment accumulated in ∆UmRrm75 strains displayed typical characteristics of melanin such as brown coloration, insolubility in organic compounds, and was soluble at 100 °C in KOH alkaline solution (Supplementary Table 2). In the second approach spectroscopic methods were employed, which agreed with the melanin nature of U. maydis pigments; for example, UV-Vis spectroscopy revealed maximum absorption between 210–220 nm for ∆UmRrm75 mutants at 28 °C and parental strains at 37 °C (Supplementary Fig. 1). Infrared analyses showed that ∆UmRrm75 mutants at 28 °C, and parental strains at 37 °C, have bands representing phenolic groups (3400-3100 and 1260-1240 cm−1), methyl or methylene groups (2980-2850 cm−1), and -NH groups (3300-3260 and 1650-1630 cm−1). Unique bands between 2980-2850 cm−1 were observed in the pigment purified from UmRrm75 mutants. These peaks could be explained as being specific to the U. maydis melanin (Fig. 4A–B). Finally, the analysis through 1H NMR of melanins from ∆UmRrm75 at 28 °C and parental strains at 37 °C displayed two signals at δH 7.70 and 2.48 ppm, which can be assigned to CH=C and –NH groups of the indole moiety. Additional signals at δH 3.24, 3.13, and 0.45 ppm were detected in ∆UmRrm75 mutant’s melanin (Fig. 4C,D). The melanin content in parental strains at 28 °C and 37 °C was not detected by subsequent spectrometric analyses. The ESI-MS analysis revealed that melanins produced by UmRrm75 mutants at 28 °C were closely similar to those obtained for synthetic melanin with fragment losses of 150 amu (Supplementary Fig. 2). These spectroscopic and spectrometric data indicate that the pigment produced by ΔUmRrm75 mutant and parental strains under stress conditions was melanin of the eumelanine type, consisting mainly of the 5,6-dihydoxyindole (DHI) building block14,15.

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.

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The ΔUmRrm75 mutant strains accumulate H2O2

The presence of melanin in ΔUmRrm75 mutants incubated at 28 °C or 37 °C, and in parental strains at 37 °C, suggested changes in H2O2 content. We evaluated H2O2 production using 2′,7′-dichlorofluorescein diacetate dye using Epi-fluorescence microscopy. Under optimal growth conditions (28 °C), H2O2 signal was clearly observed as a green fluorescent signal in ΔUmRrm75 mutant strains, while in parental strains no signal was detected (Fig. 5A). However, when cells were grown at 15 °C or 37 °C, the green fluorescent signal was observed in both parental and ΔUmRrm75 strains (Fig. 5A). H2O2 was quantified in FB2 parental and 1/46 mutant strains grown for 10, 12 and 24 h at 28 °C, respectively. Our data indicated high levels of H2O2 in the 1/46 mutant for all tested times, whereas the FB2 showed basal or no detectable H2O2 (Fig. 5B). In addition, ΔUmRrm75 mutants were subjected to exogenous H2O2 treatment in an agar diffusion test for 6 days at 28 °C. We observed that the growth inhibition halo was wider in all ΔUmRrm75 strains in comparison to the parental strains (Fig. 6A). After 6 days of the H2O2 diffusion test, the characteristic brown pigment was only observed in the ΔUmRrm75 strains (Fig. 6B). Our results indicated that ΔUmRrm75 mutant strains accumulate H2O2, even under optimal conditions, which made them more sensitive to the application of exogenous H2O2.

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.

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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.

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The ΔUmRrm75 mutants show accumulation of peroxisomes

Peroxisomes play an important role in the protection of cells from reactive oxygen species16. We focused on peroxisomes visualization of ΔUmRrm75 mutant and parental strains using an ultrastructural cytochemical staining approach (DAB-oxidation). The ∆UmRrm75 mutants and parental cells were grown on minimal medium (MM) at 28 °C for 24 h. Images of cells from parental strains did not display the staining signal from the DAB-oxidation reaction (Fig. 7A). Conversely, cells from the ΔUmRrm75 mutant strains clearly showed a positive DAB-oxidation reaction in peroxisomes (Fig. 7A). As a control, we also analyzed the effect of the exogenous application of 1 mM H2O2 on DAB-staining in parental and mutant strains. As expected, both mutant and parental cells presented an intense signal of the DAB-reaction product in the peroxisomes (Fig. 7A). Subsequently, we quantified the transcript expression of the peroxisome membrane biogenesis factor UmPex3 gene17 in FB2 parental and 1/46 mutant strains. Both strains were grown in liquid MM for 4 and 6 h at 28 °C. Our results revealed that the UmPex3 gene was induced in the 1/46 mutant 1.3-fold at 4 h and 0.5-fold at 6 h relative to FB2 (Fig. 7B). These results suggest a peroxisome proliferation in ΔUmRrm75 mutant strains that can be explained as a consequence of H2O2 accumulation.

