摘要
病原菌与植物互作过程中分泌的细胞壁降解酶具有致病和激发植物免疫的双重功能。BcGs1是从灰葡萄孢菌代谢物中分离的葡聚糖-1,4-α糖苷酶,能激发番茄和烟草的免疫防御反应,提高抗病性。BcGs1包含糖苷水解酶家族15(GH15)和糖结合模块家族20(CBM20)两个结构域,其激发植物防御反应的作用方式及功能结构域尚不清楚。本文测定了BcGs1不同诱导时间对提高番茄抗灰葡萄孢菌的效果、体外糖苷酶活性,并比较了BcGs1不同结构域截短蛋白与全长蛋白引起细胞坏死活性的差异。结果表明,BcGs1蛋白诱导番茄72 h可将灰葡萄孢菌引起的病斑面积减少23.25%;BcGs1能水解淀粉和海带多糖,但不能水解羟甲基纤维素和微晶纤维素;BcGs1全长、GH15和CBM20截短蛋白均能在烟草细胞中正常瞬时表达,但表达GH15的烟草叶片枯斑面积明显小于表达全长蛋白BcGs1的枯斑面积,表达CBM20截短蛋白的叶片没有产生枯斑反应;为了进一步明确截短蛋白GH15和全长蛋白BcGs1激发烟草细胞坏死活性的差异,用大肠杆菌表达并获得了纯化的重组蛋白GH15,叶片浸润法测定结果表明,截断蛋白GH15的细胞坏死活性仍然低于BcGs1全长蛋白的坏死活性,说明BcGs1诱导烟草细胞坏死活性需要GH15和CBM20结构域的共同参与。本文研究结果为进一步探讨BcGs1诱导植物抗病机制奠定了基础。
Cell wall-degrading enzymes are secreted by pathogenic fungi in infection process and has the functions of both pathogenicity and elicitor activity. BcGs1, a cell wall-degrading enzyme from B. cinerea, is a glucan-1,4-alpha-glucosidase that contains a Glyco-hydro 15 （GH15） domain and a CBM20_glucoamylase （CBM20） domain. Previous study showed BcGs1 could induce necrosis and disease resistance against B. cinerea and Tobacco mosaic virus （TMV）. In this study, we investigated the appropriate induction period of BcGs1 for disease resistance against B. cinerea in tomato. Glycosidic enzyme activity was also measured in vitro and functional domain of BcGs1 for triggering plant cell death was determined through protein truncation assay. The results showed that the most appropriate induction period is at 72 h post infiltration with 0.25 μM concentration BcGs1 and the lesion area was decreased by 23.25% compared with control treatment. BcGs1 could hydrolyze starch and laminarin, but could not hydrolyze polysaccharide and hydroxymethyl cellulose in vitro. The proteins of BcGs1, truncated GH15 and CBM20 were all expressed in Nicotiana benthamiana leaves via Agrobacterium-mediated transient expression system. Necrotic response induced by GH15 was weaker than that induced by BcGs1, while necrotic response was invisible in CBM20-induced tobacco leaves. For further verification, we purified the recombinant protein GH15 from E. coli, and found that GH15 induced weaker necrotic activity than BcGs1 in tobacco leaves with the same concentration. These results indicated that both domains GH15 and CBM20 are required for full elicitor activity of BcGs1. Taken together, our findings provide a new clue for further study on mechanism of BcGs1-induced plant immunity.
出处
《植物病理学报》
CAS
CSCD
北大核心
2017年第5期612-618,共7页
Acta Phytopathologica Sinica
基金
北京市科技计划(D151100003915003)
关键词
灰葡萄孢
激发子BcGs1
抗病性
细胞坏死
功能结构域
Botrytis cinerea
protein elicitor BcGs1
disease resistance
cell necrosis
functional domain
