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Journal of Integrative Agriculture  2021, Vol. 20 Issue (7): 1858-1870    DOI: 10.1016/S2095-3119(20)63353-6
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Extracellular superoxide dismutase VdSOD5 is required for virulence in Verticillium dahliae
TIAN Li1, HUANG Cai-min1, ZHANG Dan-dan2, LI Ran2, CHEN Jie-yin2, SUN Wei-xia1, QIU Nian-wei1, DAI Xiao-feng
1 College of Life Science, Qufu Normal University, Qufu 273165, P.R.China
2 State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
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产生活性氧(reactive oxygen species, ROS)是植物防御病原菌的重要途径。为对抗这种攻击,病原菌通常表达超氧化物歧化酶(superoxide dismutases, SODs)来清除寄主植物产生的ROS。大丽轮枝菌(Verticillium dahliae)是引发寄主植物黄萎病的重要土传性病原真菌,前期研究发现大丽轮枝菌Vd991在寄主棉花组织诱导下,胞外超氧化物歧化酶VdSOD5丰度显著提高。暗示超氧化物歧化酶在大丽轮枝菌侵染寄主过程中发挥重要作用,但其是否具有清除ROS及致病功能尚不清楚。因此,本研究拟系统解析大丽轮枝菌超氧化物歧化酶VdSOD5在清除寄主ROS的功能及其在致病性方面发挥的作用。序列分析显示VdSOD5编码蛋白为仅具有Cu2+结合位点的超氧化物歧化酶。酵母信号肽捕获系统证明VdSOD5蛋白N端肽段具有介导蛋白分泌的功能。利用同源重组技术构建了VdSOD5基因缺失突变体(ΔVdSOD5-1/2)。氯化硝基四氮唑蓝(p-Nitro-Blue Tetrazolium, NBT)还原法检测表明ΔVdSOD5培养液和菌体的SOD活性较野生型菌株分别下降了74%和28%。ΔVdSOD5对胞内活性氧产生剂甲萘醌的敏感性与野生型菌株相似。NBT染色表明VdSOD5在病原侵染过程中具有降解棉花根系超氧化物的能力。VdSOD5的转录在侵染棉花根部的早期显著上调。VdSOD5缺失不影响大丽轮枝菌营养生长、碳源利用和产孢量。蘸根接种实验表明ΔVdSOD5 对棉花的致病力较野生型菌株显著下降,定量PCR显示ΔVdSOD5在寄主棉花体内的生物量较野生型菌株降低了30%。VdSOD5是大丽轮枝菌带有Cu2+结合位点的胞外超氧化物歧化酶。VdSOD5缺失不影响大丽轮枝菌生长发育,其在侵染过程中通过降解寄主ROS而发挥解毒功能,从而促进大丽轮枝菌对寄主棉花的侵染。

Plants produce reactive oxygen species (ROS) to defend pathogens.  To counteract this attack, certain pathogens express superoxide dismutases (SODs) to scavenge host-derived ROS.  However, the roles of SODs in Verticillium dahliae, an important vascular pathogen, are not clear.  Our previous study has shown that a putative extracellular SOD (VdSOD5) of V. dahliae is significantly induced by culturing in cotton tissues, suggesting that VdSOD5 may play an important role in host–pathogen interactions and virulence.  Here, we showed that VdSOD5 encoded a superoxide dismutase with a co-factor copper-binding site and a functional signal peptide that can conduct protein secretion in an invertase-mutated yeast strain.  The mutations in VdSOD5 (ΔVdSOD5) did not change the normal vegetative growth and conidial production but reduced the virulence of V. dahliae on susceptible host cotton.  Further studies showed that the transcription of VdSOD5 was significantly up-regulated during the early stage of infection, and the loss-of-function of VdSOD5 decreased culture filtrate and fungal tissue SOD activities of V. dahliae by 74 and 28%, respectively.  Compared to the wild-type strain Vd991, the ΔVdSOD5 showed the same sensitivity to the intracellular ROS generator menadione.  Furthermore, nitroblue tetrazolium (NBT) staining demonstrated that VdSOD5 functioned in the detoxification of superoxides generated by host roots during infection.  These results suggest that VdSOD5 of V. dahliae is an important virulence factor, secreted out of cells to combat host-derived ROS. 
Keywords:  Verticillium dahliae        superoxide dismutase        secretion        virulence        ROS detoxification  
Received: 08 March 2020   Accepted:
Fund: This work was supported by the National Natural Science Foundation of China (31501588, 31972228, and 31970142).
