The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
권호 표지
DOI QR코드

DOI QR Code

A Small GTPase RHO2 Plays an Important Role in Pre-infection Development in the Rice Blast Pathogen Magnaporthe oryzae

  • Fu, Teng (Division of Bioresource Sciences, and Bioherb Research Institute, Kangwon National University) ;
  • Kim, Joon-Oh (Division of Bioresource Sciences, and Bioherb Research Institute, Kangwon National University) ;
  • Han, Joon-Hee (Division of Bioresource Sciences, and Bioherb Research Institute, Kangwon National University) ;
  • Gumilang, Adiyantara (Division of Bioresource Sciences, and Bioherb Research Institute, Kangwon National University) ;
  • Lee, Yong-Hwan (Department of Agricultural Biotechnology, and Center for Fungal Genetic Resources, Seoul National University) ;
  • Kim, Kyoung Su (Division of Bioresource Sciences, and Bioherb Research Institute, Kangwon National University)
  • Received : 2018.04.18
  • Accepted : 2018.08.21
  • Published : 2018.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

The rice blast pathogen Magnaporthe oryzae is a global threat to rice production. Here we characterized RHO2 gene (MGG_02457) that belongs to the Rho GTPase family, using a deletion mutant. This mutant ${\Delta}Morho2$ exhibited no defects in conidiation and germination but developed only 6% of appressoria in response to a hydrophobic surface when compared to the wild-type progenitor. This result indicates that MoRHO2 plays a role in appressorium development. Furthermore, exogenous cAMP treatment on the mutant led to appressoria that exhibited abnormal morphology on both hydrophobic and hydrophilic surfaces. These outcomes suggested the involvement of MoRHO2 in cAMP-mediated appressorium development. ${\Delta}Morho2$ mutation also delayed the development of appressorium-like structures (ALS) at hyphal tips on hydrophobic surface, which were also abnormally shaped. These results suggested that MoRHO2 is involved in morphological development of appressoria and ALS from conidia and hyphae, respectively. As expected, ${\Delta}Morho2$ mutant was defective in plant penetration, but was still able to cause lesions, albeit at a reduced rate on wounded plants. These results implied that MoRHO2 plays a role in M. oryzae virulence as well.

Keywords

E1PPBG_2018_v34n6_470_f0001.png 이미지

Fig. 1. Domain structure and conserved amino acids of RHO GTPase in M. oryzae. (A) Schematic structure of the small GTP-binding protein domain (IPR005225) in the RHO protein family. The domain structure was predicted using InterProScan. (B) The conserved amino acid sequence alignment of Rho GTPase. G1, G2, G3, G4, G5, switch I, and switch II denote special motifs in the small GTPbinding protein domain. The identity of each protein BLAST search with MoRHO2 is followed by its name.

E1PPBG_2018_v34n6_470_f0002.png 이미지

Fig. 2. Phylogenetic analysis and conserved amino acid sequence alignment of MoRHO2 and homologues from other organisms. (A) Phylogenetic analysis among MoRHO2 and homologues. A phylogenetic tree was generated using a neighbor-joining method based on comparing MoRHO2 and its homologues. (B) The conserved amino acid sequence alignment among MoRHO2 and homologues. The identity of each protein BLAST search with MoRHO2 is followed by its name.

E1PPBG_2018_v34n6_470_f0003.png 이미지

Fig. 3. The expression profile and targeted gene deletion of MoRHO2. (A) The expression profile of MoRHO2 in different developmental stages of M. oryzae. The expression of MoRHO2 was measured during five stages including mycelia (MY), conidia (CO), germinated conidia (GC), appressoria (AP), and infectious hyphae stages in rice leaves (IP). The results were normalized to β-tubulin and presented with a relative value of 1 in MY. (B) The targeted gene knockout of MoRHO2. The knockout strategy used the HPH cassette to replace MoRHO2. (C) The conformation of the MoRHO2 deletion using southern blot analysis. The genomic DNA was digested with HindIII and hybridized with specific probes. (D) Reverse transcription-PCR was used to check the expression of MoRHO2. The total RNA was extracted from wild type, ΔMorho2, and Morho2c samples.

E1PPBG_2018_v34n6_470_f0004.png 이미지

Fig. 4. Appressorium formation on artificial surfaces. (A) Statistical analysis of appressoria formed on the hydrophobic and hydrophilic surface. Appressorium formation was assessed at 48 h after inoculation. (B) The appressorium morphology on a hydrophobic surface. Appressoria were observed after a 6 h incubation. Scale bar = 20 μm.

