摘要
自噬是寄主响应病毒侵染的一种重要防御机制。为明确柑橘褪绿矮缩病毒(Citrus chlorotic dwarf-associated virus,CCDaV)侵染柑橘是否引起细胞自噬反应,运用透射电镜、实时荧光定量检测、GFP-NbATG8f荧光标记和蛋白印记等方法,结果发现,CCDaV感染尤力克柠檬15 d后,在尤力克柠檬叶片细胞质内观察到自噬小体结构的形成,自噬相关基因(ClATG3、ClATG5、ClATG7、ClATG8a、ClATG8d、ClATG8f、ClATG8g、ClATG8i、ClBeclin1、ClPI3K和ClNBRI)的表达量显著上调,且ATG8的含量是对照组的1.99倍。将CCDaV编码的6个病毒蛋白分别与GFP-NbATG8f共表达于本生烟48 h后,通过共聚焦观察到V3和V4蛋白分别与GFP-NbATG8f共表达时诱导产生的自噬小体数量显著高于对照,且游离GFP的含量也显著增加。表明CCDaV侵染可诱导尤力克柠檬产生细胞自噬反应,且病毒编码的V3和V4蛋白可能是诱导自噬的重要因子。
Autophagy is an important defense mechanism of host in response to virus infection.To clarify whether the infection of Citrus chlorotic dwarf-associated virus(CCDaV)could induce autophagic response in plant,in this study,transmission electron microscope(TEM),quantitative real-time PCR,confocal microscopy analysis of enhanced green fluorescent protein-tagged Nicotiana benthamiana ATG8f(GFP-NbATG8f)and western blot were used to investigate the formation of autophagic structure,the transcript levels of autophagy related genes and ATG8 content in CCDaV-infected Eureka lemon,respectively.The results showed that the formation of autophagic structure was observed in the cytoplasm of CCDaV-infected Eureka lemon leaf cell by TEM at 15 days post inoculation(dpi).Compared with the healthy control,the expression levels of autophagy related genes(ClATG3,ClATG5,ClATG7,ClATG8a,ClATG8d,ClATG8f,ClATG8g,ClATG8i,ClBeclin1,ClPI3K and ClNBRI)in CCDaV-infected Eureka lemon leaves were significantly up-regulated,and ATG8 content was increased by 1.99 times.After co-expression of the six viral proteins encoded by CCDaV with GFP-NbATG8f in N.benthamiana for 48 h,the number of autophagosomes induced by co-expression of V3 and V4 proteins with GFP-NbATG8f was significantly higher than that of the control,and the content of free GFP was also significantly increased.These results indicated that CCDaV infection induced autophagic response in Eureka lemon,and V3 and V4 proteins may be important factors to induce autophagy.
作者
余玲
申世凯
王甲军
柏自琴
陈力
胡德玉
周彦
YU Ling;SHEN Shikai;WANG Jiajun;BAI Ziqin;CHEN Li;HU Deyu;ZHOU Yan(Citrus Research Institute,Southwest University/National Citrus Engineering Research Center,Chongqing 400712,China;Fruit Research Institute,Guizhou Academy of Agricultural Sciences,Guiyang 550000,China;Economic Crop Development Center of Wanzhou District,Wanzhou Chongqing 404100,China)
出处
《西南大学学报(自然科学版)》
北大核心
2025年第3期37-45,共9页
Journal of Southwest University(Natural Science Edition)
基金
国家现代农业产业技术体系项目(CARS-26-05B)
重庆市自然科学基金面上项目(CSTB2024NSCQ-MSX0311)
重庆市万州区农业技术研发服务项目(柑橘)。
关键词
柑橘褪绿矮缩病毒
尤力克柠檬
细胞自噬
病毒蛋白
Citrus chlorotic dwarf-associated virus
Eureka lemon
autophagy
viral proteins
