Current Genetics

, Volume 65, Issue 5, pp 1185–1197 | Cite as

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

  • Songlin Xie
  • Yufu Wang
  • Wei Wei
  • Chongyang Li
  • Yi Liu
  • Jinsong Qu
  • Qianghong Meng
  • Yang Lin
  • Weixiao YinEmail author
  • Yinong Yang
  • Chaoxi Luo
Original Article


Bax inhibitor-1 (BI-1), an evolutionarily conserved protein, is a suppressor of cell death induced by the proapoptotic protein Bax and is involved in the response to biotic and abiotic stress in animals, plants and yeast. Rice false smut caused by Ustilaginoidea virens is one of the destructive rice diseases worldwide. Although BI-1 proteins are widely distributed across filamentous fungi, few of them are functionally characterized. In this study, we identified a BI-1 protein in U. virens, UvBI-1, which contains a predicted Bax inhibitor-1-like family domain and could suppress the cell death induced by Bax. By co-transformation of the CRISPR/Cas9 construct along with donor DNA fragment containing the hygromycin resistance gene, we successfully generated Uvbi-1 deletion mutants. The UvBI-1 deletion showed an increase in mycelia vegetative growth and conidiation, suggesting this gene acts as a negative regulator of the growth and conidiation. In addition, the Uvbi-1 mutants exhibited higher sensitivity to osmotic and salt stress, hydrogen peroxide stress, and cell wall or membrane stress than the wild-type strain. Furthermore, UvBI-1 deletion was found to cause increased production of secondary metabolites and loss of pathogenicity of U. virens. Taken together, our results demonstrate that UvBI-1 plays a negative role in mycelial growth and conidiation, and is critical for stress tolerance, cell wall integrity, secondary metabolites production and pathogenicity of U. virens. Therefore, this study provides new evidence on the conserved function of BI-1 among fungal organisms and other species.


Bax inhibitor-1 Ustilaginoidea virens Hyphal growth Conidiation Stress response Secondary metabolites Pathogenicity 



We thank JinRong Xu from Northwest A&F University, Yangling, China, and Purdue University, West Lafayette, IN, the United States, for the CRISPR-Cas9 system plasmid, Yuanchao Wang from Nanjing Agricultural University for the pGR107 vector and Bax and Daohong Jiang from Huazhong Agricultural University for the complemented vector PCETNS4. The National Natural Science Foundation of China (No. 31701736), the National Key Research and Development Program (2016YFD0300700) and the Fundamental Research Funds for the Central Universities (No. 2662017JC003 and 2662018JC051) supported this research.

Supplementary material

294_2019_970_MOESM1_ESM.docx (361 kb)
Supplementary material 1 (DOCX 360 kb)