E1PPBG_2018_v34n6_470_f0005.png 이미지

Fig. 5. An appressorium-like structure (ALS) formed on hyphal tips. Hyphal plugs (5 mm in diameter) of wild type, ΔMorho2, and Morho2c samples were placed on hydrophobic surfaces. Photographs were taken after 24 and 36 h. Scale bar = 50 μm.

E1PPBG_2018_v34n6_470_f0006.png 이미지

Fig. 6. Plant pathogenic assays. (A) The spray assays. The conidial suspension was sprayed onto rice leaves and the leaves were incubated for 7 days. (B) The influence of wounding on disease development. Conidial drops or hyphal plugs (6 mm in diameter) were inoculated onto rice leaves with or without wounding and the leaves were incubated for 2 days.

E1PPBG_2018_v34n6_470_f0007.png 이미지

Fig. 7. Penetration assays. A conidial suspension of the indicated strains was dropped on rice sheath cells. Photographs were taken at 2 days after inoculation. Scar bar = 50 μm.

Table 1. List of primers used in this study.

E1PPBG_2018_v34n6_470_t0001.png 이미지

References

  1. An, B., Li, B., Qin, G. and Tian, S. 2015. Function of small GTPase Rho3 in regulating growth, conidiation and virulence of Botrytis cinerea. Fungal Genet. Biol. 75:46-55. https://doi.org/10.1016/j.fgb.2015.01.007
  2. Arellano, M., Coll, P. M. and Perez, P. 1999. RHO GTPases in the control of cell morphology, cell polarity, and actin localization in fission yeast. Microsc. Res. Tech. 47:51-60. https://doi.org/10.1002/(SICI)1097-0029(19991001)47:1<51::AID-JEMT5>3.0.CO;2-3
  3. Choi, J., Kim, K. S., Rho, H. S. and Lee, Y. H. 2011. Differential roles of the phospholipase C genes in fungal development and pathogenicity of Magnaporthe oryzae. Fungal Genet. Biol. 48:445-455. https://doi.org/10.1016/j.fgb.2011.01.001
  4. Dean, R. A. 1997. Signal pathways and appressorium morphogenesis. Annu. Rev. Phytopathol. 35:211-234. https://doi.org/10.1146/annurev.phyto.35.1.211
  5. Dean, R. A., Talbot, N. J., Ebbole, D. J., Farman, M. L., Mitchell, T. K., Orbach, M. J., Thon, M., Kulkarni, R., Xu, J.-R., Pan, H., Read, N. D., Lee, Y.-H., Carbone, I., Brown, D., Oh, Y. Y., Donofrio, N., Jeong, J. S., Soanes, D. M., Djonovic, S., Kolomiets, E., Rehmeyer, C., Li, W., Harding, M., Kim, S., Lebrun, M.-H., Bohnert, H., Coughlan, S., Butler, J., Calvo, S., Ma, L.-J., Nicol, R., Purcell, S., Nusbaum, C., Galagan, J.-E. and Birren, B. W. 2005. The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986. https://doi.org/10.1038/nature03449
  6. Du, Y., Shi, Y., Yang, J., Chen, X., Xue, M., Zhou, W. and Peng, Y.-L. 2013. A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae. Curr. Genet. 59:33-41. https://doi.org/10.1007/s00294-012-0385-3
  7. Etienne-Manneville, S. and Hall, A. 2002. Rho GTPases in cell biology. Nature 420:629-635. https://doi.org/10.1038/nature01148
  8. Guest, G. M., Lin, X. and Momany, M. 2004. Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis. Fungal Genet. Biol. 41:13-22. https://doi.org/10.1016/j.fgb.2003.08.006
  9. Hamer, J. E. and Talbot, N. J. 1998. Infection-related development in the rice blast fungus Magnaporthe grisea. Curr. Opin. Microbiol. 1:693-697. https://doi.org/10.1016/S1369-5274(98)80117-3
  10. Han, J. H., Lee, H. M., Shin, J. H., Lee, Y. H. and Kim, K. S. 2015. Role of the MoYAK1 protein kinase gene in Magnaporthe oryzae development and pathogenicity. Environ. Microbiol. 17:4672-4689. https://doi.org/10.1111/1462-2920.13010
  11. Hanna, S. and El-Sibai, M. 2013. Signaling networks of Rho GTPases in cell motility. Cell. Signal. 25:1955-1961. https://doi.org/10.1016/j.cellsig.2013.04.009