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).

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ΔUmRrm75 mutant strains show increased catalase activity

According to the previous data, we quantified the catalase (CAT) activity in 1/46 mutant and FB2 parental strains at 28 °C grown for 10, 12, and 24 h. No changes in CAT activity were observed between the 1/46 mutant and FB2 at 10 or 12 h. After 24 h of growth, CAT activity was increased in the 1/46 mutant (3-fold) in comparison to the FB2 strain (Fig. 8A), showing that the unusual H2O2 accumulation in the 1/46 mutant strain is activating the detoxification system when grown at an optimal temperature. These data suggest that the sustained oxidative stress in 1/46 mutant is due to the increased levels of H2O2 generation.

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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Exogenous application of catalase alleviates H2O2 accumulation in ΔUmRrm75 mutant strains

As a defense mechanism during oxidative stress, cells produce antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). These enzymes are responsible for converting reactive oxygen species (ROS) into harmless products18,19. In order to explore if the application of exogenous CAT enzyme may reduce H2O2 accumulation in ΔUmRrm75 mutants under control conditions (28 °C), we incubated null mutant cells with 0 or 250 U/mL of CAT with 2′,7′-dichlorofluorescein diacetate dye. We did not observe fluorescent signals due to H2O2 accumulation from ΔUmRrm75 cells after the application of 250 U/mL CAT (Fig. 8B). Thus, this result confirmed H2O2 accumulation in ΔUmRrm75 mutants, and revealed that this accumulation can be scavenged by the application of exogenous CAT.

UmYap1 gene is induced in ΔUmRrm75 mutant (1/46) under optimal conditions

The UmYap1 transcription factor plays an essential role in the detoxification of U. maydis cells by acting as a redox sensor20. In order to evaluate the expression of the UmYap1 gene in response to H2O2 accumulation, we analyzed the FB2 parental and 1/46 mutant strains using qRT-PCR. Both strains were grown in liquid MM at 28 °C for 4 and 6 h. The results showed that the UmYap1 gene was highly induced in the 1/46 mutant: 13-fold at 4 h and 7-fold at 6 h in comparison to FB2 strain (Fig. 8C). The high transcriptional levels of UmYap1 supported the notion that the redox sensor was not affected in the ΔUmRrm75 mutant, and that the mutant is working to counteract the accumulation of H2O2 under normal growth conditions.

Discussion

Our data showed that the UmRrm75 protein had RNA chaperone activity in an E. coli heterologous system, providing relevant evidence about the possible role of this protein in U. maydis as a RNA chaperone. In bacteria, there is evidence that cold shock proteins (CSPs) with RNA binding domains have RNA chaperone activity under stress conditions. It has been reported that CSPs play important roles in response to low-temperature, post-transcriptional machinery regulation, adaptation, and survival21,22. In plants, there are homologs of CSPs, which contain an N-terminal cold shock domain and also glycine rich domains23. These glycine-rich RNA-binding proteins are related to freezing stress tolerance in Arabidopsis24. In fungi, the RNA binding proteins (RBPs) have been related to growth, development, morphology, pathogenicity processes, and stress response25,26,27. It is well known that many fungi are able to adapt and overcome extreme temperatures28,29. However, the molecular mechanisms, and particularly the role of RNA binding proteins under temperature stress, have not been fully explored27,30. Here, we analyzed the growth capacity of the ΔUmRrm75 mutant strains under stress temperatures, 15 °C and 37 °C. We found that ΔUmRrm75 mutant strains were affected in their growth capacity (even at an optimal temperature of 28 °C) in contrast to the parental strains. These data correlated with the induction of UmRrm75 gene in the FB2 parental strain that was subjected to 15 °C and 37 °C. Fang & St Leger27 reported that two RNA binding proteins (Crp1 and Crp2) of Metarhizium anisopliae fungus were also capable of melting RNA secondary structure in an E. coli heterologous system. Moreover, when M. anisopliae was subjected to abiotic stress conditions and non-optimal temperatures stress, a high expression level of Crp1 was observed under all stress conditions.