  1. Abbas H, Shier W, Cartwright R, Sciumbato G (2014) Ustilaginoidea virens infection of rice in arkansas toxicity of false smut galls, their extracts and the ustiloxin fraction. Am J Plant Sci 5:3166–3176CrossRefGoogle Scholar
  2. Ameisen JC (2002) On the origin, evolution, and nature of programmed cell death: a timeline of four billion years. Cell Death Differ 9:367–393CrossRefGoogle Scholar
  3. Bickle M, Delley PA, Schmidt A, Hall MN (1998) Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J 17:2235–2245CrossRefGoogle Scholar
  4. Borner C (2003) The Bcl-2 protein family: sensors and checkpoints for life-or-death decisions. Mol Immunol 39:615–647CrossRefGoogle Scholar
  5. Cebulski J, Malouin J, Pinches N, Cascio V, Austriaco N (2011) Yeast Bax inhibitor, Bxi1p, is an ER-localized protein that links the unfolded protein response and programmed cell death in Saccharomyces cerevisiae. Plos One 6(6):e20882CrossRefGoogle Scholar
  6. Chae HJ, Ke N, Kim HR, Chen SR, Godzik A, Dickman M, Reed JC (2003) Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 homologs from animals, plants, and yeast. Gene 323:101–113CrossRefGoogle Scholar
  7. Chae HJ, Kim HR, Xu CY, Bailly-Maitre B, Krajewska M, Krajewski S, Banares S, Cui J, Digicaylioglu M, Ke N, Kitada S, Monosov E, Thomas M, Kress CL, Babendure JR, Tsien RY, Lipton SA, Reed JC (2004) BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Mol Cell 15:355–366CrossRefGoogle Scholar
  8. Chen YX, Duan ZB, Chen PL, Shang YF, Wang CS (2015) The Bax inhibitor MrBI-1 regulates heat tolerance, apoptotic-like cell death, and virulence in Metarhizium robertsii. Sci Rep. 5:10625CrossRefGoogle Scholar
  9. Danial NN, Korsmeyer SJ (2004) Cell death: critical control points. Cell 116:205–219CrossRefGoogle Scholar
  10. Du ZQ, Lan JF, Weng YD, Zhao XF, Wang JX (2013) BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish, Procambarus clarkii. Fish Shellfish Immun 35:46–53CrossRefGoogle Scholar
  11. Gaguancela OA, Zuniga LP, Arias AV, Halterman D, Flores FJ, Johansen IE, Wang A, Yamaji Y, Verchot J (2016) The IRE1/bZIP60 Pathway and Bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants. Mol Plant Microbe Interact 29:750–766CrossRefGoogle Scholar
  12. Greenberg JT, Yao N (2004) The role and regulation of programmed cell death in plant-pathogen interactions. Cell Microbiol 6:201–211CrossRefGoogle Scholar
  13. Grzmil M, Thelen P, Hemmerlein B, Schweyer S, Voigt S, Mury D, Burfeind P (2003) Bax Inhibitor-1 is overexpressed in prostate cancer and its specific down-regulation by rna interference leads to cell death in human prostate carcinoma cells. Am J Pathol 163:543–552CrossRefGoogle Scholar
  14. Grzmil M, Kaulfuss S, Thelen P, Hemmerlein B, Schweyer S, Obenauer S, Kang TW, Burfeind P (2006) Expression and functional analysis of Bax inhibitor-I in human breast cancer cells. J Pathol 208:340–349CrossRefGoogle Scholar
  15. Guo M, Guo W, Chen Y, Dong S, Zhang X, Zhang H, Song W, Wang W, Wang Q, Lv R, Zhang Z, Wang Y, Zheng X (2010) The basic leucine zipper transcription factor Moatf1 mediates oxidative stress responses and is necessary for full virulence of the rice blast fungus Magnaporthe oryzae. Mol Plant Microbe Interact 23:1053–1068CrossRefGoogle Scholar
  16. Hamann A, Brust D, Osiewacz HD (2008) Apoptosis pathways in fungal growth, development and ageing. Trends Microbiol 16:276–283CrossRefGoogle Scholar
  17. Heath MC (1998) Apoptosis, programmed cell death and the hypersensitive response. Eur J Plant Pathol 104:117–124CrossRefGoogle Scholar
  18. Henke N, Lisak DA, Schneider L, Habicht J, Pergande M, Methner A (2011) The ancient cell death suppressor BAX inhibitor-1. Cell Calcium 50:251–260CrossRefGoogle Scholar
  19. Huckelhoven R (2004) BAX Inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryotic relatives. Apoptosis 9:299–307CrossRefGoogle Scholar
  20. Jia Q, Lv B, Guo MY, Luo CX, Zheng L, Hsiang T, Huang JB (2015) Effect of rice growth stage, temperature, relative humidity and wetness duration on infection of rice panicles by Villosiclava virens. Eur J Plant Pathol 141:15–25CrossRefGoogle Scholar
