Extracellular superoxide dismutase VdSOD5 is required for virulence in Verticillium dahliae

doi:10.1016/S2095-3119(20)63353-6

摘要/Abstract

摘要：

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。

Abstract:

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.

Key words: Verticillium dahliae , superoxide dismutase , secretion , virulence , ROS detoxification

. [J]. Journal of Integrative Agriculture, 2021, 20(7): 1858-1870.

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

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