Please wait a minute...
Journal of Integrative Agriculture  2021, Vol. 20 Issue (1): 147-158    DOI: 10.1016/S2095-3119(20)63233-6
Special Issue: 植物病理合辑Plant Protection—Plant Pathology 植物细菌真菌合辑Plant Bacteria/Fungus
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
StKU80, a component in the NHEJ repair pathway, is involved in mycelial morphogenesis, conidiation, appressorium development, and oxidative stress reactions in Exserohilum turcicum
GONG Xiao-dong1*, LIU Yu-wei1*, BI Huan-huan3, YANG Xiao-rong1, HAN Jian-min1, DONG Jin-gao1, 2, GU Shou-qin
1 State Key Laboratory of North China Crop Improvement and Regulation/Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, College of Life Sciences, Hebei Agricultural University, Baoding 071001, P.R.China
2 College of Plant Protection, Hebei Agricultural University, Baoding 071001, P.R.China
3 Botou Vocational College, Cangzhou 062150, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      

同源重组(homologous recombination, HR)和非同源末端连接(nonhomologous end joining, NHEJ)是真核生物两种主要的双链断裂(DSB)修复方法。通常抑制NHEJ中关键组分的活性能够提高靶基因敲除的效率或者影响真核生物的生长和发育。然而,在玉米大斑病菌(Exserohilum turcicum)有关NHEJ途径的作用了解甚少。为了研究玉米大斑病菌中编码NHEJ途径关键组分蛋白Ku80基因的功能,我们在玉米大斑病菌鉴定并分析了该基因对病菌生长发育及致病性调控作用。方法:本研究通过利用同源比对的方法,在玉米大斑病菌中鉴定到与酵母Ku80同源的基因,命名为StKU80,并对该基因进行了相关生物信息分析;利用农杆菌介导的遗传转化技术(ATMT)获得了两株稳定的StKU80基因敲除突变体,并对基因的功能进行了分析。结果:保守结构域分析表明,StKu80包含真核生物的KU70p / KU80p蛋白典型的结构域VWA,Ku78和Ku-PK-bind;进一步的系统发育分析表明,StKu80与来自小麦颖枯病菌(Parastagonospora nodorum)的Ku80(XP_001802136.1)亲缘关系较近,其次是来自红曲霉(Monascus ruber)的Ku80(AGF90044.1);突变体与野生型(WT)菌株相比,突变体的菌丝间隔变的更长,细胞壁较薄,在细胞壁表面的物质变的变少以及细胞中线粒体的含量变多;对突变体致病相关的结构进行分析表明,突变体不产生分生孢子和成熟的附着胞,但是突变体的HT毒素活性与WT类似,表明StKU80影响了病菌了侵染过程,但并未影响病菌的致病力;对StKU80能否参与调控胁迫响应反应分析发现,突变体对由H2O2产生的氧化反应高度敏感,但是对紫外不敏感。结论:StKU80在调控玉米大斑病菌的生长发育、致病性及胁迫响应过程中发挥着重要的作用

Homologous recombination (HR) and nonhomologous end joining (NHEJ) are considered the two main double-strand break (DSB) repair approaches in eukaryotes.  Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes.  However, little is known about the roles of the NHEJ pathway in foliar pathogens.  Here we identified a gene designated StKU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum.  Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in StKu80.  Phylogenetic analysis indicated that StKu80 is most closely related to Ku80 (XP_001802136.1) from Parastagonospora nodorum, followed by Ku80 (AGF90044.1) from Monascus ruber.  Furthermore, the gene knockout mutants ΔStKU80-1 and ΔStKU80-2 were obtained.  These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type (WT) strain but similar HT-toxin activity.  The mutants did not produce conidia and mature appressoria.  On the other hand, the mutants were highly sensitive to H2O2, but not to ultraviolet radiation.  In summary, the StKU80 plays devious roles in regulating the development of E.?turcicum.
Keywords:  Exserohilum turcicum        pathogenicity        gene knockout        growth and development  
Received: 19 January 2020   Accepted:
Fund: This work was supported by the National Natural Science Foundation of China (31701741 and 31671983) and the Natural Science Foundation of Hebei Province, China (C2016204164 and C2019204211).
