拟南芥抗寄生疫霉菌突变体的筛选及其初步分析
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摘要
卵菌(Oomycetes)包括腐霉菌(Pythium)、霜霉菌(downy mildews)和疫霉菌(Phytophthora)等,多数是世界上毁灭性植物病原菌,在世界范围内每年造成数百亿美元的经济损失。研究卵菌与寄主植物互作的机理,可望为建立卵菌类植物病原菌防治新途径提供理论依据。植物与病原物互作的结果可分为两类:亲和互作和非亲和互作,两者都是植物抗性因子/易感因子与病原物无毒蛋白/毒性因子复杂互作的结果。近年来人们对非亲和互作的认识突飞猛进,大量的无毒基因和抗性基因被鉴别,但相对于非亲和互作,人们对亲和互作的研究比较少。为了解植物-卵菌亲和互作的机理,本研究在已建立了拟南芥(Arabidopsis thaliana)-寄生疫霉菌(Phytophthora parasitica)亲和互作体系的基础上,通过筛选拟南芥的一个包括12000个独立的T-DNA插入的化学诱导激活型突变体库,寻找抗寄生疫霉菌的拟南芥突变体,以期为了解拟南芥-卵菌亲和互作的机制奠定基础。
通过寄生疫霉菌游动孢子接菌离体的拟南芥叶片,我们从约12000个独立转化株的37808个T3代植株中,筛选到失活型突变体165株,激活型突变体145株,其中失活型抗病突变体267-31的抗病性表型十分稳定;将267-31 T4代收种子后,用皿内活体接菌的方法验证其抗病性,结果与T4代离体接菌一致;对野生型拟南芥、抗病突变体接种H1111游动孢子后进行细胞学观察发现,接菌2h后在抗病植株上与野生型上游动孢子萌发率没有明显差异,但在抗病突变体上附着胞产生率明显低,菌丝扩展也明显较慢。通过Tail-PCR法和Southern blot法分析表明,267-31抗病突变体上有3个T-DNA插入位点。
Oomycetes including Pythium, downy mildews, Phytophthora, most of which are devastating plant pathogens, cause an annual loss of tens of billions dollars worldwide. An understanding of the compatible interaction between oomycetes and their host plants is crucial to the development of efficient control measures for plant diseases caused by oomycete pathogens. The outcome of interactions between plants and pathogens can be grouped generally into two types: incompatible and compatible, both are the results of complex interplay between many factors of both plant and pathogen origin. Compared with our knowledge of incompatible interaction in which many of both pathogen avirulence genes and plant disease resistance genes have been cloned and characterized, little is known about the nature of compatible plant-pathogen interaction. To understand the mechanism of compatible interaction between plants and pathogens, in this study we took advantage of our established compatible plant-pathogen interaction system in which the model plant Arabidopsis thaliana and oomycete pathogen Phytophthora parasitica are employed to screen a large collection of A. thaliana T-DNA insertional, chemical activated mutants for P. parasitica-resistant mutants. The identification and characterization of disease resistant mutants will aid the understanding of compatible interaction between P. parasitica and A. thaliana.
Inoculation of detached leaves with P. parasitica zoospores led to the identification of 165 degenerate mutants and 145 activated mutants that were likely resistant to P. parasitica infection, from 37808 T3 A. thaliana plants representing ~12000 independent T-DNA insertional events. One of the degenerate mutants named 267-31 has been shown to be stably resistant to P. parasitica infection. Root infection assay using our established method in petri dishes confirmed the resistant phenotype. Cytological characterization showed that the zoospore germination had no significant difference between the wild-type and the resistant mutant 267-31. However, fewer appressoria-like structures were formed and the hyphal growth and expanding were slower on the resistant mutant than the wild-type. Southern blot and Tail-PCR analysis showed that the resistant mutant 267-31 had three T-DNA insertion sites.
通过寄生疫霉菌游动孢子接菌离体的拟南芥叶片,我们从约12000个独立转化株的37808个T3代植株中,筛选到失活型突变体165株,激活型突变体145株,其中失活型抗病突变体267-31的抗病性表型十分稳定;将267-31 T4代收种子后,用皿内活体接菌的方法验证其抗病性,结果与T4代离体接菌一致;对野生型拟南芥、抗病突变体接种H1111游动孢子后进行细胞学观察发现,接菌2h后在抗病植株上与野生型上游动孢子萌发率没有明显差异,但在抗病突变体上附着胞产生率明显低,菌丝扩展也明显较慢。通过Tail-PCR法和Southern blot法分析表明,267-31抗病突变体上有3个T-DNA插入位点。
Oomycetes including Pythium, downy mildews, Phytophthora, most of which are devastating plant pathogens, cause an annual loss of tens of billions dollars worldwide. An understanding of the compatible interaction between oomycetes and their host plants is crucial to the development of efficient control measures for plant diseases caused by oomycete pathogens. The outcome of interactions between plants and pathogens can be grouped generally into two types: incompatible and compatible, both are the results of complex interplay between many factors of both plant and pathogen origin. Compared with our knowledge of incompatible interaction in which many of both pathogen avirulence genes and plant disease resistance genes have been cloned and characterized, little is known about the nature of compatible plant-pathogen interaction. To understand the mechanism of compatible interaction between plants and pathogens, in this study we took advantage of our established compatible plant-pathogen interaction system in which the model plant Arabidopsis thaliana and oomycete pathogen Phytophthora parasitica are employed to screen a large collection of A. thaliana T-DNA insertional, chemical activated mutants for P. parasitica-resistant mutants. The identification and characterization of disease resistant mutants will aid the understanding of compatible interaction between P. parasitica and A. thaliana.
Inoculation of detached leaves with P. parasitica zoospores led to the identification of 165 degenerate mutants and 145 activated mutants that were likely resistant to P. parasitica infection, from 37808 T3 A. thaliana plants representing ~12000 independent T-DNA insertional events. One of the degenerate mutants named 267-31 has been shown to be stably resistant to P. parasitica infection. Root infection assay using our established method in petri dishes confirmed the resistant phenotype. Cytological characterization showed that the zoospore germination had no significant difference between the wild-type and the resistant mutant 267-31. However, fewer appressoria-like structures were formed and the hyphal growth and expanding were slower on the resistant mutant than the wild-type. Southern blot and Tail-PCR analysis showed that the resistant mutant 267-31 had three T-DNA insertion sites.
引文
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