Dow J M, Fan M J, Newman M A, Daniels M J
Sainsbury Laboratory, John Innes Centre for Plant Science Research, Norwich, United Kingdom.
Appl Environ Microbiol. 1993 Dec;59(12):3996-4003. doi: 10.1128/aem.59.12.3996-4003.1993.
Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.
野油菜黄单胞菌致病变种辣根致病变种和萝卜致病变种可引起十字花科植物叶斑病，它们在液体培养中会产生一种主要的胞外蛋白酶，并对其进行了部分纯化。该蛋白酶（PRT 3）是一种需要锌的金属酶，通过β-酪蛋白的降解模式和对抑制剂的敏感性，很容易与野油菜黄单胞菌野油菜致病变种先前鉴定的两种蛋白酶（PRT 1和PRT 2）区分开来。PRT 3在维管束十字花科病原菌野油菜黄单胞菌野油菜致病变种（测试了5个菌株）中低水平产生，其中PRT 1和PRT 2占主导地位。相比之下，在六个测试的叶斑叶肉病原菌菌株中未检测到丝氨酸蛋白酶PRT 1的表达。然而，所有这些菌株都有与prtA探针杂交的DNA片段，这些片段可能携带功能性的prtA（PRT 1的结构基因）。通过在蛋白酶缺陷型野油菜黄单胞菌野油菜致病变种菌株中筛选野油菜黄单胞菌萝卜致病变种的基因组文库，克隆了PRT 3的结构基因（prtC）。亚克隆和Tn5诱变将结构基因定位到1.2 kb的DNA上。在所有测试的十字花科攻击型野油菜黄单胞菌致病变种菌株以及一些其他致病变种中都发现了与结构基因杂交的DNA片段。通过将克隆基因导入异源致病变种来操纵致病变种蛋白酶产生模式的实验表明，蛋白酶基因表达模式与（维管束或叶肉）致病模式之间不存在决定性关系。