Corresponding Authors:  Correspondence DONG Jin-gao, Tel: +86-312-7528266, E-mail:; GU Shou-qin, Tel: +86-312-7528876, E-mail:    
About author:  GONG Xiao-dong, E-mail:; LIU Yu-wei, E-mail:; * These authors contributed equally to this study.

Cite this article: 

GONG Xiao-dong, LIU Yu-wei, BI Huan-huan, YANG Xiao-rong, HAN Jian-min, DONG Jin-gao, GU Shou-qin. 2021. StKU80, a component in the NHEJ repair pathway, is involved in mycelial morphogenesis, conidiation, appressorium development, and oxidative stress reactions in Exserohilum turcicum. Journal of Integrative Agriculture, 20(1): 147-158.

Aylon Y, Kupiec M. 2004. DSB repair: The yeast paradigm. DNA Repair, 3, 797–815.
Chan D W, Ye R, Veillette C J, Lees-Miller S P. 1999. DNA-dependent protein kinase phosphorylation sites in Ku 70/80 heterodimer. Biochemistry, 38, 1819–1828.
Choquer M, Robin G, Le Pêcheur P, Giraud C, Levis C, Viaud M. 2008. Ku70 or Ku80 deficiencies in the fungus Botrytis cinerea facilitate targeting of genes that are hard to knock out in a wild-type context. FEMS Microbiology Letters, 289, 225–232.
Daley J M, Palmbos P L, Wu D, Wilson T E. 2005. Nonhomologous end joining in yeast. Annual Review of Genetics, 39, 431–451.
Downs J A, Jackson S P. 2004. A means to a DNA end: The many roles of Ku. Nature Reviews Molecular Cell Biology 5, 367–368.
Fell V L, Schild-Poulter C. 2015. The Ku heterodimer: Function in DNA repair and beyond. Mutation Research/Reviews in Mutation Research, 763, 15–29.
Feng J, Li W, Hwang S F, Gossen B D, Strelkov S E. 2012. Enhanced gene replacement frequency in KU70 disruption strain of Stagonospora nodorum. Microbiological Research,  167, 173–178.
Fisher T S, Zakian V A. 2005. Ku: A multifunctional protein involved in telomere maintenance. DNA Repair, 4, 1215–1226.
Gandía M, Xu S, Font C, Marcos J F. 2016. Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum. Fungal Biology, 120, 317–323.
van Gent D C, Hoeijmakers J H, Kanaar R. 2001. Chromosomal stability and the DNA double-stranded break connection. Nature Reviews Genetics, 2, 196–206.
Gong C, Bongiorno P, Martins A, Stephanou N C, Zhu H, Shuman S, Glickman M S. 2005. Mechanism of nonhomologous end-joining in mycobacteria: A low-fidelity repair system driven by Ku, ligase D and ligase C. Nature Structural & Molecular Biology, 12, 304–312.
Gong X D, Feng S Z, Zhao J, Tang C, Tian L, Fan Y S, Cao Z Y, Hao Z M, Jia H, Zang J P, Zhang Y F, Han J M, Gu S Q, Dong J G. 2017. StPBS2, a MAPK kinase gene, is involved in determining hyphal morphology, cell wall development, hypertonic stress reaction as well as the production of secondary metabolites in northern corn leaf blight pathogen Setosphaeria turcica. Microbiological Research, 201, 30–38.
Grigoriev I V, Nordberg H, Shabalov I, Aerts A, Cantor M, Goodstein D, Kuo A, Minovitsky S, Nikitin R, Ohm R A, Otillar R, Poliakov A, Ratnere I, Riley R, Smirnova T, Rokhsar D, Dubchak I. 2012. The genome portal of the department of energy joint genome institute. Nucleic Acids Research, 40, D26–D32.
Gu S Q, Li P, Wu M, Hao Z M, Gong X D, Zhang X Y, Tian L, Zhang P, Wang Y, Cao Z Y, Fan Y S, Han J M, Dong J G. 2014. StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration. Microbiological Research,  169, 817–823.
Gu Y, Seidl K J, Rathbun G A, Zhu C, Manis J P, van der Stoep N, Davidson L, Cheng H L, Sekiguchi J M, Frank K, Stanhope-Baker P, Schlissel M S, Roth D B, Alt F W. 1997. Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity, 7, 653–665.
Haarmann T, Lorenz N, Tudzynski P. 2008. Use of a nonhomologous end joining deficient strain (Δku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Genetics and Biology, 45, 35–44.
