Abstract
In eukaryotic organisms, the 5-oxoprolinase is one of the six key enzymes in the γ-glutamyl cycle that is involved in the biosynthetic pathway of glutathione (GSH, an antioxidative tripeptide counteracting the oxidative stress). To date, little is known about the biological functions of the 5-oxoprolinase in filamentous phytopathogenic fungi. In this study, we investigated the 5-oxoprolinase in Fusarium graminearum for the first time. In F. graminearum, two paralogous genes (FgOXP1 and FgOXP2) were identified to encode the 5-oxoprolinase while only one homologous gene encoding the 5-oxoprolinase could be found in other filamentous phytopathogenic fungi or Saccharomyces cerevisiae. Deletion of FgOXP1 or FgOXP2 in F. graminearum led to significant defects in its virulence on wheat. This is likely caused by an observed decreased deoxynivalenol (DON, a mycotoxin) production in the gene deletion mutant strains as DON is one of the best characterized virulence factors of F. graminearum. The FgOXP2 deletion mutant strains were also defective in conidiation and sexual reproduction while the FgOXP1 deletion mutant strains were normal for those phenotypes. Double deletion of FgOXP1 and FgOXP2 led to more severe defects in conidiation, DON production and virulence on plants, suggesting that both FgOXP1 and FgOXP2 play a role in fungal development and plant colonization. Although transformation of MoOXP1into ΔFgoxp1 was able to complement ΔFgoxp1, transformation of MoOXP1 into ΔFgoxp2 failed to restore its defects in sexual development, DON production and pathogenicity. Taken together, these results suggest that FgOXP1 and FgOXP2 are likely to have been functionally diversified and play significant roles in fungal development and full virulence in F. graminearum.
References
Avalos J, Carmen Limon M (2015) Biological roles of fungal carotenoids. Curr Genet 61:309–324. doi:10.1007/s00294-014-0454-x
Canovas D, Marcos JF, Marcos AT, Strauss J (2016) Nitric oxide in fungi: is there NO light at the end of the tunnel? Curr Genet 62:513–518. doi:10.1007/s00294-016-0574-6
Cappellini RA, Peterson JL (1965) Macroconidium formation in submerged cultures by a non-sporulating strain of Gibberella zeae. Mycologia 57:962–966. doi:10.2307/3756895
Catlett LN, Lee BN, Yoder OC, Turgeon BG (2002) Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genet News 49:9–11
Chen X, Schecter RL, Griffith OW, Hayward MA, Alpert LC, Batist G (1998) Characterization of 5-oxo-l-prolinase in normal and tumor tissues of humans and rats: a potential new target for biochemical modulation of glutathione. Clin Cancer Res Off J Am Assoc Cancer Res 4:131–138
Choi YE, Xu JR (2010) The cAMP signaling pathway in Fusarium verticillioides is important for conidiation, plant infection, and stress responses but not fumonisin production. Mol Plant Microbe Interact 23:522–533. doi:10.1094/MPMI-23-4-0522
Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD (2012) The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430. doi:10.1111/j.1364-3703.2011.00783.x
Deng YZ, Qu Z, He Y, Naqvi NI (2012) Sorting nexin Snx41 is essential for conidiation and mediates glutathione-based antioxidant defense during invasive growth in Magnaporthe oryzae. Autophagy 8:1058–1070. doi:10.4161/auto.20217
Desjardins AE (1996) Reduced virulence of trichothecene-nonproducing mutants of Gibberella zeaein wheat field tests. Mol Plant Microbe Interact 9:775. doi:10.1094/mpmi-9-0775
Ding S, Mehrabi R, Koten C, Kang Z, Wei Y, Seong K, Kistler HC, Xu JR (2009) Transducin beta-like gene FTL1 is essential for pathogenesis in Fusarium graminearum. Eukaryot Cell 8:867–876. doi:10.1128/EC.00048-09
Gardiner DM, Kazan K, Manners JM (2009) Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum. Fungal Genet Biol 46:604–613. doi:10.1016/j.fgb.2009.04.004
Holmes AR, Collings A, Farnden KJ, Shepherd MG (1989) Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase. J Gen Microbiol 135:1423–1430. doi:10.1099/00221287-135-6-1423
Hou Z, Xue C, Peng Y, Katan T, Kistler HC, Xu JR (2002) A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection. Mol Plant Microbe Interact 15:1119–1127. doi:10.1094/MPMI.2002.15.11.1119
Jaspers CJ, Gigot D, Penninckx MJ (1985) Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae. Phytochemistry 24:703–707. doi:10.1016/S0031-9422(00)84880-3
Jiang J, Yun Y, Yang Q, Shim WB, Wang Z, Ma Z (2011) A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum. PLoS ONE 6:e25311. doi:10.1371/journal.pone.0025311
Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36:W5–W9. doi:10.1093/nar/gkn201
Kumar A, Bachhawat AK (2010) OXP1/YKL215c encodes an ATP-dependent 5-oxoprolinase in Saccharomyces cerevisiae: functional characterization, domain structure and identification of actin-like ATP-binding motifs in eukaryotic 5-oxoprolinases. FEMS Yeast Res 10:394–401. doi:10.1111/j.1567-1364.2010.00619.x
Kumar A, Bachhawat AK (2012) Pyroglutamic acid: throwing light on a lightly studied metabolite. Curr Sci 102:288–297
Letunic I, Doerks T, Bork P (2015) SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43:D257–D260. doi:10.1093/nar/gku949
Liu X, Xu J, Wang J, Ji F, Yin X, Shi J (2015) Involvement of threonine deaminase FgIlv1 in isoleucine biosynthesis and full virulence in Fusarium graminearum. Curr Genet 61:55–65. doi:10.1007/s00294-014-0444-z
Mayatepek E, Hoffmann GF, Larsson A, Becker K, Bremer HJ (1995) 5-Oxoprolinase deficiency associated with severe psychomotor developmental delay, failure to thrive, microcephaly and microcytic anaemia. J Inherit Metab Dis 18:83–84
Mazelis M, Creveling RK (1978) 5-Oxoprolinase (l-pyroglutamate hydrolase) in higher plants. Partial Purif Charact Wheat Germ Enzym 62:798–801. doi:10.1104/pp.62.5.798
McMullen M, Jones R, Gallenberg D (1997) Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Dis 81:1340–1348. doi:10.1094/pdis.1997.81.12.1340
Meister A (1973) On the enzymology of amino acid transport. Science 180:33–39. doi:10.1126/science.180.4081.33
Mooz ED, Wigglesworth L (1976) Evidence for the γ-glutamyl cycle in yeast. Biochem Biophys Res Commun 68:1066–1072. doi:10.1016/0006-291X(76)90304-1
Njalsson R, Norgren S (2005) Physiological and pathological aspects of GSH metabolism. Acta Paediatr 94:132–137. doi:10.1080/08035250410025285
Orlowski M, Meister A (1970) The gamma-glutamyl cycle: a possible transport system for amino acids. Proc Natl Acad Sci USA 67:1248–1255. doi:10.1073/pnas.67.3.1248
Richman PG, Meister A (1975) Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 250:1422–1426
Robaczewska J, Kedziora-Kornatowska K, Kozakiewicz M, Zary-Sikorska E, Pawluk H, Pawliszak W, Kedziora J (2016) Role of glutathione metabolism and glutathione-related antioxidant defense systems in hypertension. J Physiol Pharmacol Off J Polish Physiol Soc 67:331–337
Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42:W320–W324. doi:10.1093/nar/gku316
Saunders DG, Aves SJ, Talbot NJ (2010a) Cell cycle-mediated regulation of plant infection by the rice blast fungus. Plant Cell 22:497–507. doi:10.1105/tpc.109.072447
Saunders DG, Dagdas YF, Talbot NJ (2010b) Spatial uncoupling of mitosis and cytokinesis during appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae. Plant Cell 22:2417–2428. doi:10.1105/tpc.110.074492
Shalaby S, Horwitz BA (2015) Plant phenolic compounds and oxidative stress: integrated signals in fungal-plant interactions. Curr Genet 61:347–357. doi:10.1007/s00294-014-0458-6
Starkey DE, Ward TJ, Aoki T, Gale LR, Kistler HC, Geiser DM, Suga H, Toth B, Varga J, O’Donnell K (2007) Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity. Fungal Genet Biol 44:1191–1204. doi:10.1016/j.fgb.2007.03.001
Talbot NJ (2003) On the trail of a cereal killer: exploring the biology of Magnaporthe grisea. Annu Rev Microbiol 57:177–202. doi:10.1146/annurev.micro.57.030502.090957
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. doi:10.1093/molbev/msr121
Tang W, Ru Y, Hong L, Zhu Q, Zuo R, Guo X, Wang J, Zhang H, Zheng X, Wang P, Zhang Z (2015) System-wide characterization of bZIP transcription factor proteins involved in infection-related morphogenesis of Magnaporthe oryzae. Environ Microbiol 17:1377–1396. doi:10.1111/1462-2920.12618
Teng PS, Klein-Gebbinck HW, Pinnschmidt H (1991) An analysis of the blast pathosystem to guide modeling and forecasting. International Rice Research Institute (IRRI), Manila, pp 1–30
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. doi:10.1093/nar/22.22.4673
Trail F (2009) For blighted waves of grain: Fusarium graminearum in the postgenomics era. Plant Physiol 149:103–110. doi:10.1104/pp.108.129684
Trail F, Xu H, Loranger R, Gadoury D (2002) Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum). Mycologia 94:181–189. doi:10.1080/15572536.2003.11833223
Van Der Werf P, Stephani RA, Orlowski M, Meister A (1973) Inhibition of 5-oxoprolinase by 2-imidazolidone-4-carboxylic acid. Proc Natl Acad Sci 70:759–761. doi:10.1073/pnas.70.3.759
Xu X, Nicholson P (2009) Community ecology of fungal pathogens causing wheat head blight. Annu Rev Phytopathol 47:83–103. doi:10.1146/annurev-phyto-080508-081737