Corresponding Authors:  Correspondence QIU Nian-wei, Tel: +86-537-4456415, E-mail:; DAI Xiao-feng, Tel: +86-10-62813566, E-mail:   
About author:  TIAN Li, E-mail:

Cite this article: 

TIAN Li, HUANG Cai-min, ZHANG Dan-dan, LI Ran, CHEN Jie-yin, SUN Wei-xia, QIU Nian-wei, DAI Xiao-feng. 2021. Extracellular superoxide dismutase VdSOD5 is required for virulence in Verticillium dahliae. Journal of Integrative Agriculture, 20(7): 1858-1870.

Abreu I A, Cabelli D E. 2010. Superoxide dismutases - a review of the metal-associated mechanistic variations. Biochimica et Biophysica Acta, 1804, 263–274.
Adachi T, Yamamoto M, Hara H, Masuda K, Mitsui N, Oh-ishi T, Okazaki M. 2001. Extracellular-superoxide dismutase in cerebrospinal fluid from infants/children. Clinica Chimica Acta, 308, 191–193.
Apel K, Hirt H. 2004. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology, 55, 373–399.
Armenteros J J A, Tsirigos K D, Sonderby C K, Petersen T N, Winther O, Brunak S, von Heijne G, Nielsen H. 2019. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nature Biotechnology, 37, 420–423.
Beauchamp C, Fridovich I. 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, 44, 276–287.
Benov L, Chang L Y, Day B, Fridovich I. 1995. Copper, zinc superoxide dismutase in Escherichia coli: Periplasmic localization. Archives of Biochemistry and Biophysics, 319, 508–511.
Bournonville C F, Diaz-Ricci J C. 2011. Quantitative determination of superoxide in plant leaves using a modified NBT staining method. Phytochemical Analysis, 22, 268–271.
Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.
Broxton C N, Culotta V C. 2016. SOD enzymes and microbial pathogens: surviving the oxidative storm of infection. PLoS Pathogens, 12, e1005295.
Chen J Y, Liu C, Gui Y J, Si K W, Zhang D D, Wang J, Short D P G, Huang J Q, Li N Y, Liang Y, Zhang W Q, Yang L, Ma X F, Li T G, Zhou L, Wang B L, Bao Y M, Subbarao K V, Zhang G Y, Dai X F. 2018. Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium. New Phytologist, 217, 756–770.
Chen J Y, Xiao H L, Gui Y J, Zhang D D, Li L, Bao Y M, Dai X F. 2016. Characterization of the Verticillium dahliae exoproteome involves in pathogenicity from cotton-containing medium. Frontiers in Microbiology, 7, 1709.
Chmielowska J, Veloso J, Gutierrez J, Silvar C, Diaz J. 2010. Cross-protection of pepper plants stressed by copper against a vascular pathogen is accompanied by the induction of a defence response. Plant Science, 178, 176–182.
Fradin E F, Thomma B P. 2006. Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum. Molecular Plant Pathology, 7, 71–86.
Fridovich I. 1995. Superoxide radical and superoxide dismutases. Annual Review of Biochemistry, 64, 97–112.
Frohner I E, Bourgeois C, Yatsyk K, Majer O, Kuchler K. 2009. Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance. Molecular Microbiology, 71, 240–252.
Gayoso C, Pomar F, Novo-Uzal E, Merino F, de Ilarduya O M. 2010. The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression. BMC Plant Biology, 10, 232.
Giles S S, Batinic-Haberle I, Perfect J R, Cox G M. 2005. Cryptococcus neoformans mitochondrial superoxide dismutase: An essential link between antioxidant function and high-temperature growth. Eukaryotic Cell, 4, 46–54.
Gleason J E, Galaleldeen A, Peterson R L, Taylor A B, Holloway S P, Waninger-Saroni J, Cormack B P, Cabelli D E, Hart P J, Culotta V C. 2014. Candida albicans SOD5 represents the prototype of an unprecedented class of Cu-only superoxide dismutases required for pathogen defense. Proceedings of the National Academy of Sciences of the United States of America, 111, 5866–5871.
Gui Y J, Chen J Y, Zhang D D, Li N Y, Li T G, Zhang W Q, Wang X Y, Short D P G, Li L, Guo W, Kong Z Q, Bao Y M, Subbarao K V, Dai X F. 2017. Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1.
Environmental Microbiology, 19, 1914–1932.