Haber J E. 2006. Transpositions and translocations induced by site-specific double-strand breaks in budding yeast. DNA Repair, 5, 998–1009.
He Y, Liu Q, Shao Y C, Chen F S. 2013. Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7. Applied Microbiology and Biotechnology, 97, 4965–4976.
Hefferin M L, Tomkinson A E. 2005. Mechanism of DNA double-strand break repair by non-homologous end joining. DNA Repair, 4, 639–648.
Hoff B, Kamerewerd J, Sigl C, Zadra I, Kück U. 2010. Homologous recombination in the antibiotic producer Penicillium chrysogenum: Strain ΔPcku70 shows up-regulation of genes from the HOG pathway. Applied Microbiology and Biotechnology, 85, 1081–1094.
Jackson S P, Bartek J. 2009. The DNA-damage response in human biology and disease. Nature, 461, 1071–1078.
Kito H, Fujikawa T, Moriwaki A, Tomono A, Izawa M, Kamakura T, Ohashi M, Sato H, Abe K, Nishimura M. 2008. MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea. Fungal Genetics and Biology, 45, 1543–1551.
Koh C M J, Liu Y B, Moehninsi, Du M G, Ji L H. 2014. Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides. BMC Microbiology, 14, 50.
Kotze R G, van der Merwe C F, Crampton B G, Kritzinger Q. 2019. A histological assessment of the infection strategy of Exserohilum turcicum in maize. Plant Pathology, 68, 504–512.
Krappmann S. 2007. Gene targeting in filamentous fungi: The benefits of impaired repair. Fungal Biology Reviews, 21, 25–29.
Kück U, Hoff B. 2010. New tools for the genetic manipulation of filamentous fungi. Applied Microbiology and Biotechnology,  86, 51–62.
Li P, Gong X D, Jia H, Fan Y S, Zhang Y F, Cao Z Y, Hao Z M, Han J M, Gu S Q, Dong J G. 2016. MAP kinase gene STK1 is required for hyphal, conidial, and appressorial development, toxin biosynthesis, pathogenicity, and hypertonic stress response in the plant pathogenic fungus Setosphaeria turcica. Journal of Integrative Agriculture, 15, 2786–2794.
Lieber M R. 2008. The mechanism of human nonhomologous DNA end joining. The Journal of Biological Chemistry, 283, 1–5.
Lieber M R. 2010. The mechanism of double-strand dna break repair by the nonhomologous dna end-joining pathway. Annual Review of Biochemistry, 79, 181–211.
Lu G Q, Duan J Z, Shu S , Wang X X, Gao L L, Guo J, Zhang Y. 2016. Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining. Proceedings of the National Academy of Sciences of the United States of America, 113, 1256–1260.
Maruyama J I, Kitamoto K. 2008. Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (ΔligD) in Aspergillus oryzae. Biotechnology Letters, 30, 1811–1817.
Nakazawa T, Ando Y, Kitaaki K, Nakahori K, Kamada T. 2011. Efficient gene targeting in ΔCc.ku70 or ΔCc.lig4 mutants of the agaricomycete Coprinopsis cinerea. Fungal Genetics and Biology, 48, 939–946.
Ninomiya Y, Suzuki K, Ishii C, Inoue H. 2004. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proceedings of the National Academy of Sciences of the United States of America, 101, 12248–12253.
Ohm R A, Feau N, Henrissat B, Schoch C L, Horwitz B A, Barry K W, Condon B J, Copeland A C, Dhillon B, Glaser F, Hesse C N, Kosti I, LaButti K, Lindquist E A, Lucas S, Salamov A A, Bradshaw R E, Ciuffetti L, Hamelin C R, Kema H J G, et al. 2012. Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi. PLoS Pathogens, 8, e1003037.
Pastink A, Eeken J C, Lohman P H. 2001. Genomic integrity and the repair of double-strand DNA breaks. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 480, 37–50.
Ruiz-Roldán M C, Maier F J, Schäfer W. 2001. PTK1, a mitogen-activated-protein kinase gene, is required for conidiation, appressorium formation, and pathogenicity of pyrenophora teres on barley. Molecular Plant-Microbe Interactions, 14, 116–125.
