Functional studies of the bacterial avirulence protein AvrPto by mutational analysis
Functional studies of the bacterial avirulence protein AvrPto by mutational analysis
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DOI：
10.1094/mpmi.2001.14.4.451
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发表时间：
2001-04-01
影响因子：
3.5
通讯作者：
Michelmore, RW
中科院分区：
文献类型：
--
作者：
Chang, JH;Tobias, CM;Michelmore, RW
表达avrPto的假单胞菌致病变种对表达抗性基因Pto的植物是无毒的。用多种策略产生了超过85个avrPto突变体，并使用几种测定来表征AvrPto功能。在酵母双杂交系统中，164个残基中只有95个氨基酸的核心足以结合Pto。只有9的65个突变蛋白的AvrPto的氨基酸取代，在植物和体外，没有与Pto的Gal 4酵母双杂交系统中，这表明AvrPto可以容忍许多非保守性取代，仍然与Pto相互作用。进一步表征AvrPto的这9个和12个另外的置换突变体。这21个AvrPto突变体引起过敏反应的能力和对植物致病性的影响与Pto在酵母双杂交系统中的识别密切相关。对具有取代H54 P或D52 G/L 65 P的两种蛋白质的分析表明，这些残基可能分别是递送到宿主细胞中和蛋白质在细菌细胞质中稳定性所需的。预测不再与Pto相互作用且在植物中具有修饰活性的突变体在其二级结构上具有变化。
Pseudomonas syringae pathovars expressing avrPto are avirulent on plants expressing the resistance gene Pto, Over 85 mutants of avrPto were generated with multiple strategies, and several assays were used to characterize AvrPto function. Only a core of 95 amino acids of the 164 residues was sufficient for binding Pto in the yeast two-hybrid system. Only nine of 65 mutant proteins of AvrPto with amino acid substitutions, created in planta and in vitro, did not interact with Pto in the Gal4 yeast two-hybrid system, suggesting that AvrPto can tolerate many nonconservative substitutions and still interact with Pto. These nine and 12 additional substitution mutants of AvrPto were characterized further. The ability to elicit a hypersensitive response and the effect on pathogenesis in planta for these 21 mutants of AvrPto were strongly correlated with recognition by Pto in the yeast two-hybrid system. Analyses of two proteins with substitutions H54P or D52G/L65P indicated that these residues may be required for delivery into the host cell and protein stability in the bacterial cytoplasm, respectively. The mutants that no longer interacted with Pto and had modified activities in planta were predicted to have changes in their secondary structure.