Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be up-regulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results indicate that pKFDV02 did not keep its promises as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

中文翻译：

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一种提高苹果火疫病抗性的解淀粉欧文氏菌诱导型启动子

内发生是转基因的重要替代方法，以产生仅含有天然 DNA 的修饰植物。开发这种策略的一个关键点是控制感兴趣基因表达的调控序列的可用性。为了找到可由火疫病病原体解淀粉欧文氏菌 (Ea)诱导或适度组成型的苹果基因启动子，我们专注于多酚氧化酶基因 ( PPO )。这些基因编码参与许多生理过程的氧化酶，并且先前已显示在Ea感染过程中上调。我们在苹果基因组中发现了 10 个 PPO 和 2 个 PPO 样序列，并对MdPPO16 ( pPPO16 ) 和MdKFDV02 PPO样 ( pKFDV02 ) 分别作为Ea诱导型和低组成型调节序列的潜力。在各种处理后量化了与这些启动子融合并在苹果中瞬时或稳定表达的报告基因的表达水平。与表现出可变活性的 pKFDV02 不同，pPPO16允许转基因在Ea感染后以 3 型分泌系统依赖性方式在苹果中快速而强烈地表达。总之，我们的结果表明pKFDV02没有履行其作为组成型和弱启动子的承诺，而pPPO16是第一个Ea-从苹果克隆的诱导型启动子，可以作为基因内策略的有用组成部分，以创建抗火疫病苹果基因型。