Abstract
Ustilaginoidea virens is an important fungus causing rice false smut, a devastating disease on spikelets of rice. In this study, we identified and characterized two CMGC (CDK/MAPK/GSK3/CLK) kinase genes, UvPmk1 and UvCDC2, in U. virens. Although UvPmk1 and UvCDC2 are, respectively, homologous to Fus3/Kss1 mitogen-activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs), they all have a conserved serine/threonine protein kinase domain. The qRT-PCR analysis of the relative expression of UvPmk1 and UvCDC2 during the infection of U. virens showed that these two genes were highly expressed during infection. UvPmk1 and UvCDC2 knockout mutants exhibited no significant changes in mycelial vegetative growth but decreases in conidiation. In addition, both UvPmk1 and UvCDC2 knockout mutants showed increases in tolerance to hyperosmotic and cell wall stresses, but they, respectively, exhibited decreases and increases in tolerance to oxidative stress compared with the wild-type strain HWD-2. Pathogenicity and infection assays demonstrated the defective growth of infection hyphae and significant loss of virulence in UvPmk1 and UvCDC2 knockout mutants. Taken together, our results demonstrate that UvPmk1 and UvCDC2 play important roles in the conidiation, stress response, and pathogenicity of U. virens.
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References
Ashizawa T, Takahashi M, Arai M, Arie T (2012) Rice false smut pathogen, Ustilaginoidea virens, invades through small gap at the apex of a rice spikelet before heading. J Gen Plant Pathol 78:255–259. https://doi.org/10.1007/s10327-012-0389-3
Booher R, Beach D (1986) Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe. Mol Cell Biol 6(10):3523–3530. https://doi.org/10.1128/MCB.6.10.3523
Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, Read ND, Seiler S, Bell-Pedersen D, Paietta J, Plesofsky N et al (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68(1):1–108. https://doi.org/10.1128/mmbr.68.1.1-108.2004
Bruno KS, Tenjo F, Li L, Hamer JE, Xu JR (2004) Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryot Cell 3(6):1525–1532. https://doi.org/10.1128/EC.3.6.1525-1532.2004
Di Pietro A, García-Maceira FI, Méglecz E, Roncero MIG (2001) A MAP kinase of the vascular wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis. Mol Microbiol 39(5):1140–1152. https://doi.org/10.1111/j.1365-2958.2001.02307.x
Fan J, Yang J, Wang YQ, Li GB, Li Y, Huang F, Wang WM (2016) Current understanding on Villosiclava virens, a unique flower-infecting fungus causing rice false smut disease. Mol Plant Pathol 17(9):1321–1330. https://doi.org/10.1111/mpp.12362
Fan J, Du N, Li L, Li GB, Wang YQ, Zhou YF, Hu XH, Liu J, Zhao JQ, Li Y et al (2019) A core effector UV_1261 promotes Ustilaginoidea virens infection via spatiotemporally suppressing plant defense. Phytopathol Res 1(1):11. https://doi.org/10.1186/s42483-019-0019-5
Fang AF, Gao H, Zhang N, Zheng XH, Qiu SS, Li YJ, Zhou S, Cui FH, Sun WX (2019) A novel effector gene SCRE2 contributes to full virulence of Ustilaginoidea virens to rice. Front Microbiol 10:845. https://doi.org/10.3389/fmicb.2019.00845
Guo WW, Gao YY, Yu ZM, Xiao YH, Zhang ZG, Zhang HF (2019) The adenylate cyclase UvAc1 and phosphodiesterase UvPdeH control the intracellular cAMP level, development, and pathogenicity of the rice false smut fungus Ustilaginoidea virens. Fungal Genet Biol. https://doi.org/10.1016/j.fgb.2019.04.017
Hamel LP, Nicole MC, Duplessis S, Ellis BE (2012) Mitogen-activated protein kinase signaling in plant-interacting fungi: distinct messages from conserved messengers. Plant Cell 24:1327–1351. https://doi.org/10.1105/tpc.112.096156
Hu M, Luo L, Wang S, Liu Y, Li J (2014) Infection processes of Ustilaginoidea virens during artificial inoculation of rice panicles. Euro J Plant Pathol 139:67–77. https://doi.org/10.1007/s10658-013-0364-7
Jia Q, Lv B, Guo MY, Luo CX, Zheng L, Hsiang T, Huang JB (2015a) Effect of rice growth stage, temperature, relative humidity and wetness duration on infection of rice panicles by Villosiclava virens. Eur J Plant Pathol 141:15–25. https://doi.org/10.1007/s10658-014-0516-4