The accumulation of dark-brown pigments in many fungi is associated to environmental stress response31,32,33,34. ΔUmRrm75 mutant strains after 6 days at 28 °C accumulated a dark-brown pigment. When strains were challenged to heat stress (37 °C), parental strains also showed accumulation of this pigment. At a low temperature (15 °C), no pigmentation was observed, neither in parental nor in ΔUmRrm75 mutant strains. Therefore, this pigmentation correlated with the deletion of the UmRrm75 gene, and also as a response to heat.

Dark-brown pigments produced by ΔUmRrm75 mutants and parental strains were characterized by spectroscopic and spectrometric analyses. Our data showed that this pigment has the same chemical properties of those reported for fungal melanins35. In addition, UV-Vis spectra of ΔUmRrm75 mutant at 28 °C and parental strains at 37 °C displayed absorption profiles similar to a synthetic melanin employed as a reference. The log of optical density of melanin solution when plotted against wavelength produces a linear curve with negative slopes35,36. The IR spectrum of synthetic melanin showed the same bands produced by ΔUmRrm75 mutant at 28 °C and parental strains at 37 °C (except the bands between 2980-2850 cm−1), and also to those observed in other melanins isolated from fungi and plants15,37. The ESI-MS analyses of melanin from ΔUmRrm75 mutant strains were carried out in an m/z ranging from 100–2000 amu. Mass spectra showed molecular ions at m/z 1679 (melanin reference at m/z 1677), and subsequent fragments with losses of multiples of 150 amu (m/z 1529, 1379 and 1231), suggesting that 5,6-dihydroxyindole (DHI) is the main building block for this melanin. This fragmentation pattern was consistent with those described for other melanins, i.e. those containing 3,4-dihydoxyphenylalanine and p-coumaric acid as monomeric units14,15. These data were supported by the 1H NMR spectra of melanins from ∆UmRrm75 mutants and parental strains. The signals at δH 7.70 and 2.48 ppm also indicated that melanins consisted of 5,6-dihydroxyindole (DHI) as the main building block. Additional signals at δH 3.24, 3.13, and 0.45 were observed. The first two signals could be attributed to other –NH groups of building blocks described for melanins, such as pyrrole-2,3-dicarboxylic acid or pyrrole-2,3,5-tricarboxylic acid38, and the last signal could be attributed to methylene groups of another kind of pyrroles moiety39. In summary, spectral comparison of melanins derived from ∆UmRrm75 mutant and parental strains with those described for natural and semi-synthetic melanins from fungal origin, isolated from Lachnum species, showed a great similarity and supported our data on the structure of melanins from U. maydis37,40.

Melanin is described as a dark-brown pigment formed by polymeric macromolecules of hydrophobic character with a negative charge, such as phenolic or indole rings41,42,43. Melanin is a multifunctional pigment that is found in all biological kingdoms, and is involved in the defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation44. In fungi, it is well documented that melanin contributes to the ability of survival in harsh environments, and tolerance to desiccation and extreme temperatures, as well as chemo-protector absorbing free radicals, protecting against oxidative stress and UV radiation45,46,47,48. Particularly, Rita & Pombeiro-Sponchiado49 reported that the melanin from Aspergillus nidulans has a potential activity as HOCl and H2O2 scavenger. Our data show that the UmRrm75 gene deletion affects H2O2 and melanin content, which suggest that ΔUmRrm75 mutants are stressed even under normal conditions.