Gui Y J, Zhang W Q, Zhang D D, Zhou L, Short D P G, Wang J, Ma X F, Li T G, Kong Z Q, Wang B L, Wang D, Li N Y, Subbarao K V, Chen J Y, Dai X F. 2018. A Verticillium dahliae extracellular cutinase modulates plant immune responses. Molecular Plant–Microbe Interactions, 31, 260–273.
Hodgkinson V, Petris M J. 2012. Copper homeostasis at the host–pathogen interface. Journal of Biological Chemistry, 287, 13549–13555.
Jacobs K A, Collins-Racie L A, Colbert M, Duckett M, Golden-Fleet M, Kelleher K, Kriz R, LaVallie E R, Merberg D, Spaulding V, Stover J, Williamson M J, McCoy J M. 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene, 198, 289–296.
Kawamura F, Hirashima N, Furuno T, Nakanishi M. 2006. Effects of 2-methyl-1,4-naphtoquinone (menadione) on cellular signaling in RBL-2H3 cells. Biological & Pharmaceutical Bulletin, 29, 605–607.
Khang C H, Park S Y, Lee Y H, Kang S. 2005. A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genetics and Biology, 42, 483–492.
Klosterman S J, Atallah Z K, Vallad G E, Subbarao K V. 2009. Diversity, pathogenicity, and management of verticillium species. Annual Review of Phytopathology, 47, 39–62.
Kuo W Y, Huang C H, Liu A C, Cheng C P, Li S H, Chang W C, Weiss C, Azem A, Jinn T L. 2013. CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts. New Phytologist, 197, 99–110.
Lambou K, Lamarre C, Beau R, Dufour N, Latge J P. 2010. Functional analysis of the superoxide dismutase family in Aspergillus fumigatus. Molecular Microbiology, 75, 910–923.
Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J, Higgins D G. 2007. Clustal W and clustal X version 2.0. Bioinformatics, 23, 2947–2948.
Li F, Shi H Q, Ying S H, Feng M G. 2015. Distinct contributions of one Fe- and two Cu/Zn-cofactored superoxide dismutases to antioxidation, UV tolerance and virulence of Beauveria bassiana. Fungal Genetics and Biology, 81, 160–171.
Li J J, Zhou L, Yin C M, Zhang D D, Klosterman S J, Wang B L, Song J, Wang D, Hu X P, Subbarao K V, Chen J Y, Dai X F. 2019. The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection. Environmental Microbiology, 21, 4852–4874.
Liu J, Guan T, Zheng P, Chen L, Yang Y, Huai B, Li D, Chang Q, Huang L, Kang Z. 2016. An extracellular Zn-only superoxide dismutase from Puccinia striiformis confers enhanced resistance to host-derived oxidative stress. Environmental Microbiology, 18, 4118–4135.
Liu S Y, Chen J Y, Wang J L, Li L, Xiao H L, Adam S M, Dai X F. 2013. Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae. Gene, 529, 307–316.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25, 402–408.
Lyons T W, Reinhard C T, Planavsky N J. 2014. The rise of oxygen in Earth’s early ocean and atmosphere. Nature, 50, 307–315.
Martchenko M, Alarco A M, Harcus D, Whiteway M. 2004. Superoxide dismutases in Candida albicans: Transcriptional regulation and functional characterization of the hyphal-induced SOD5 gene. Molecular Biology of the Cell, 15, 456–467.
Miller A F. 2012. Superoxide dismutases: Ancient enzymes and new insights. FEBS Letters, 586, 585–595.
Moore S, de Vries O M, Tudzynski P. 2002. The major Cu, Zn SOD of the phytopathogen Claviceps purpurea is not essential for pathogenicity. Molecular Plant Pathology, 3, 9–22.
Mullins E D, Chen X, Romaine P, Raina R, Geiser D M, Kang S. 2001. Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 91, 173–180.
Narasipura S D, Chaturvedi V, Chaturvedi S. 2005. Characterization of Cryptococcus neoformans variety gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence. Molecular Microbiology, 55, 1782–1800.
Okado-Matsumoto A, Fridovich I. 2001. Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu, Zn-SOD in mitochondria. Journal of Biological Chemistry, 276, 38388–38393.
Pierleoni A, Martelli P L, Casadio R. 2008. PredGPI: A GPI-anchor predictor. BMC Bioinformatics, 9, 392.
Potter S Z, Zhu H, Shaw B F, Rodriguez J A, Doucette P A, Sohn S H, Durazo A, Faull K F, Gralla E B, Nersissian A M, Valentine J S. 2007. Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein. Journal of the American Chemical Society, 129, 4575–4583.