Yadav AK, Desai PR, Rai MN, Kaur R, Ganesan K, Bachhawat AK (2011) Glutathione biosynthesis in the yeast pathogens Candida glabrata and Candida albicans: essential in C. glabrata, and essential for virulence in C. albicans. Microbiology 157:484–495. doi:10.1099/mic.0.045054-0
Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Dominguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981. doi:10.1016/j.fgb.2004.08.001
Zheng W, Zhao X, Xie Q, Huang Q, Zhang C, Zhai H, Xu L, Lu G, Shim WB, Wang Z (2012) A conserved homeobox transcription factor Htf1 is required for phialide development and conidiogenesis in Fusarium species. PLoS ONE 7:e45432. doi:10.1371/journal.pone.0045432
Zheng W, Zheng H, Zhao X, Zhang Y, Xie Q, Lin X, Chen A, Yu W, Lu G, Shim WB, Zhou J, Wang Z (2016) Retrograde trafficking from the endosome to the trans-Golgi network mediated by the retromer is required for fungal development and pathogenicity in Fusarium graminearum. New Phytol 210:1327–1343. doi:10.1111/nph.13867
Acknowledgements
This work was supported by the National Natural Science Foundation of China under Grant no. 31670142 and the China Postdoctoral Science Foundation under Grant 2016M590587. We thank Drs. Xu Zhao, Guanghui Wang, Lianhu Zhang, Huawei Zheng and Yi Lou for fruitful discussions and suggestions.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Communicated by M. Kupiec.
Electronic supplementary material
Below is the link to the electronic supplementary material.
294_2017_747_MOESM1_ESM.docx
Fig. S1 Sequence comparison of 5-oxoprolinase from different species. ClustalW (Thompson, et al. 1994)assisted multiple sequence alignments of 5-oxoprolinase of NcOxp1 (N. crassa), SmOxp1 (S. macrospora), MoOxp1 (M. oryzae), FfOxp1 (F. fujikuroi), FoOxp1 (F. oxysporum), FvOxp1 (F. verticillioides), FgOxp1 (F. graminearum), TrOxp1 (T. reesei), BcOxp1 (B. cinerea), ScOxp1 (S. cerevisiae), CgOxp1 (C. gloeosporioides), TrOxp2 (T. reesei), FfOxp2 (F. fujikuroi), FoOxp2 (F. oxysporum), FvOxp2 (F. verticillioides), FgOxp2 (F. graminearum), CgOxp2 (C. gloeosporioides), AtOxp1 (A. thaliana), HsOxp1 (H. sapiens). Box shade was used for graphical rendering and presentation. Residues that are the same in all four sequences are boxed in red. Fig. S2 Relative mycelial growth rate, hyphal tip growth and branching patterns of each strain of F. graminearum. Images of each strain were registered after 3 days of incubation at 25°C on CMII plates (9 cm in diameter). Scale bar = 100 μm. Fig. S3 Conidial morphology of each strain of F. graminearum. Conidia of each strain were stained with CFW to visualize the cell wall and septa and with DAPI to visualize the cell nucleus. Conidia were observed via differential interference contrast (DIC, left) and fluorescence microscopy (UV, middle). MERGE images were constructed by the microscopic software of “NIS-Elements” (right). Scale bar = 10μm. Fig. S4 Conidiation without or with 0.5mM GSH of each strain of F. graminearum. Fresh mycelia of each strain were inoculated in liquid CMC culture without or with 0.5mM GSH and shaking at 28°C at 180 rpm for 3 days, 6 days, 9 days and 15 days, respectively. Means and standard deviations were calculated from data of three biological replicates. The experiment was repeated three times with similar results. Asterisk indicates a significant difference of conidiation between the mutant strain and the wild-type (PH-1) (P≤0.05, Duncan’s test). Fig. S5 DON production without or with 0.5mM GSH of each strain of F. graminearum. DON production (μg g−1 dry weight) was tested for each strain incubated in LTB liquid culture for 7 days at 28 °C in the dark without shaking. The blank control for DON quantification was 0. Error bars denote the standard deviation of three biological replicates. The experiment was repeated three times with similar results. Asterisk indicates a significant difference of DON production between the mutant strain and the wild-type (PH-1) (P≤0.05, Duncan’s test). Table. S1 Wild-type and mutant strains used in this study. Table. S2 PCR Primers used in this study. Supplementary material 1 (DOCX 10909 kb)
Rights and permissions
About this article
Cite this article
Yang, P., Chen, Y., Wu, H. et al. The 5-oxoprolinase is required for conidiation, sexual reproduction, virulence and deoxynivalenol production of Fusarium graminearum . Curr Genet 64, 285–301 (2018). https://doi.org/10.1007/s00294-017-0747-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-017-0747-y