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. 2016 Feb 22;11(2):e0149606.
doi: 10.1371/journal.pone.0149606. eCollection 2016.

Characterization of Three New Glutaredoxin Genes in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis: Putative Role of RiGRX4 and RiGRX5 in Iron Homeostasis

Elisabeth Tamayo  1 , Karim Benabdellah  2 , Nuria Ferrol  1
Affiliations

Characterization of Three New Glutaredoxin Genes in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis: Putative Role of RiGRX4 and RiGRX5 in Iron Homeostasis

Elisabeth Tamayo et al. PLoS One. .

Abstract

Glutaredoxins (GRXs) are small ubiquitous oxidoreductases involved in the regulation of the redox state in living cells. In an attempt to identify the full complement of GRXs in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, three additional GRX homologs, besides the formerly characterized GintGRX1 (renamed here as RiGRX1), were identified. The three new GRXs (RiGRX4, RiGRX5 and RiGRX6) contain the CXXS domain of monothiol GRXs, but whereas RiGRX4 and RiGRX5 belong to class II GRXs, RiGRX6 belongs to class I together with RiGRX1. By using a yeast expression system, we observed that the newly identified homologs partially reverted sensitivity of the GRX deletion yeast strains to external oxidants. Furthermore, our results indicated that RiGRX4 and RiGRX5 play a role in iron homeostasis in yeast. Gene expression analyses revealed that RiGRX1 and RiGRX6 were more highly expressed in the intraradical (IRM) than in the extraradical mycelium (ERM). Exposure of the ERM to hydrogen peroxide induced up-regulation of RiGRX1, RiGRX4 and RiGRX5 gene expression. RiGRX4 expression was also up-regulated in the ERM when the fungus was grown in media supplemented with a high iron concentration. These data indicate the two monothiol class II GRXs, RiGRX4 and RiGRX5, might be involved in oxidative stress protection and in the regulation of fungal iron homeostasis. Increased expression of RiGRX1 and RiGRX6 in the IRM suggests that these GRXs should play a key role in oxidative stress protection of R. irregularis during its in planta phase.

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Conflict of interest statement

Competing Interests: Karim Benabdellah (KB) is affiliated with GENYO (Centre for Genomic and Oncological Research), a public center co-financed by Pfizer, the University of Granada and the Andalusian Regional Government. KB is financed by the Andalusian Regional Government and does not have any contract with Pfizer. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Therefore all materials presented in the manuscript will be freely available to the scientific community although not for commercial purposes.