In fungi, like in many other aerobic organisms, one of the first cell detoxification responses is against ROS accumulation, which includes an increase in the activities of the principal antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), which play key roles in ROS scavenging50,51,52. Herein, we reported that CAT activity was higher in the 1/46 mutant strain than FB2 parental strain. The increase in CAT activity in the 1/46 mutant strain could indicate that the null mutant cells are trying to degrade the H2O2 over-accumulation. Despite this induction in CAT activity, the 1/46 mutant was not able to maintain the H2O2 homeostasis inside cells. Sokolovsky & Belozerskaya53 suggested that higher CAT and SOD activities in fungus are associated with resistance to oxidative stress factors such as H2O2, which is the most stable species of ROS found inside the cell. However, when we applied exogenous CAT enzyme to the ΔUmRrm75 mutant strains, H2O2 levels were reduced.

It is interesting that we observed peroxisomes in mutant strains when they were grown at 28 °C; this phenotype was only achieved in the parental strains when exogenous H2O2 was added. Furthermore, we analyzed the UmPex3 gene, which encodes a peroxisomal membrane biogenesis factor17. We found that UmPex3 gene showed higher transcript levels in the 1/46 mutant than FB2, which suggests de novo peroxisome biogenesis is occurring in the ΔUmRrm75 mutant strains. The DAB staining evidences that H2O2 is accumulating inside the peroxisomes. Schrader and Dariush54 describe that this organelle participates in both the production and the scavenging of ROS, particular H2O2. Peroxisomes can proliferate in response to nutritional and extracellular environmental stimuli; this response is usually accomplished by the induction of peroxisomal enzymes55, as was observed in our study. We propose that this peroxisomal proliferation in ΔUmRrm75 mutant strain is a part of the U. maydis scavenging response to ROS accumulation.

In U. maydis, the transcription factor UmYap1 controls the cells detoxification pathway; this gene functions as a redox sensor, and is essential for virulence20. Finally, we studied how the UmYap1 gene is expressed in the ΔUmRrm75 (1/46) mutant strain. Thus, the high expression level of UmYap1 in the 1/46 mutant confirms that the detoxification system is active, and could be regulating H2O2 accumulation, but is not enough to alleviate the oxidative stress exhibited in the ΔUmRrm75 mutant strains.

In summary, this study provides novel data about the UmRrm75 protein. The transcription anti-termination assay demonstrated that UmRrm75 has an RNA chaperone activity. We found that ΔUmRRm75 mutant strains accumulate H2O2, peroxisomes, and melanin. Consequently, ΔUmRrm75 mutant strains showed an increased level of UmYap1 transcript and CAT activity. These findings could explain the previously observed phenotype of slow growth and reduced virulence in the Ustilago maydis ΔUmRRm75 mutant strains10.

Materials and Methods

Strains, media and growth conditions

The Ustilago maydis FB2, 1/2 and SG200 parental and ∆UmRrm75 1/46, 1/40 and 1/53 null mutant strains10, and Escherichia coli RL211 strain were used (genotypes are listed in Supplementary Table 1). For U. maydis growth, the complete medium (CM; 0.1% yeast extract, 0.5% casein peptone, 6.25% salt solution, 0.15% KNO3, 1.5% agar and 1% glucose), minimal medium (MM; 2% glucose, 3% KNO3 and 6.25% salt solution)56, and YEPD medium (1% yeast extract, 2% peptone, and 2% glucose) were used. For E. coli RL211 growth, Luria broth (LB; 1% peptone, 0.5% yeast extract, 1% NaCl, 500 μM IPTG) was used. These strains were stored at −70 °C in 50% glycerol (v/v). The U. maydis strains were recovered in YEPD at 28 °C, and the E. coli strain was recovered in LB medium at 37 °C.

Thermal stress assay

The U. maydis parental and UmRRm75 null mutant strains were grown overnight in YEPD medium. Cells were adjusted to an OD600 of 0.3 with fresh YEPD medium, and incubated at 28 °C until an OD600 of 0.8–1.0 was reached. Cells were collected by centrifugation, and pellets were washed twice with sterile distilled water. Subsequently, 2 µL of each suspension were spotted at four serial dilutions (1 × 102–1 × 105) in Petri dishes containing solid CM. The inoculated plates were incubated at 15 °C, 28 °C or 37 °C for 3 or 6 days. Images shown are representative of the experiment conducted with three biological replicates. This experiment was repeated at least 3 times with similar results.