  12. Huh, A., Dubey, A., Kim, S., Jeon, J. and Lee, Y. H. 2017. MoJMJ1, encoding a histone demethylase containing JmjC domain, is required for pathogenic development of the rice blast fungus, Magnaporthe oryzae. Plant Pathol. J. 33:193-205. https://doi.org/10.5423/PPJ.OA.11.2016.0244
  13. Karnoub, A. E., Symons, M., Campbell, S. L. and Der, C. J. 2004. Molecular basis for Rho GTPase signaling specificity. Breast Cancer Res. Treat. 84:61-71. https://doi.org/10.1023/B:BREA.0000018427.84929.5c
  14. Kim, H. J., Han, J. H., Kim, K. S. and Lee, Y. H. 2014. Comparative functional analysis of the velvet gene family reveals unique roles in fungal development and pathogenicity in Magnaporthe oryzae. Fungal Genet. Biol. 66:33-43. https://doi.org/10.1016/j.fgb.2014.02.011
  15. Kim, K. S. and Lee, Y. H. 2012. Gene expression profiling during conidiation in the rice blast pathogen Magnaporthe oryzae. PLoS ONE 7:e43202. https://doi.org/10.1371/journal.pone.0043202
  16. Kim, S., Park, S. Y., Kim, K. S., Rho, H. S., Chi, M. H., Choi, J., Park, J., Kong, S., Park, J., Goh, J. and Lee, Y. H. 2009. Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae. PLoS Genet. 5:e1000757. https://doi.org/10.1371/journal.pgen.1000757
  17. Kong, L. A., Li, G. T., Liu, Y., Liu, M. G., Zhang, S. J., Yang, J., Zhou, X. Y., Peng, Y. L. and Xu, J. R. 2013. Differences between appressoria formed by germ tubes and appressoriumlike structures developed by hyphal tips in Magnaporthe oryzae. Fungal Genet. Biol. 56:33-41. https://doi.org/10.1016/j.fgb.2013.03.006
  18. Kwon, M. J., Arentshorst, M., Roos, E. D., van den Hondel, C. A., Meyer, V. and Ram, A. F. 2011. Functional characterization of Rho GTPases in Aspergillus niger uncovers conserved and diverged roles of Rho proteins within filamentous fungi. Mol. Microbiol. 79:1151-1167. https://doi.org/10.1111/j.1365-2958.2010.07524.x
  19. Madaule, P. and Axel, R. 1985. A novel ras-related gene family. Cell 41:31-40. https://doi.org/10.1016/0092-8674(85)90058-3
  20. Mahlert, M., Leveleki, L., Hlubek, A., Sandrock, B. and Bolker, M. 2006. Rac1 and Cdc42 regulate hyphal growth and cytokinesis in the dimorphic fungus Ustilago maydis. Mol. Microbiol. 59:567-578. https://doi.org/10.1111/j.1365-2958.2005.04952.x
  21. Martinez-Rocha, A. L., Roncero, M. I., Lopez-Ramirez, A., Marine, M., Guarro, J., Martinez-Cadena, G. and Di Pietro, A. 2008. Rho1 has distinct functions in morphogenesis, cell wall biosynthesis and virulence of Fusarium oxysporum. Cell. Microbiol. 10:1339-1351. https://doi.org/10.1111/j.1462-5822.2008.01130.x
  22. Matia-Gonzalez, A. M. and Rodriguez-Gabriel, M. A. 2011. Slt2 MAPK pathway is essential for cell integrity in the presence of arsenate. Yeast 28:9-17. https://doi.org/10.1002/yea.1816
  23. Nakano, K., Mutoh, T., Arai, R. and Mabuchi, I. 2003. The small GTPase Rho4 is involved in controlling cell morphology and septation in fission yeast. Genes Cells 8:357-370. https://doi.org/10.1046/j.1365-2443.2003.00639.x
  24. Park, J., Park, B., Jung, K., Jang, S., Yu, K., Choi, J., Kong, S., Park, J., Kim, S. Kim, H., Kim, S., Kim, J. F., Blair, J. E., Lee, K., Kang, S. and Lee, Y. H. 2007. CFGP: a web-based, comparative fungal genomics platform. Nucleic Acids Res. 36:D562-D571. https://doi.org/10.1093/nar/gkm758
  25. Rasmussen, C. G. and Glass, N. L. 2005. A Rho-type GTPase, rho-4, is required for septation in Neurospora crassa. Eukaryot. Cell 4:1913-1925. https://doi.org/10.1128/EC.4.11.1913-1925.2005