Jia Q, Gu QN, Zheng L, Hsiang T, Luo CX, Huang JB (2015b) Genetic analysis of the population structure of the rice false smut fungus, Villosiclava virens, in China using microsatellite markers mined from a genome assembly. Plant Pathol 64(6):1440–1449. https://doi.org/10.1111/ppa.12373
Jiang C, Zhang X, Liu H, Xu JR (2018) Mitogen-activated protein kinase signaling in plant pathogenic fungi. PLoS Pathog 14(3):e1006875. https://doi.org/10.1371/journal.ppat.1006875
Koiso Y, Li Y, Iwasaki S, Hanaka K, Kobayashi T, Sonoda R, Fujita Y, Yaegashi H, Sato Z (1994) Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens. J Antibiot 47:765–773. https://doi.org/10.7164/antibiotics.47.765
Koyama K, Natori S (1988) Further characterization of seven bis (naphtho-gpyrone) congeners of ustilaginoidins, coloring matters of Claviceps virens (Ustilaginoidea virens). Chem Pharm Bull 36:146–152. https://doi.org/10.1248/cpb.36.146
Koyama K, Ominato K, Natori S, Tashiro T, Tsuruo T (1988) Cytotoxicity and antitumor activities of fungal bis (naphtho-gamma-pyrone) derivatives. J Pharmacobiodyn 11:630–635. https://doi.org/10.1248/bpb1978.11.630
Li YJ, Wang M, Liu ZH, Zhang K, Cui FH, Sun WX (2019) Towards understanding the biosynthetic pathway for ustilaginoidin mycotoxins in Ustilaginoidea virens. Environ Microbiol. https://doi.org/10.1111/1462-2920.14572
Liang Y, Han Y, Wang CF, Jiang C, Xu JR (2018) Targeted deletion of the USTA and UvSLT2 genes efficiently in Ustilaginoidea virens with the CRISPR-Cas9 system. Front Plant Sci 9:699. https://doi.org/10.3389/fpls.2018.00699
Liu HQ, Zhang SJ, Ma JW, Dai YF, Li CH, Lyu XL, Wang CF, Xu JR (2015) Two Cdc2 kinase genes with distinct functions in vegetative and infectious hyphae in Fusarium graminearum. PLoS Pathog 11(6):e1004913. https://doi.org/10.1371/journal.ppat.1004913
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lv B, Zheng L, Liu H, Tang JT, Hsiang T, Huang JB (2016) Use of random T-DNA mutagenesis in identification of gene UvPRO1, a regulator of conidiation, stress response, and virulence in Ustilaginoidea virens. Front Microbiol 7:2086. https://doi.org/10.3389/fmicb.2016.02086
Madrid M, Soto T, Khong HK, Franco A, Vicente J, Pérez P, Gacto M, Cansado J (2006) Stress-induced response, localization, and regulation of the Pmk1 cell integrity pathway in Schizosaccharomyces pombe. J Biol Chem 281(4):2033–2043. https://doi.org/10.1074/jbc.m506467200
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298(5600):1912–1934. https://doi.org/10.1126/science.1075762
Mendenhall MD, Hodge AE (1998) Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62(4):1191–1243
Mey G, Oeser B, Lebrun MH, Tudzynski P (2002) The biotrophic, non-appressorium-forming grass pathogen Claviceps purpurea needs a Fus3/Pmk1 homologous mitogen-activated protein kinase for colonization of rye ovarian tissue. Mol Plant Microbe Interact 15(4):303–312. https://doi.org/10.1094/MPMI.2002.15.4.303
Osmani AH, van Peij N, Mischke M, O’Connell MJ, Osmani SA (1994) A single p34cdc2 protein kinase (encoded by nimXcdc2) is required at G1 and G2 in Aspergillus nidulans. J Cell Sci 107(6):1519–1528
Qiu JH, Meng S, Deng YZ, Kou YJ (2019) Ustilaginoidea Virens: A fungus infects rice flower and threats world rice production. Rice Sci 26:199–206
Rispail N, Soanes DM, Ant C, Czajkowski R, Grunler A, Czajkowski R, Grünler A, Huguet R, Perez-Nadales E, Poli A et al (2009) Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi. Fungal Genet Biol 46:287–298. https://doi.org/10.1016/j.fgb.2009.01.002
Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42(W1):W320–W324. https://doi.org/10.1093/nar/gku316
Sakulkoo W, Osés-Ruiz M, Garcia EO, Soanes DM, Littlejohn GR, Hacker C, Correia A, Valent B, Talbot NJ (2018) A single fungal MAP kinase controls plant cell-to-cell invasion by the rice blast fungus. Science 359(6382):1399–1403. https://doi.org/10.1126/science.aaq0892