In vivo transcription anti-terminator assay

The open reading frame (ORF) of UmRrm75 gene was amplified by PCR using Phusion High-fidelity DNA polymerase (ThermoFisher, Carlsbad, CA, USA) and cloned between the XbaI/BamH1 restrictions sites present in the pINIII plasmid. The pINIII:UmRrm75 construct was confirmed by sequencing, and transformed in the E. coli RL211 mutant strain11. As a positive control, RL211 strain was transformed with the pINIII-CspA plasmid, and as a negative control the pINIII empty vector was used. The RL211 strains carrying the various vectors were grown in LB liquid medium, and then spotted in serial dilutions (1 × 102–1 × 105) on LB plates supplemented with 8 µg/L or 10 µg/L chloramphenicol (Cm). Plates were incubated at 37 °C for 72 h. Photographs shown are representative of the experiment conducted with three biological replicates. This experiment was repeated at least 3 times with similar results.

Expression analysis of UmRrm75 transcript under abiotic stress conditions

For all treatments, the FB2 parental strain was grown overnight in YEPD liquid medium at 28 °C. Then, the cell culture was adjusted to an OD600 of 0.3 using fresh liquid MM. For the thermal stress assay, cells were grown at 15 °C, 28 °C or 37 °C for 24 h. For osmotic and oxidative stresses, 1 M sorbitol and 1 mM H2O2, were independently added to the FB2 parental cells culture and then subsequently grown for 24 h. After cells were collected, pellets of each condition were stored at −70 °C for subsequent RNA extraction.

Analysis of UmYap1 and UmPex3 transcripts in parental and null mutant strains

The FB2 parental and 1/46 null mutant strains were grown at 28 °C in liquid MM and collected at 4 and 6 h. Then, the pellets for each strain were stored at −70 °C for a subsequent RNA extraction. The RNA extraction method was conducted as described by Collart and Oliviero57. The genomic DNA was removed using TURBO DNAse enzyme (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. For synthesis and quantification of cDNA, the One-Step Kit and Power SYBR Green RNA-to-CT kit (Applied Biosystems, USA) were used. The qRT-PCR was performed as described previously in Rodríguez-Hernández et al.58 and Ortega-Amaro et al.59. The UmRrm75, UmPex3, and UmYap1 gene expression were analyzed by the 2−ΔΔCT method60 and the data were normalized against the UmGAPDH gene. The designed primers are listed in Supplementary Table 3. For each sample, three biological replicates (n = 3) were analyzed with their respective technical replicate.

Extraction and isolation of melanin

Parental (FB2 and SG200) and ΔUmRrm75 mutant (1/46 and 1/53) strains of U. maydis were grown in liquid MM at 37 °C and 28 °C respectively, for 10 days. Afterwards, each culture was centrifuged and washed twice with sterile deionized water. Each cellular pellet was dissolved in 1 M NaOH and heated to 120 °C for 20 min; then acidified with 6 M HCl and heated to 100 °C for 3 h, and then centrifuged for 10 min. The pellet was dissolved in 0.1 M KOH. Concentrated HCl was added to the aqueous portion to precipitate the brown pigment. The precipitate material was washed with distilled water and dried in a SpeedVac Concentrator (SAVANT, SPD131DDA) with a refrigerated vapor trap (RVT405DDA) at RT for 2 h. The obtained powder was used for spectroscopic and spectrometric analysis.

Experimental procedures for spectroscopic and spectrometric characterization of melanins

UV-Vis spectra of aqueous solution of brown pigment at a concentration of 10 μg/mL in 0.1 M KOH were recorded using a Thermo Scientific Aquamate 9423AQA2700E UV-Vis Spectrophotometer in the wavelength range 200–899 nm. IR spectra were obtained using the ATR sampling technique in a Thermo Nicolet 6700 FT-IR spectrometer. The IR and UV-Vis data were visualized using Origin Pro 8.0 software. 1H NMR (400 MHz) experiments were performed with a Varian Inova spectrometer. Chemical shifts were referenced relative to TMS, and J values are given in Hz. The 1H NMR spectra were acquired by dissolving 6–8 mg of melanin in 0.8 mL NaOD in 40% D2O at 60 °C. The NMR data were processed and visualized using MestReNova software. HRESIMS data were recorded on a Thermo Q Exactive Plus mass spectrometer in positive detection mode. For this analysis, each melanin sample was dissolved in 300 μL of a mixture of 2 M KOH in MeOH/saturated NH4Cl aqueous/DMSO 1:1:1. Samples were directly infused in an Orbitrap instrument (Thermo Fisher Scientific).