De Sena-Tomás C, Yu E Y, Calzada A, Holloman W K, Lue N F, Pérez-Martín J. 2015. Fungal Ku prevents permanent cell cycle arrest by suppressing DNA damage signaling at telomeres. Nucleic Acids Research, 43, 2138–2151.
Shen S, Hao Z M, Gu S Q, Wang J J, Cao Z Y, Li Z Y, Wang Q, Li P, Hao J, Dong J G. 2013. The catalytic subunit of cAMP-dependent protein kinase A StPKA-c contributes to conidiation and early invasion in the phytopathogenic fungus Setosphaeria turcica. FEMS Microbiology Letters, 343, 135–144.
Shrivastav M, De Haro L P, Nickoloff J A. 2007. Regulation of DNA double-strand break repair pathway choice. Cell Research, 18, 134.
Shuman S, Glickman M S. 2007. Bacterial DNA repair by non-homologous end joining. Nature Reviews Microbiology, 5, 852–861.
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. Molecular Biology and Evolution, 28, 2731–2739.
Thompson L H. 2012. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography. Mutation Research/Reviews in Mutation Research, 751, 158–246.
Ushimaru T, Terada H, Tsuboi K, Kogou Y, Sakaguchi A, Tsuji G, Kubo Y. 2010. Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and agrobacterium tumefaciens-mediated gene transfer. Molecular Genetics and Genomics, 284, 357–371.
Villalba F, Collemare J, Landraud P, Lambou K, Brozek V, Cirer B, Morin D, Bruel C, Beffa R, Lebrun M H. 2008. Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining. Fungal Genetics and Biology, 45, 68–75.
Vogel H, Lim D S, Karsenty G, Finegold M, Hasty P. 1999. Deletion of Ku86 causes early onset of senescence in mice. Proceedings of the National Academy of Sciences of the United States of America, 96, 10770–10775.
Walker J R, Corpina R A, Goldberg J. 2001. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature, 412, 607.
Wang N Y, Yang S L, Lin C H, Chung K R. 2011. Gene inactivation in the citrus pathogenic fungus Alternaria alternata defect at the Ku70 locus associated with non-homologous end joining. World Journal of Microbiology and Biotechnology, 27, 1817–1826.
Weld R J, Plummer K M, Carpenter M A, Ridgway H J. 2006. Approaches to functional genomics in filamentous fungi. Cell Research, 16, 31–44.
Woods D S, Sears C R, Turchi J J. 2015. Recognition of DNA termini by the C-terminal region of the Ku80 and the DNA-dependent protein kinase catalytic subunit. PLoS ONE,  10, e0127321.
Xu Q, Zhu C Y, Wang M S, Sun X P, Li H Y. 2014. Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum. Journal of Zhejiang University (Science B: Biomedicine & Biotechnology), 15, 116–124.
Zhang S R, Hao Z M, Wang L H, Shen S, Cao Z Y, Xin Y Y, Hou M L, Gu S Q, Han J M, Dong J G. 2012. StRas2 regulates morphogenesis, conidiation and appressorium development in Setosphaeria turcica. Microbiological Research, 167, 478–486.
[1] LIU Na, LIAN Sen, ZHOU Shan-yue, WANG Cai-xia, REN Wei-chao, LI Bao-hua. Involvement of the autophagy-related gene BdATG8 in development and pathogenicity in Botryosphaeria dothidea[J]. >Journal of Integrative Agriculture, 2022, 21(8): 2319-2328.
[2] ZHANG Li-mei, CHEN Shu-ting, QI Min, CAO Xue-qi, LIANG Nan, LI Qian, TANG Wei, LU Guo-dong, ZHOU Jie, YU Wen-ying, WANG Zong-hua, ZHENG Hua-kun. The putative elongator complex protein Elp3 is involved in asexual development and pathogenicity by regulating autophagy in the rice blast fungus[J]. >Journal of Integrative Agriculture, 2021, 20(11): 2944-2956.
[3] ZHANG Yong-sheng, LU Dan, LIU Yu-wen, YI Guo-qiang, TANG Zhong-lin . The untold story between enhancers and skeletal muscle development[J]. >Journal of Integrative Agriculture, 2020, 19(9): 2137-2149.
[4] PAN Li-jun, LU Lin, LIU Yu-ping, WEN Sheng-xian, ZHANG Zeng-yan. The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat[J]. >Journal of Integrative Agriculture, 2020, 19(8): 2044-2055.
