致病疫霉效应蛋白Pi16275的功能研究

Toxicity of effector Pi16275 of Phytophthora infestans

摘要:

【目的】分析致病疫霉效应蛋白Pi16275的超量表达对病原菌致病性的影响，明确Pi16275的亚细胞定位，筛选Pi16275在植物中的互作靶标蛋白及靶标蛋白在抵御病原菌侵染过程中的作用，初步揭示Pi16275在病原菌侵染植物过程中的作用机制。【方法】利用农杆菌介导的烟草瞬时表达系统在烟草叶片表皮细胞中瞬时表达Pi16275并观察其亚细胞定位，同时在瞬时表达部位接种致病疫霉游动孢子并统计病斑面积；通过酵母核系统cDNA文库及酵母双杂交技术筛选、验证，确定Pi16275在马铃薯中的靶标蛋白；运用病毒介导的基因沉默技术在植物中沉默靶标蛋白基因，探究基因沉默是否影响植物对病原菌的抗性。【结果】在烟草叶片中瞬时表达Pi16275显著促进致病疫霉的侵染；Pi16275定位在植物细胞核、细胞质及细胞膜中；初步筛选到3个与Pi16275互作的马铃薯蛋白：40S核糖体亚基蛋白S5（StRPS5）、粘蛋白2（StMUC2）、V型ATP酶E亚基类似蛋白（StVAEL）；沉默StRPS5的同源基因后显著降低了烟草对致病疫霉的抗性。【结论】Pi16275在致病疫霉侵染植物过程中发挥重要作用。

Abstract:

[Objective] Our aim was to analyze the effect of overexpressing the effector Pi16275 on the pathogenicity of Phytophthora infestans, and to clarify the subcellular localization of Pi16275. We screened the plant proteins that interacted with Pi16275 and explored their role in plant disease resistance, and finally revealed the role of Pi16275 in the process of pathogen infecting plants.[Methods] We used the Agrobacterium-mediated method to transiently overexpress Pi16275 in the epidermal cells of tobacco leaves, and then observed the subcellular location of Pi16275 under a fluorescence microscope. After inoculating the zoospores of P. infestans at the transient expression site, we measured the area of the diseased spot. The yeast nuclear cDNA library and yeast two-hybrid assay were used to mine and verify the target protein of Pi16275 in potato. In order to explore whether gene silencing affects the plant resistance to pathogens, we used virus-mediated gene silencing technology to silence the homologous gene of the target protein gene in tobacco and inoculated the tobacco leaves with zoospores of P. infestans.[Results] The transient expression of Pi16275 in tobacco leaves significantly promoted the infection of P. infestans, and Pi16275 was located in plant cell nucleus, cytoplasm, and cell membrane. Three target proteins of potato interacted with Pi16275, including 40S ribosomal protein S5 (StRPS5), mucin 2 (StMUC2), and V-type proton ATPase subunit E-like protein (StVAEL). Silencing the homologous gene of StRPS5 significantly weakened the resistance of tobacco to P. infestans.[Conclusion] Pi16275 plays a role in the infection of P. infestans in plants.