Figures

Fig 1
Fig 1
A. Domain organization of the R. irregularis GRXs. Glutaredoxin domains are represented by black boxes. The thioredoxin-like (Trx) domain of RiGRX4, the mitochondrial location signal (MLS) of RiGRX5 and the domain of unknown function of RiGRX6 (white box) are also indicated. Numbers correspond to the position of the first cysteine in the active site in the GRX domains, the first glycine of the glutathione binding domains and the total length of the proteins. The position of the cysteine in the Trx domain of RiGRX4 is also indicated. B. Unrooted Nieghbor-Joining tree of the GRX family in fungi. Organisms: An, Aspergillus niger; Bc, Botrytis cinerea; Cc, Coprinopsis cinerea; Cn, Cryptococcus neoformans; Lb, Laccaria bicolor; Mg, Magnaporthe grisea; Nc, Neurospora crassa; Pc, Phanerochaete chrysosporium; Pg, Puccinia graminis; Ri, Rhizophagus irregularis; Ro, Rhizopus oryzae; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tm, Tuber melanosporum; Um, Ustilago maydis. R. irregularis GRXs are emphasized in bold. Protein JGI identification numbers are indicated. R. oryzae sequences were retrieved from the Broad Institute databases (http://www.broad.mit.edu/annotation/). Bootstrap values above 70 and supporting a node are indicated.
Fig 2
Fig 2. Relative expression of the RiGRX genes in extraradical mycelia (ERM) and intraradical (IRM) mycelia of R. irregularis.
RiGRX gene expression was assessed in ERM developed in monoxenic cultures (ERM), R. irregularis-colonized carrot roots grown in monoxenic cultures and lacking ERM (IRM(C)) and R. irregularis-colonized rice roots grown in pot cultures and devoid of ERM (IRM(R)). Samples were normalized using the housekeeping gene RiTEF. Relative expression levels were calculated by the 2-ΔCT method. Data are means +/- standard error. Asterisks show statistically significant differences (p<0.05) relative to the ERM, according to the Fisher’s LSD test.
Fig 3
Fig 3. Complementation of the sensitivity to external oxidants of the grx yeast mutants by the R. irregularis GRX genes.
A. Effect of RiGRX4, RiGRX5 and RiGRX6 expression on the sensitivity of the Δgrx3Δgrx4 strain to 1 mM hydrogen peroxide (H2O2). B. Effect of RiGRX4, RiGRX5 and RiGRX6 expression on the sensitivity of the Δgrx5 strain to 0.1 mM menadione (Md). The photographs were taken after 3 days of growth at 30°C. C. Effect of RiGRX6 expression on the sensitivity of Δgrx6Δgrx7 to 500 mM CaCl2 (40 h). Data are means of three independent experiments +/- standard error and represent the growth yield ratio between treated and untreated cultures and then made relative to this ratio in cells expressing the S. cerevisiae Grx6. Asterisks show statistically significant differences (p<0.05) relative to the strain transformed with the empty vector, according to the Fisher’s LSD test.
Fig 4
Fig 4. Localization of R. irregularis GRXs in S. cerevisiae.
Soluble GFP (A-C) and C-terminal GFP-tagged versions of RiGRX4 (D-F) and RiGRX5 (G-J) were expressed in the Δgrx3Δgrx4 cells. C-terminal GFP-tagged version of RiGRX6 (K-M) was expressed in the Δgrx6Δgrx7 yeast mutant cells. Cells were grown to mid-logarithmic phase and localization of fusion proteins was visualized by fluorescence microscopy (A, D, G and K). Mitochondria in the RiGRX6-GFP expressing cells were stained with MitoTracker® Red and visualized by fluorescence microscopy (H). Bright field (B, E, J and L) and merged (C, F, I and M) images.
Fig 5
Fig 5. Analysis of the in vivo role of RiGRX5 in the biogenesis of Fe-S clusters in yeast.
A. Δgrx5 cells transformed with the empty vector or expressing ScGrx5, RiGRX5 or RiGRX4 were plated on SD medium with or without lysine. Plates were incubated at 30°C for 3 days. B. The activities of a Fe-S protein (aconitase) and a non-Fe-S protein (malate dehydrogenase) were determined in lysates of the Δgrx5 cells transformed with the different constructs. Data are means +/- standard error. Asterisks show statistically significant differences (p<0.05) relative to the activities of the strain transformed with the empty vector, according to the Fisher’s LSD test.
Fig 6
Fig 6. Analysis of the in vivo role of RiGRX4 and RiGRX5 in intracellular iron accumulation in Δgrx3Δgrx4 (A) and Δgrx5 (B) yeast mutant strains.
Intracellular iron concentrations were determined in lysates of cells transformed with the empty vector or expressing ScGrx4, ScGrx5, RiGRX4 or RiGRX5, with a Quantichron™ Iron Assay Kit. Data are means +/- standard error. Asterisks show statistically significant differences (p<0.1) relative to the values of the strain transformed with the empty vector (white columns), according to the Fisher’s LSD test.
Fig 7
Fig 7. Effect of hydrogen peroxide on the expression of the R. irregularis GRX genes.
R. irregularis ERM grown in M-C medium was exposed for different periods of time to 0.1 mM H2O2 (grey columns) or 1 mM H2O2 (black columns). RiGRX1 (A), RiGRX4 (B), RiGRX5(C), RiGRX6 (D) and GintPDX1 (E) gene expression. Data were normalized using the housekeeping gene RiTEF. Relative expression levels were calculated by the 2-ΔΔCT method. Data are means +/- standard error. Asterisks show statistically significant differences (p<0.05) compared to the control value, according to the Fisher’s LSD test.
Fig 8
Fig 8. Effect of iron on the expression of the R. irregularis GRX genes.
R. irregularis was grown in M-C media containing 45 μM Fe (control) or supplemented with 4.5 mM Fe or 45 mM Fe medium for 2 weeks. RiGRX1 (A), RiGRX4 (B), RiGRX5 (C) and RiGRX6 (D) gene expression. Data were normalized using the housekeeping gene RiTEF. Relative expression levels were calculated by the 2-ΔΔCT method. Data are means +/- standard error. Asterisks show statistically significant differences (p<0.05) compared to the control value, according to the Fisher’s LSD test.
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This research was supported by the Spanish Ministry of Economy and Competitivity (Projects AGL2012-35611 and AGL2015-67098-R). Elisabeth Tamayo was supported by a PhD contract (I3P) from the Spanish National Research Council (CSIC) (JAEPre_2010_00977). Karim Benabdellah is employed by Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government. GENYO is a public center co-financed by Pfizer, but Pfizer did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section.