  26. Richthammer, C., Enseleit, M., Sanchez-Leon, E., Marz, S., Heilig, Y., Riquelme, M. and Seiler, S. 2012. RHO1 and RHO2 share partially overlapping functions in the regulation of cell wall integrity and hyphal polarity in Neurospora crassa. Mol. Microbiol. 85:716-733. https://doi.org/10.1111/j.1365-2958.2012.08133.x
  27. Ridley, A. J. 2001. Rho family proteins: coordinating cell responses. Trends Cell Biol. 11:471-477. https://doi.org/10.1016/S0962-8924(01)02153-5
  28. Ryder, L. S. and Talbot, N. J. 2015. Regulation of appressorium development in pathogenic fungi. Curr. Opin. Plant Biol. 26:8-13.
  29. Saleh, D., Milazzo, J., Adreit, H., Fournier, E. and Tharreau, D. 2014. South-East Asia is the center of origin, diversity and dispersion of the rice blast fungus, Magnaporthe oryzae. New Phytol. 201:1440-1456. https://doi.org/10.1111/nph.12627
  30. Schaefer, A., Reinhard, N. R. and Hordijk, P. L. 2014. Toward understanding RhoGTPase specificity: structure, function and local activation. Small GTPases 5:e968004. https://doi.org/10.4161/21541248.2014.968004
  31. Smithers, C. C. and Overduin, M. 2016. Structural mechanisms and drug discovery prospects of Rho GTPases. Cells 5:26. https://doi.org/10.3390/cells5020026
  32. Talbot, N. J., Ebbole, D. J. and Hamer, J. E. 1993. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5:1575-1590.
  33. Valent, B. 1990. Rice blast as a model system for plant pathology. Phytopathology 80:33-36. https://doi.org/10.1094/Phyto-80-33
  34. Vasara, T., Salusjarvi, L., Raudaskoski, M., Keranen, S., Penttila, M. and Saloheimo, M. 2001. Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei. Mol. Microbiol. 42:1349-1361.
  35. Wittinghofer, A. and Vetter, I. R. 2011. Structure-function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem. 80:943-971. https://doi.org/10.1146/annurev-biochem-062708-134043
  36. Xu, J. R. and Hamer, J. E. 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. https://doi.org/10.1101/gad.10.21.2696
  37. Xu, J. R., Staiger, C. J. and Hamer, J. E. 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. https://doi.org/10.1073/pnas.95.21.12713
  38. Xu, X., Wang, Y., Tian, C. and Liang, Y. 2016. The Colletotrichum gloeosporioides RhoB regulates cAMP and stress response pathways and is required for pathogenesis. Fungal Genet. Biol. 96:12-24. https://doi.org/10.1016/j.fgb.2016.09.002
  39. Yu, J. H., Hamari, Z., Han, K. H., Seo, J. A., Reyes-Dominguez, Y. and Scazzocchio, C. 2004. Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet. Biol. 41:973-981. https://doi.org/10.1016/j.fgb.2004.08.001
  40. Zhang, C., Wang, Y., Wang, J., Zhai, Z., Zhang, L., Zheng, W., Zheng, W., Yu, W., Zhou, J., Lu, G., Shim, W. B. and Wang, Z. 2013. Functional characterization of Rho family small GTPases in Fusarium graminearum. Fungal Genet. Biol. 61:90-99. https://doi.org/10.1016/j.fgb.2013.09.001
  41. Zheng, W., Chen, J., Liu, W., Zheng, S., Zhou, J., Lu, G. and Wang, Z. 2007. A Rho3 homolog is essential for appressorium development and pathogenicity of Magnaporthe grisea. Eukaryot. Cell 6:2240-2250. https://doi.org/10.1128/EC.00104-07
  42. Zhu, L., Zhu, J., Liu, Z., Wang, Z., Zhou, C. and Wang, H. 2017. Host-Induced Gene Silencing of Rice Blast Fungus Magnaporthe oryzae Pathogenicity Genes Mediated by the Brome Mosaic Virus. Genes 8:241. https://doi.org/10.3390/genes8100241