Sambrook J, Frisch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Saunders DG, Aves SJ, Talbot NJ (2010) Cell cycle–mediated regulation of plant infection by the rice blast fungus. Plant Cell 22:497–507. https://doi.org/10.1105/tpc.109.072447
Singh B, Wu PYJ (2019) Regulation of the program of DNA replication by CDK: new findings and perspectives. Curr Genet 65:79. https://doi.org/10.1007/s00294-018-0860-6
Song JH, Wei W, Lv B, Lin Y, Yin WX, Peng YL, Schnabel G, Huang JB, Jiang DH, Luo CX (2016) Rice false smut fungus hijacks the rice nutrients supply by blocking and mimicking the fertilization of rice ovary. Environ Microbiol 18:3840–3849. https://doi.org/10.1111/1462-2920.13343
Tang YX, Jin J, Hu DW, Yong ML, Xu Y, He LP (2013) Elucidation of the infection process of Ustilaginoidea virens (teleomorph: Villosiclava virens) in rice spikelets. Plant Pathol 62:1–8. https://doi.org/10.1111/j.1365-3059.2012.02629.x
Turrà D, Segorbe D, Di Pietro A (2014) Protein kinases in plant-pathogenic fungi: conserved regulators of infection. Annu Rev Phytopathol 52:267–288. https://doi.org/10.1146/annurev-phyto-102313-050143
Ubersax JA, Woodbury EL, Quang PN, Paraz M, Blethrow JD, Shah K, Shokat KM, Morgan DO (2003) Targets of the cyclin-dependent kinase Cdk1. Nature 425(6960):859. https://doi.org/10.1038/nature02062
Xie SL, Wang YF, Wei W, Li CY, Liu Y, Qu JS, Meng QH, Lin Y, Yin WX, Luo CX (2019) The bax inhibitor UvBI-1, a negative regulator of mycelial growth and conidiation, mediates stress response and is critical for pathogenicity of the rice false smut fungus Ustilaginoidea virens. Curr Genet. https://doi.org/10.1007/s00294-019-00970-2
Xu JR, Hamer JE (1996) MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. Gene Dev 10(21):2696–2706. https://doi.org/10.1101/gad.10.21.2696
Yin WX, Cui P, Wei W, Lin Y, Luo CX (2017) Genome-wide identification and analysis of the basic leucine zipper (bZIP) transcription factor gene family in Ustilaginoidea virens. Genome 60(12):1051–1059. https://doi.org/10.1139/gen-2017-0089
Yu MN, Yu JJ, Hu JK, Huang L, Wang YH, Yin XL, Nie YF, Meng XK, Wang WD, Liu YF (2015) Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis. Fungal Genet Biol 76:10–19. https://doi.org/10.1016/j.fgb.2015.01.004
Zhang Y, Zhang K, Fang AF, Han YQ, Yang J, Xue MF, Bao JD, Hu WD, Zhou B, Sun XY et al (2014) Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics. Nat Commun 5:3849. https://doi.org/10.1038/ncomms4849
Zhao X, Kim Y, Park G, Xu JR (2005) A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea. Plant Cell 17:1317–1329. https://doi.org/10.1105/tpc.104.029116
Zhao X, Mehrabi R, Xu JR (2007) Mitogen-activated protein kinase pathways and fungal pathogenesis. Eukaryot Cell 6:1701–1714. https://doi.org/10.1128/EC.00216-07
Zheng L, Campbell M, Murphy J, Lam S, Xu JR (2000) The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea. Mol Plant Microbe Interact 13(7):724–732. https://doi.org/10.1094/MPMI.2000.13.7.724
Zheng DW, Wang Y, Han Y, Xu JR, Wang CF (2016) UvHOG1 is important for hyphal growth and stress responses in the rice false smut fungus Ustilaginoidea virens. Sci Rep 6:24824. https://doi.org/10.1038/srep24824
Zheng MT, Ding H, Huang L, Wang YH, Yu MN, Zheng R, Yu JJ, Liu YF (2017) Low-affinity iron transport protein Uvt3277 is important for pathogenesis in the rice false smut fungus Ustilaginoidea virens. Curr Genet 63(1):131–144. https://doi.org/10.1007/s00294-016-0620-4
Acknowledgements
We are grateful to Prof. Wenxian Sun at China Agricultural University for providing the gene deletion vectors pGKO. This work was supported by the National Key Research and Development Program (2017YFD0301400, 2016YFD0300700) and the Fundamental Research Funds for the Central Universities of China (2662018JC051).
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Tang, J., Bai, J., Chen, X. et al. Two protein kinases UvPmk1 and UvCDC2 with significant functions in conidiation, stress response and pathogenicity of rice false smut fungus Ustilaginoidea virens. Curr Genet 66, 409–420 (2020). https://doi.org/10.1007/s00294-019-01029-y
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DOI: https://doi.org/10.1007/s00294-019-01029-y