摘要
【目的】以玉米穗腐病优势病原菌和伏马毒素主要产毒菌拟轮枝镰孢(Fusarium verticillioides)为研究对象,构建组成型表达绿色荧光蛋白GFP、红色荧光蛋白DsRED、过氧化物酶体靶向标记蛋白DsRED-PTS1和细胞骨架F-actin结合蛋白LifeAct-GFP的荧光菌株,观察拟轮枝镰孢侵染玉米须表皮的穿透结构,明确拟轮枝镰孢侵染结构形成的调控机制和影响因素。【方法】通过农杆菌介导的遗传转化方法将荧光表达载体pCOM-GFP、pCOM-DsRED、pCOM-DsRED-PTS1和pCOM-LifeAct-GFP分别导入拟轮枝镰孢野生型菌株D85-2中,PCR鉴定含有目标基因的转化子,激光共聚焦显微镜观察各荧光蛋白的表达和分布;接种玉米须观察侵染结构;赛璐酚膜模拟植物表皮穿透试验,观察穿透结构和F-actin的动态组装;使用F-actin聚合抑制剂Latrunculin A、三环唑和活性氧抑制剂DPI(diphenyleneiodonium chloride)检测穿透结构形成的影响因素。【结果】建立了以遗传霉素G418为抗性标记的遗传转化方法;FV-GFP和FV-DsRED菌株分别具有清晰、明亮的绿色荧光和红色荧光,FV-DsRED-PTS1菌株中红色荧光呈圆点状分布,符合真菌中过氧化物酶体的分布特征,FV-LifeAct-GFP菌株孢子和菌丝内部具有丝絮状绿色荧光,符合真菌中F-actin蛋白的分布特征;玉米须表皮侵染过程中观察到菌丝顶端膨大类似附着枝侵染结构;赛璐酚膜模拟穿透试验观察到穿透结构中F-actin蛋白的成环组装;药物胁迫试验表明,穿透结构的形成受F-actin蛋白聚合和活性氧调控。同时,三环唑能够抑制穿透。【结论】构建了4种荧光标记菌株,细胞定位清晰,具有正常的生长表型和致病力,能够满足致病机制相关研究需求;明确了拟轮枝镰孢在侵染玉米时能够形成一种菌丝顶端膨大的类似附着枝状的侵染结构;确定了F-actin成环聚集、细胞骨架组装和活性氧调控是影响拟轮枝镰孢侵染结构形成的关键因素。
【Objective】Taking the dominant pathogen Fusarium verticillioides causing maize ear rot and producing fumonisin as the research object,the objective of this study is to construct fluorescent strains that constitutively express green fluorescent protein GFP,red fluorescent protein DsRED,peroxisome targeted marker protein DsRED-PTS1 and F-actin cytoskeleton binding protein LifeAct-GFP,observe the penetration structure of F.verticillioides infecting maize silk epidermis,and to clarify the regulatory mechanisms and influencing factors regulating the infection structure formation of F.verticillioides.【Method】Fluorescent expression vectors pCOM-GFP,pCOM-DsRED,pCOM-DsRED-PTS1,and pCOM-LifeAct-GFP were transformed into the wild-type strain D85-2 of F.verticillioides through Agrobacterium-mediated genetic transformation.The transformants containing the target genes were identified by PCR,and the expression and distribution of various fluorescent proteins were observed under laser confocal microscopy.Maize was inoculated to observe the infection structure.The penetration structure and dynamic assembly of F-actin were observed by simulating the plant epidermal penetration experiment with cellophane membrane.The influencing factors of penetration structure formation were detected using F-actin polymerization inhibitor Latruculin A,tricyclazole,and reactive oxygen species(ROS)inhibitor DPI(diphenyleneiodonium chloride).【Result】A genetic transformation method using geneticin G418 as a resistance marker was established.FV-GFP and FV-DsRED strains have clear and bright green fluorescence and red fluorescence,respectively.The red fluorescence in FV-DsRED-PTS1 strain is distributed as round dots in conidia and hypha,which is consistent with the distribution characteristics of peroxisome in fungi.FV-LifeAct-GFP strain has filamentous green fluorescence in conidia and hypha,which is consistent with the distribution characteristics of F-actin protein in fungi.During the infection process of maize silk epidermis,an infection structure resembling a hyphopodium was observed at the top of the hyphae.The ring-forming assembly of F-actin protein in the penetration structure was observed in the mimic penetration experiment on the cellophane membrane.Drug stress experiments have shown that the formation of penetrating structures is regulated by F-actin protein polymerization and ROS.Meanwhile,tricyclazole can inhibit penetration.【Conclusion】Four fluorescent labeled strains were constructed,with clear cell localization and normal growth phenotype and pathogenicity,which can meet the research needs related to pathogenic mechanisms.F.verticillioides can form a hyphopodium like infection structure with swollen hyphae at the top when infecting maize.It is confirmed that F-actin cyclization and polymerization,cytoskeleton assembly and ROS regulation are the key factors affecting the formation of infection structure of F.verticillioides.
作者
哈丹丹
郑红霞
张振昊
竺利红
刘浩
王教瑜
周雷
HA DanDan;ZHENG HongXia;ZHANG ZhenHao;ZHU LiHong;LIU Hao;WANG JiaoYu;ZHOU Lei(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457;Institute of Agro-Product Safety and Nutrition,Zhejiang Academy of Agricultural Sciences/State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products,Hangzhou 310021;Institute of Plant Protection and Microbiology,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021)
出处
《中国农业科学》
CAS
CSCD
北大核心
2023年第18期3556-3573,共18页
Scientia Agricultura Sinica
基金
浙江省自然科学基金(LZ20C1400001)。
关键词
拟轮枝镰孢
荧光标记
过氧化物酶体
侵染结构
细胞骨架
活性氧
Fusarium verticillioides
fluorescent labeling
peroxisome
infection structure
F-actin
reactive oxygen species(ROS)