  21. Kawai-Yamada M, Jin LH, Yoshinaga K, Hirata A, Uchimiya H (2001) Mammalian Bax-induced plant cell death can be down-regulated by overexpression of Arabidopsis Bax Inhibitor-1 (AtBl-1). Proc Natl Acad Sci USA 98:12295–12300CrossRefGoogle Scholar
  22. Kawai-Yamada M, Ohori Y, Uchimiya H (2004) Dissection of Arabidopsis Bax inhibitor-1 suppressing Bax-, hydrogen peroxide-, and salicylic acid-induced cell death. Plant Cell 16:21–32CrossRefGoogle Scholar
  23. Kotsafti A, Farinati F, Cardin R, Burra P, Bortolami M (2010) Bax Inhibitor-1 down-regulation in the progression of chronic liver diseases. BMC Gastroenterol 10:35CrossRefGoogle Scholar
  24. Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Phys 48:251–275CrossRefGoogle Scholar
  25. Lehmann S, Serrano M, L’Haridon F, Tjamos SE, Metraux JP (2015) Reactive oxygen species and plant resistance to fungal pathogens. Phytochemistry 112:54–62CrossRefGoogle Scholar
  26. Letunic I, Doerks T, Bork P (2015) SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43:D257–D260CrossRefGoogle Scholar
  27. Liang YF, 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 9:699CrossRefGoogle Scholar
  28. Lisak DA, Schacht T, Enders V, Habicht J, Kiviluoto S, Schneider J, Henke N, Bultynck G, Methner A (2015) The transmembrane Bax inhibitor motif (TMBIM) containing protein family: tissue expression, intracellular localization and effects on the ER CA2+-filling state. Biochim Biophys Acta Mol Cell Res 1853:2104–2114CrossRefGoogle Scholar
  29. Lisbona F, Rojas-Rivera D, Thielen P, Zamorano S, Todd D, Martinon F, Glavic A, Kress C, Lin JH, Walter P, Reed JC, Glimcher LH, Hetz C (2009) BAX inhibitor-1 is a negative regulator of the ER stress sensor IRE1 alpha. Mol Cell 33:679–691CrossRefGoogle Scholar
  30. Lu PP, Yu TF, Zheng WJ, Chen M, Zhou YB, Chen J, Ma YZ, Xi YJ, Xu ZS (2018) The wheat Bax inhibitor-1 protein interacts with an aquaporin TaPIP1 and enhances disease resistance in Arabidopsis. Front Plant Sci 9:20CrossRefGoogle Scholar
  31. 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 7:2086CrossRefGoogle Scholar
  32. Marchler-Bauer A, Bo Y, Han LY, He JE, Lanczycki CJ, Lu SN, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang ZX, Yamashita RA, Zhang DC, Zheng CJ, Geer LY, Bryant SH (2017) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45:D200–D203CrossRefGoogle Scholar
  33. Matsumura H, Nirasawa S, Kiba A, Urasaki N, Saitoh H, Ito M, Kawai-Yamada M, Uchimiya H, Terauchi R (2003) Overexpression of Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza sativa L.) cells. Plant J 33:425–434CrossRefGoogle Scholar
  34. Meng J, Sun W, Mao Z, Xu D, Wang X, Lu S, Lai D, Liu Y, Zhou L, Zhang G (2015) Main ustilaginoidins and their distribution in rice false smut balls. Toxins 7:4023–4034CrossRefGoogle Scholar
  35. Qi JS, Wang JL, Gong ZZ, Zhou JM (2017) Apoplastic ROS signaling in plant immunity. Curr Opin Plant Biol 38:92–100CrossRefGoogle Scholar
  36. Robinson KS, Clements A, Williams AC, Berger CN, Frankel G (2011) Bax Inhibitor 1 in apoptosis and disease. Oncogene 30:2391–2400CrossRefGoogle Scholar
  37. Roncero C, Duran A (1985) Effect of calcofluor white and congo red on fungal cell-wall morphogenesis—invivo activation of chitin polymerization. J Bacteriol 163:1180–1185Google Scholar
  38. Rush MC, Shahjahan AKM, Jones JP, Groth DE (2000) Outbreak of false smut of rice in Louisiana. Plant Dis 84:100–100CrossRefGoogle Scholar
  39. Scorrano L, Korsmeyer SJ (2003) Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochem Bioph Res Co. 304:437–444CrossRefGoogle Scholar
  40. Sharon A, Finkelstein A, Shlezinger N, Hatam I (2009) Fungal apoptosis: function, genes and gene function. FEMS Microbiol Rev 33:833–854CrossRefGoogle Scholar
  41. 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 18:3840–3849CrossRefGoogle Scholar
  42. Sun X, Kang S, Zhang Y, Tan X, Yu Y, He H, Zhang X, Liu Y, Wang S, Sun W, Cai L, Li S (2013) Genetic diversity and population structure of rice pathogen Ustilaginoidea virens in China. PLoS One 8:e76879CrossRefGoogle Scholar
