Skip to main content

Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5′-phosphate synthesis and pathogenesis of Magnaporthe oryzae

Abstract

Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5′-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  • Carroll AM, Sweigard JA, Valent B (1994) Improved vectors for selecting resistance to hygromycin. Fungal Genet Newslett 41:22

  • Chen Y, Mo HZ, Zheng MY, Xian M, Qi ZQ, Li YQ, Hu LB, Chen J, Yang LF (2014a) Selenium inhibits root elongation by repressing the generation of endogenous hydrogen sulfide in Brassica rapa. PLoS ONE 9:e110904

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen Y, Zuo RF, Zhu Q, Sun Y, Li MY, Dong YH, Ru YY, Zhang HF, Zheng XB, Zhang ZG (2014b) MoLys2 is necessary for growth, conidiogenesis, lysine biosynthesis, and pathogenicity in Magnaporthe oryzae. Fungal Genet Biol 67:51–57

    CAS  Article  PubMed  Google Scholar 

  • Ding SL, Liu WD, Lliuk A, Ribot C, Vallet J, Tao A, Wang Y, Lebrun MH, Xu JR (2010) The Tig1 histone deacetylase complex regulates infectious growth in the rice blast fungus Magnaporthe oryzae. Plant Cell 22:2495–2508

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Dou XY, Wang Q, Qi ZQ, Song WW, Wang W, Guo M, Zhang HF, Zhang ZG, Wang P, Zheng XB (2011) MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae. PLoS ONE 6:e16439

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Du Y, Zhang HF, Hong L, Wang JM, Zheng XB, Zhang ZG (2013) Acetolactate synthases MoIlv2 and MoIlv6 are required for infection-related morphogenesis in Magnaporthe oryzae. Mol Plant Pathol 14:870–884

    CAS  Article  PubMed  Google Scholar 

  • Ebbole DJ (2007) Magnaporthe as a model for understanding host-pathogen interactions. Annu Rev Phytopathol 45:437–456

    CAS  Article  PubMed  Google Scholar 

  • Fernandez J, Yang KT, Cornwell KM, Wright JD, Wilson RA (2013) Growth in rice cells requires de novo purine biosynthesis by the blast fungus Magnaporthe oryzae. Sci Rep 3:2398

    Article  PubMed  PubMed Central  Google Scholar 

  • Fernandez J, Marroquin-Guzman M, Wilson RA (2014) Evidence for a transketolase-mediated metabolic checkpoint governing biotrophic growth in rice cells by the blast fungus Magnaporthe oryzae. PLoS Pathog 10:e1004354

    Article  PubMed  PubMed Central  Google Scholar 

  • Foster AJ, Jenkinson JM, Talbot NJ (2003) Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J 22:225–235

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Geigenberger P, Regierer B, Nunes-Nesi A, Leisse A, Urbanczyk-Wochniak E, Springer F, van Dongen JT, Kossmann J, Fernie AR (2005) Inhibition of de novo pyrimidine synthesis in growing potato tubers leads to a compensatory stimulation of the pyrimidine salvage pathway and a subsequent increase in biosynthetic performance. Plant Cell 17:2077–2088

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Ghosh AC, Shimell MJ, Leof ER, Haley MJ, O’Connor MB (2015) UPRT, a suicide-gene therapy candidate in higher eukaryotes, is required for Drosophila larval growth and normal adult lifespan. Sci Rep 5:13176

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360

    CAS  Article  PubMed  Google Scholar 

  • Grubmeyer C, Segura E, Dorfman R (1993) Active site lysines in orotate phosphoribosyltransferase. J Biol Chem 268:20299–20304

    CAS  PubMed  Google Scholar 

  • Guo M, Gao F, Zhu XL, Nie X, Pan YM, Gao ZM (2015) MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae. Appl Microbiol Biot 99:8075–8088

    CAS  Article  Google Scholar 

  • Hamer JE, Howard RJ, Chumley FG, Valent B (1988) A mechanism for surface attachment in spores of a plant pathogenic fungus. Science 239:288–290

    CAS  Article  PubMed  Google Scholar 

  • Howard RJ, Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci U S A 88:11281–11284

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Jeon J, Goh J, Yoo S, Chi MH, Choi J, Rho HS, Park J, Han SS, Kim BR, Park SY, Kim S, Lee YH (2008) A putative MAP kinase kinase kinase, MCK1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae. Mol Plant-Microbe Interact 21:525–534

    CAS  Article  PubMed  Google Scholar 

  • Jong JCD, Mccormack BJ, Smirnoff N, Talbot NJ (1997) Glycerol generates turgor in rice blast. Nature 389:244–244

    Article  Google Scholar 

  • Kawamura C, Moriwaki J, Kimura N, Fujita Y, Fuji S, Hirano T, Koizumi S, Tsuge T (1997) The melanin biosynthesis genes of Alternaria alternata can restore pathogenicity of the melanin-deficient mutants of Magnaporthe grisea. Mol Plant-Microbe Interact 10:446–453