[5] Lü Wu-yun, YANG Nan, XU Zhe, DAI Han, TANG Shuai, WANG Zheng-yi. FgHAT2 is involved in regulating vegetative growth, conidiation, DNA damage repair, DON production and virulence in Fusarium graminearum[J]. >Journal of Integrative Agriculture, 2020, 19(7): 1813-1824.
[6] Bongekile NGOBESE, Oliver Tendayi ZISHIRI, Mohamed Ezzat EL ZOWALATY. Molecular detection of virulence genes in Campylobacter species isolated from livestock production systems in South Africa[J]. >Journal of Integrative Agriculture, 2020, 19(6): 1656-1670.
[7] CHEN Bin, TIAN Yan-li, ZHAO Yu-qiang, WANG Jia-nan, XU Zhi-gang, LI Xiang, HU Bai-shi. Bleeding canker of pears caused by Dickeya fangzhongdai: Symptoms, etiology and biology[J]. >Journal of Integrative Agriculture, 2020, 19(4): 889-897.
[8] SONG Su-qin, Lü Zhuo, WANG Jing, ZHU Jing, GU Mei-ying, TANG Qi-yong, ZHANG Zhi-dong, WANG Wei, ZHANG Li-juan, WANG Bo. First report of a new potato disease caused by Galactomyces candidum F12 in China[J]. >Journal of Integrative Agriculture, 2020, 19(10): 2470-2476.
[9] WU Kai-li, CHEN Wei-zhong, YANG Shuai, WEN Ya, ZHENG Yu-ru, Wilfred Mabeche Anjago, YUN Ying-zi, WANG Zong-hua.
Isolation and identification of Fusarium oxysporum f. sp. cubense in Fujian Province, China
[J]. >Journal of Integrative Agriculture, 2019, 18(8): 1905-1913.
[10] CHANG Ji-tao, YU De-bin, LIANG Jian-bin, CHEN Jia, WANG Jian-fa, WANG Fang, JIANG Zhi-gang, HE Xi-jun, WU Rui, YU Li. Mycoplasma leachii causes bovine mastitis: Evidence from clinical symptoms, histopathology and immunohistochemistry[J]. >Journal of Integrative Agriculture, 2019, 18(1): 160-168.
[11] JIA Xiao-hui, FU Jun-fan, WANG Wen-hui, CUI Jian-chao, DU Yan-min, ZHOU Ru-jun, SUN Pingping. First report of Athelia bombacina causing postharvest fruit rot on pear[J]. >Journal of Integrative Agriculture, 2018, 17(11): 2596-2599.
[12] WANG Li-min, ZHANG Yi-fan, DU Zhen-lin, Kang Rui-jiao, CHEN Lin-lin, XING Xiao-ping, YUAN Hong-xia, Ding Sheng-li, LI Hong-lian. FpPDE1 function of Fsarium pseudograminearum on pathogenesis in wheat[J]. >Journal of Integrative Agriculture, 2017, 16(11): 2504-2512.
[13] WEN Chu, ZHONG Qi, ZHANG Jia-dong, LU Jian-shan, ZHANG Li-xin, YUAN Xi-min, GAN Menghou, CAI Xue-peng, ZHANG Guo-zhong. Sequence and phylogenetic analysis of chicken reoviruses in China[J]. >Journal of Integrative Agriculture, 2016, 15(8): 1846-1855.
[14] HUANG Min, LIN Li, WU Yi-xin, Honhing Ho, HE Peng-fei, LI Guo-zhi, HE Peng-bo, XIONG Guo-ru, YUAN Yuan, HE Yue-qiu. Pathogenicity of Klebsiella pneumonia (KpC4) infecting maize and mice[J]. >Journal of Integrative Agriculture, 2016, 15(7): 1510-1520.
[15] LI Po, GONG Xiao-dong, JIA Hui, FAN Yong-shan, ZHANG Yun-feng, CAO Zhi-yan, HAO Zhi-min, HAN Jian-min, GU Shou-qin, DONG Jin-gao. MAP kinase gene STK1 is required for hyphal, conidial, and appressorial development, toxin biosynthesis, pathogenicity, and hypertonic stress response in the plant pathogenic fungus Setosphaeria turcica[J]. >Journal of Integrative Agriculture, 2016, 15(12): 2786-2794.
No Suggested Reading articles found!