  43. Sun W, Dong X, Xu D, Meng J, Fu X, Wang X, Lai D, Zhou L, Liu Y (2016) Preparative separation of main ustilaginoidins from rice false smut balls by high-speed counter-current chromatography. Toxins 8(1):20CrossRefGoogle Scholar
  44. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  45. Tsukui T, Nagano N, Umemura M, Kumagai T, Terai G, Machida M, Asai K (2015) Ustiloxins, fungal cyclic peptides, are ribosomally synthesized in Ustilaginoidea virens. Bioinformatics 31:981–985CrossRefGoogle Scholar
  46. Wang S, Bai Y, Zhou Y, Yao J, Bai J (1998) The pathogen of false smut of rice. Acta Phytopathol Sin 28:19–24 (In Chinese, abstract in English) CrossRefGoogle Scholar
  47. Wang Q, Han C, Ferreira AO, Yu X, Ye W, Tripathy S, Kale SD, Gu B, Sheng Y, Sui Y, Wang X, Zhang Z, Cheng B, Dong S, Shan W, Zheng X, Dou D, Tyler BM, Wang Y (2011) Transcriptional programming and functional interactions within the Phytophthora sojae RXLR effector repertoire. Plant Cell 23:2064–2086CrossRefGoogle Scholar
  48. Wang XJ, Tang CL, Huang XL, Li FF, Chen XM, Zhang G, Sun YF, Han DJ, Kang ZS (2012) Wheat BAX inhibitor-1 contributes to wheat resistance to Puccinia striiformis. J Exp Bot 63:4571–4584CrossRefGoogle Scholar
  49. Watanabe N, Lam E (2009) Bax inhibitor-1, a conserved cell death suppressor, is a key molecular switch downstream from a variety of biotic and abiotic stress signals in plants. Int J Mol Sci 10:3149–3167CrossRefGoogle Scholar
  50. Weis C, Pfeilmeier S, Glawischnig E, Isono E, Pachl F, Hahne H, Kuster B, Eichmann R, Huckelhoven R (2013) Co-immunoprecipitation-based identification of putative BAX INHIBITOR-1-interacting proteins involved in cell death regulation and plant-powdery mildew interactions. Mol Plant Pathol 14:791–802CrossRefGoogle Scholar
  51. Wood PJ, Fulcher RG (1983) Dye interactions—a basis for specific detection and histochemistry of polysaccharides. J Histochem Cytochem 31:823–826CrossRefGoogle Scholar
  52. Xu QL, Reed JC (1998) Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol Cell 1:337–346CrossRefGoogle Scholar
  53. Xu GY, Wang SS, Han SJ, Xie K, Wang Y, Li JL, Liu YL (2017) Plant Bax inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death. Autophagy 13:1161–1175CrossRefGoogle Scholar
  54. 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 41:973–981CrossRefGoogle Scholar
  55. Yu M, Yu J, Hu J, Huang L, Wang Y, Yin X, Nie Y, Meng X, Wang W, Liu Y (2015a) Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis. Fungal Genet Biol 76:10–19CrossRefGoogle Scholar
  56. Yu Y, Xiao JF, Yang YH, Bi CW, Qing L, Tan WZ (2015b) Ss-Bi1 encodes a putative BAX inhibitor-1 protein that is required for full virulence of Sclerotinia sclerotiorum. Physiol Mol Plant Pathol 90:115–122CrossRefGoogle Scholar
  57. Yue HY, Nie SJ, Xing D (2012) Over-expression of Arabidopsis Bax inhibitor-1 delays methyl jasmonate-induced leaf senescence by suppressing the activation of MAP kinase 6. J Exp Bot 63:4463–4474CrossRefGoogle Scholar
  58. Zheng D, Wang Y, Han Y, Xu J-R, Wang C (2016) UvHOG1 is important for hyphal growth and stress responses in the rice false smut fungus Ustilaginoidea virens. Sci Rep 6:24824CrossRefGoogle Scholar
  59. 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 63:131–144CrossRefGoogle Scholar
  60. Zhou YL, Pan YJ, Xie XW, Zhu LH, Xu JL, Wang S, Li ZK (2008) Genetic diversity of rice false smut fungus, Ustilaginoidea virens and its pronounced differentiation of populations in North China. J Phytopathol 156:559–564CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Plant Pathology, College of Plant Science and Technology and the Key Lab of Crop Disease Monitoring and Safety Control in Hubei ProvinceHuazhong Agricultural UniversityWuhanChina
  2. 2.Department of Plant Pathology and Environmental Microbiology, Huck Institute of the Life SciencesThe Pennsylvania State UniversityUniversity ParkUSA

Personalised recommendations