    CAS  Article  PubMed  Google Scholar 

  • Kim S, Ahn IP, Rho HS, Lee YH (2005) MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization. Mol Microbiol 57:1224–1237

    CAS  Article  PubMed  Google Scholar 

  • Kraus PR, Fox DS, Cox GM, Heitman J (2003) The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function. Mol Microbiol 48:1377–1387

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Kronstad JW, Wang J, Covert SF, Holden DW, McKnight GL, Leong SA (1989) Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus Ustilago maydis. Gene 79:97–106

    CAS  Article  PubMed  Google Scholar 

  • Lau G, Hamer JE (1996) Regulatory genes controlling MPG1 expression and paghogenicity in the rice blast fungus Magnaporthe grisea. Plant Cell 8:771–781

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Montigny JD, Belarbi A, Hubert JC, Lacroute F (1989) Structure and expression of the URA5 gene of Saccharomyces cerevisiae. Mol Gen Genet 215:455–462

    Article  PubMed  Google Scholar 

  • Montigny JD, Kern L, Hubert JC, Lacroute F (1990) Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae. Curr Genet 17:105–111

    Article  PubMed  Google Scholar 

  • Musick WD (1981) Structural features of the phosphoribosyltransferases and their relationship to the human deficiency disorders of purine and pyrimidine metabolism. CRC Crit Rev Biochem 11:1–34

    CAS  Article  PubMed  Google Scholar 

  • Oakley BR, Rinehart JE, Mitchell BL, Oakley CE, Carmona C, Gray GL, May GS (1987) Cloning, mapping and molecular analysis of the pyrG (orotidine-5′-phosphate decarboxylase) gene of Aspergillus nidulans. Gene 61:385–399

    CAS  Article  PubMed  Google Scholar 

  • Ong HB, Sienkiewicz N, Wyllie S, Patterson S, Fairlamb AH (2013) Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy. Mol Microbiol 90:443–455

    CAS  PubMed  PubMed Central  Google Scholar 

  • Patkar RN, Xue YK, Shui G, Wenk MR, Naqvi NI (2012) Abc3-mediated efflux of an endogenous digoxin-like steroidal glycoside by Magnaporthe oryzae is necessary for host invasion during blast disease. PLoS Pathog 8:e1002888

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Prasad R, Kapoor K (2005) Multidrug resistance in yeast Candida. Int Rev Cytol 242:215–248

    CAS  Article  PubMed  Google Scholar 

  • Qi ZQ, Wang Q, Dou XY, Wang W, Zhao Q, Lv RL, Zhang HF, Zheng XB, Wang P, Zhang ZG (2012) MoSwi6, an APSES family transcription factor, interacts with MoMps1 and is required for hyphal and conidial morphogenesis, appressorial function and pathogenicity of Magnaporthe oryzae. Mol Plant Pathol 13:677–689

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Ram AF, Wolters A, Ten HR, Klis FM (1994) A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10:1019–1030

    CAS  Article  PubMed  Google Scholar 

  • Ramanujam R, Naqvi NI (2010) PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae. PLoS Pathog 6:e1000897

    Article  PubMed  PubMed Central  Google Scholar 

  • Rasmussen JB, Panaccione DG, Fang GC, Hanau RM (1992) The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis. Mol Gen Genet 235:74–80

    CAS  Article  PubMed  Google Scholar 

  • Sanglard D, Ischer F, Marchetti O, Entenza J, Bille J (2003) Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol Microbiol 48:959–976

    CAS  Article  PubMed  Google Scholar 

  • Saunders DG, Aves SJ, Talbot NJ (2010) Cell cycle-mediated regulation of plant infection by the rice blast fungus. Plant Cell 22:497–507

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Schoors S, Bruning U, Missiaen R, Queiroz KC, Borgers G, Elia I, Zecchin A, Cantelmo AR, Christen S, Goveia J, Heggermont W, Goddo L, Vinckier S, Van Veldhoven PP, Eelen G, Schoonjans L, Gerhardt H, Dewerchin M, Baes M, De Bock K, Ghesquière B, Lunt SY, Fendt SM, Carmeliet P (2015) Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature 520:192–197

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Song WW, Dou XY, Qi ZQ, Wang Q, Zhang X, Zhang HF, Guo M, Dong SM, Zhang ZG, Wang P, Zheng XB (2010) R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae. PLoS ONE 5:e13193

    Article  PubMed  PubMed Central  Google Scholar 

  • Song X, Zhang B, Zhou Y (2011) Golgi-localized UDP-glucose transporter is required for cell wall integrity in rice. Plant Signal Behav 6:1097–1100

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Talbot NJ (2003) On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea. Annu Rev Microbiol 57:177–202

    CAS  Article  PubMed  Google Scholar 

  • Talbot NJ, Ebbole DJ, Hamer JE (1993) Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5:1575–1590

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Talbot NJ, Kershaw MJ, Wakley GE, De VO, Wessels J, Hamer JE (1996) MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell 8:985–999

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Thines E, Weber RW, Talbot NJ (2000) MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell 12:1703–1718