Priya B, Premanandh J, Dhanalakshmi R T, Seethalakshmi T, Uma L, Prabaharan D, Subramanian G. 2007. Comparative analysis of cyanobacterial superoxide dismutases to discriminate canonical forms. BMC Genomics, 8, 435.
Robinett N G, Peterson R L, Culotta V C. 2018. Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains. Journal of Biological Chemistry, 293, 4636–4643.
Rolke Y, Liu S, Quidde T, Williamson B, Schouten A, Weltring K M, Siewers V, Tenberge K B, Tudzynski B, Tudzynski P. 2004. Functional analysis of H2O2-generating systems in Botrytis cinerea: The major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Molecular Plant Pathology, 5, 17–27.
Santhanam P, van Esse H P, Albert I, Faino L, Nurnberger T, Thomma B P. 2013. Evidence for functional diversification within a fungal NEP1-like protein family. Molecular Plant–Microbe Interactions, 26, 278–286.
Schatzman S S, Culotta V C. 2018. Chemical warfare at the microorganismal level: A closer look at the superoxide dismutase enzymes of pathogens. ACS Infectious Diseases, 4, 893–903.
Tamayo D, Munoz J F, Lopez A, Uran M, Herrera J, Borges C L, Restrepo A, Soares C M, Taborda C P, Almeida A J, McEwen J G, Hernandez O. 2016. Identification and analysis of the role of superoxide dismutases isoforms in the pathogenesis of Paracoccidioides spp. PLoS Neglected Tropical Diseases, 10, e0004481.
Wang B, Yang X, Zeng H, Liu H, Zhou T, Tan B, Yuan J, Guo L, Qiu D. 2012. The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco. Applied Microbiology and Biotechnology, 93, 191–201.
Wang D, Tian L, Zhang D D, Song J, Song S S, Yin C M, Zhou L, Liu Y, Wang B L, Kong Z Q, Klosterman S J, Li J J, Wang J, Li T G, Adamu S, Subbarao K V, Chen J Y, Dai X F. 2020. Functional analyses of small secreted cysteine-rich proteins identified candidate effectors in Verticillium dahliae. Molecular Plant Pathology, 21, 667–685.
Wang J, Tian L, Zhang D D, Short D P G, Zhou L, Song S S, Liu Y, Wang D, Kong Z Q, Cui W Y, Ma X F, Klosterman S J, Subbarao K V, Chen J Y, Dai X F. 2018. SNARE-encoding genes VdSec22 and VdSso1 mediate protein secretion required for full virulence in Verticillium dahliae. Molecular Plant–Microbe Interactions, 31, 651–664.
Xu L, Chen W. 2013. Random T-DNA mutagenesis identifies a Cu/Zn superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum. Molecular Plant–Microbe Interactions, 26, 431–441.
Yao S H, Guo Y, Wang Y Z, Zhang D, Xu L, Tang W H. 2016. A cytoplasmic Cu-Zn superoxide dismutase SOD1 contributes to hyphal growth and virulence of Fusarium graminearum. Fungal Genetics and Biology, 91, 32–42.
Youseff B H, Holbrook E D, Smolnycki K A, Rappleye C A. 2012. Extracellular superoxide dismutase protects Histoplasma yeast cells from host-derived oxidative stress. Plos Pathogens, 8, e1002713.
Zelko I N, Mariani T J, Folz R J. 2002. Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radical Biology & Medicine, 33, 337–349.
Zhang L, Ni H, Du X, Wang S, Ma X W, Nurnberger T, Guo H S, Hua C. 2017. The Verticillium-specific protein VdSCP7 localizes to the plant nucleus and modulates immunity to fungal infections. New Phytologist, 215, 368–381.
Zhang Y, Wang X, Rong W, Yang J, Li Z, Wu L, Zhang G, Ma Z. 2017. Histochemical analyses reveal that stronger intrinsic defenses in Gossypium barbadense than in G. hirsutum are associated with resistance to Verticillium dahliae. Molecular Plant–Microbe Interactions, 30, 984–996.
Zhao Y L, Zhou T T, Guo H S. 2016. Hyphopodium-specific VdNoxB/VdPls1-dependent ROS-Ca2+ signaling is required for plant infection by Verticillium dahliae. PLoS Pathogens, 12, e1005793.
Zhou B J, Jia P S, Gao F, Guo H S. 2012. Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae. Molecular Plant–Microbe Interactions, 25, 964–975.
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