Detection of H2O2 in U. maydis by fluorescent microscopy

The parental and UmRrm75 null mutant strains were grown in YEPD at 28 °C overnight. Cells were refreshed and grown until reaching an OD600 of 0.8–1.0. Subsequently, the strains were subjected to 15 °C, 28 °C or 37 °C for 4 h. For catalase treatment, cells were grown until reaching an OD600 of 0.8–1.0, after which 0 or 250 U/mL of CAT was added for 4 h. Then, 20 μM of 2,7-dichlorohydrofluorecein diacetate (DCFH2-DA) was added to each culture strain according to the protocol described by Fu et al.61. Cells were observed using an Axio Imager M2 microscope (Zeiss). Images shown are representative of the experiment conducted with three biological replicates. This experiment was repeated at least 3 times with similar results.

H2O2 sensitivity assay, and H2O2 quantification in parental and UmRrm75 null mutant strains

For the H2O2 sensitivity assay, the ΔUmRrm75 mutant and parental strains were plated on CM medium. Filter disks were soaked with 1 µL of H2O2 (30% v/v) and placed on the center of plates. The halo sizes were measured from four biological replicates after 6 days of incubation at 28 °C. For each sample, four biological replicates (n = 4) were analyzed. Experiments were repeated at least twice with similar results. For H2O2 quantification, one gram (fresh weight) of FB2 parental and 1/46 null mutant cells (CFW) were collected. The cell mass was homogenized in 0.1% trichloroacetic acid and collected by centrifugation. Subsequently, cells were resuspended in 10 mM phosphate buffer (pH 7.0). Finally, 0.5 mL of 1 M potassium iodide (KI) was added. The samples were measured at a wavelength of 390 nm. Dilutions of a standard H2O2 solution were read for the calibration curve. Data of each sample were interpolated with the standard H2O2 curve and were reported as μmol/g of cell fresh weight (μmol/gCFW). For each sample, three biological replicates (n = 3) were analyzed. Experiments were repeated at least twice with similar results.

Analysis of ΔUmRrm75 mutants and parental strains by transmission electron microscopy (TEM)

The parental FB2, 1/2 and SG200, as well as null mutant strains 1/46, 1/40 and 1/53, were grown in MM liquid medium at 28 °C for 24 h. For the stress condition, strains were grown in 1 mM H2O2 and the cell pellet was collected. Cells were fixed in 3% glutaraldehyde for 2 h at room temperature, then washed 3 times with PBS and incubated for 4 h at 37 °C in a freshly prepared solution (5 mL) of 10 mg 3,3′-diaminobenzidine (DAB) in 0.1 M bicarbonate buffer (pH 10.5)62. Samples were postfixed with 2% OsO4 at RT for 1 h, washed with PBS, dehydrated with ethanol, embedded in Epon 812 Resin and polymerized for 24 h at 60 °C. Ultra-thin sections were obtained and contrasted with 4% uranyl acetate and Reynold´s lead citrate. Images were acquired with a JEOL JEM 1010 electron transmission microscope at accelerating voltages of 60 kV.

Catalase enzymatic activity in FB2 and 1/46 mutant strains

The FB2 parental and 1/46 null mutant strains were grown in liquid MM at 28 °C for 10, 12 or 24 h. Protein extraction was performed as described by Hernández-Sánchez et al.63. Protein concentration was determined by the Bradford test64. Protein extract was used to quantify the enzymatic activity by the spectrophotometric method at a wavelength of 240 nm65,66. The CAT activity was normalized to the initial protein concentration and was expressed in U CAT/mg protein. For each sample, three biological replicates (n = 3) were analyzed. Experiments were repeated at least twice with similar results.

Statistical analysis

Unpaired t test, One-Way ANOVA Analysis and Tukey’s post-test analyses were performed to assess statistical significance. GraphPad Prism version 5.0b (GraphPad Software, San Diego, California, USA) was used for the analysis. Data represent the mean ± SEM. Differences at P < 0.05 were considered to be significant.