    CAS  PubMed  PubMed Central  Google Scholar 

  • Timothy MB, Richard JH (1990) In vitro development of penetration structrues in the rice blast fungus Magnaporthe grisea. Can J Bot 68:329–342

    Article  Google Scholar 

  • Valent B (1990) Rice blast as a model system for plant pathology. Phytopathology 80:33–36

    Article  Google Scholar 

  • Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot NJ (2006) Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312:580–583

    CAS  Article  PubMed  Google Scholar 

  • Wang GP, Lundegaard C, Jensen KF, Grubmeyer C (1999) Kinetic mechanism of OMP synthase: a slow physical step following group transfer limits catalytic rate. Biochemistry 38:275–283

    CAS  Article  PubMed  Google Scholar 

  • Wang ZY, Thornton CR, Kershaw MJ, Li DB, Talbot NJ (2003) The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Mol Microbiol 47:1601–1612

    CAS  Article  PubMed  Google Scholar 

  • Wang ZY, Soanes DM, Kershaw MJ, Talbot NJ (2007) Functional analysis of lipid metabolism in Magnaporthe grisea reveals a requirement for peroxisomal fatty acid beta-oxidation during appressorium-mediated plant infection. Mol Plant-Microbe Interact 20:475–491

    Article  PubMed  Google Scholar 

  • Wang JM, Du Y, Zhang HF, Zhou C, Qi ZQ, Zheng XB, Wang P, Zhang ZG (2013) The actin-regulating kinase homologue MoArk1 plays a pleiotropic function in Magnaporthe oryzae. Mol Plant Pathol 14:470–482

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Weber RW, Wakley GE, Thines E, Talbot NJ (2001) The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea. Protoplasma 216:101–112

    CAS  Article  PubMed  Google Scholar 

  • Wilson RA, Talbot NJ (2009) Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nat Rev Microbiol 7:185–195

    CAS  Article  PubMed  Google Scholar 

  • Woloshuk CP, Seip ER, Payne GA, Adkins CR (1989) Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus. Appl Environ Microbiol 55:86–90

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wood PJ, Fulcher RG (1983) Dye interactions. A basis for specific detection and histochemistry of polysaccharides. J Histochem Cytochem 31:823–826

    CAS  Article  PubMed  Google Scholar 

  • Wösten HA (2001) Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55:625–646

    Article  PubMed  Google Scholar 

  • Xu JR, Hamer JE (1996) MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. Genes Dev 10:2696–2706

    CAS  Article  PubMed  Google Scholar 

  • Xu JR, Staiger CJ, Hamer JE (1998) Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc Natl Acad Sci U S A 95:12713–12718

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Yan X, Li Y, Yue X, Wang C, Que Y, Kong D, Ma Z, Talbot NJ, Wang ZY (2011) Two novel transcriptional regulators are essential for infection-related morphogenesis and pathogenicity of the rice blast fungus Magnaporthe oryzae. PLoS Pathog 7:e1002385

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Yoshikawa K, Tanaka T, Furusawa C, Nagahisa K, Hirasawa T, Shimizu H (2009) Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae. FEMS Yeast Res 9:32–44

    CAS  Article  PubMed  Google Scholar 

  • Zhang HF, Liu KY, Zhang X, Song WW, Zhao Q, Dong YH, Guo M, Zheng XB, Zhang ZG (2010) A two-component histidine kinase, MoSLN1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae. Curr Genet 56:517–528

    CAS  Article  PubMed  Google Scholar 

  • Zhang B, Liu X, Qian Q, Liu L, Dong G, Xiong G, Zeng D, Zhou Y (2011a) Golgi nucleotide sugar transporter modulates cell wall biosynthesis and plant growth in rice. Proc Natl Acad Sci U S A 108:5110–5115

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Zhang HF, Liu KY, Zhang X, Tang W, Wang JS, Guo M, Zhao Q, Zheng XB, Wang P, Zhang ZG (2011a) Two phosphodiesterase genes, PDEL and PDEH, regulate development and pathogenicity by modulating intracellular cyclic AMP levels in Magnaporthe oryzae. PLoS ONE 6:e17241

  • Zhang HF, Tang W, Liu KY, Huang Q, Zhang XB, Yan X, Chen Y, Wang JS, Qi ZQ, Wang Z, Zheng XB, Wang P, Zhang ZG (2011b) Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae. PLoS Pathog 7:e1002450

Download references

Acknowledgments

This research was supported by the Natural Science Foundation of China (grant no. 31470248, XZ), Natural Science Foundation of China (grant no. 31271998, ZZ), and the especially appointed professorship (Jiangsu, China).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Zhengguang Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 1494 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Qi, Z., Liu, M., Dong, Y. et al. Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5′-phosphate synthesis and pathogenesis of Magnaporthe oryzae . Appl Microbiol Biotechnol 100, 3655–3666 (2016). https://doi.org/10.1007/s00253-016-7323-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7323-0

Keywords

  • Magnaporthe oryzae
  • Orotate phosphoribosyl transferase
  • UTP biosynthesis
  • Cell wall integrity
  • Pathogenicity