Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris
Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris
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DOI：
10.1111/j.1365-313x.2005.02436.x
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发表时间：
2005-07-01
期刊：
影响因子：
7.2
通讯作者：
van Kan, JAL
中科院分区：
文献类型：
--
作者：
Kars, I;Krooshof, GH;van Kan, JAL
在毕赤酵母中分别表达了5种灰葡萄孢菌内切多聚半乳糖醛酸酶（BcPGs），并进行了纯化和生化表征。虽然这五种酶的最适pH值相似（约pH 4.5），但各酶的最大活性显著不同。对于多聚半乳糖醛酸（PGA）的水解，V-max，V-app的范围为10至900 U mg（-1），而K-m，K-app的范围为0.16至0.6 mg ml（-1）。虽然所有的BcPG都是真正的内切多聚半乳糖醛酸酶，但它们显然具有不同的作用模式。BcPG 1、BcPG 2和BcPG 4对PGA的水解导致DIP < 7的寡聚体的瞬时积累，而BcPG 3和BcPG 6对PGA的水解导致单体和二聚体的立即积累。在番茄、蚕豆和拟南芥中分别测定了所有BcPGs的坏死活性（NA）。他们在这些植物上显示出不同的NA。BcPG 1和BcPG 2具有最强的NA，因为在蚕豆叶片浸润后10 min内观察到组织塌陷。BcPG 2活性位点的氨基酸（aa）D192 A取代不仅使酶活性丧失，而且使NA丧失，表明NA依赖于酶活性。此外，在B.灰霉病菌对番茄和蚕豆的毒力显著降低。原发性病变形成延迟约24小时，病变扩张率降低50- 85%。这些数据表明BcPG 2是B的重要毒力因子。灰绿色
Five Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the V-max,V-app ranged from 10 to 900 U mg(-1), while the K-m,K-app ranged from 0.16 to 0.6 mg ml(-1). Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DIP < 7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50-85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea.