Pseudomonas sytingae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter
Pseudomonas sytingae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter
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DOI：
10.1128/jb.186.2.543-555.2004
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发表时间：
2004-01-01
影响因子：
3.2
通讯作者：
Collmer, A
中科院分区：
文献类型：
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作者：
Schechter, LM;Roberts, KA;Collmer, A
紫丁香假单胞菌番茄DC3000菌株是番茄和拟南芥的病原菌。HRP-HRC编码的III型分泌系统(TTSS)将细菌效应蛋白(主要称为Hop或AVR蛋白)注入植物细胞，是致病所必需的。除了受HrpL选择性sigma因子的调控外，大多数AVR或HOP基因还编码具有N末端的蛋白质，这些蛋白质具有几个特征，包括(I)高比例的Ser残基，(Ii)第三或第四位的脂肪族氨基酸(Lie、Len或Val)或Pro，以及(Iii)前12个残基内缺乏带负电荷的氨基酸。在这里，被广泛研究的效应子AvrPto被用来优化钙调素依赖的腺苷环化酶(CyA)报告系统，用于HRP介导的紫丁香TTSS效应子到植物细胞的转位。该系统包括一个已克隆的紫丁香hrp基因簇和模式植物本氏烟草。对与CyA融合的截短的AvrPto蛋白的分析表明，AvrPto的N-端16个氨基酸和/或密码子足以引导微弱的易位进入植物细胞，而较长的N-端片段则指示逐渐强的易位。AvrB被测试，因为它在培养中被紫丁香菌HRP系统分泌得很差，被转移到植物细胞中与AvrPto一样有效。以CyA为报告基因，对DC3000候选Hop蛋白的易位进行了研究，发现了3个新的完整的Hop蛋白，分别命名为HopPtoQ、HopPtoT1和HopPtoV，以及由自然干扰基因HopPtoS4：：tnpA和HopPtoAG：：tnpA编码的两个截短的Hop蛋白。我们还证实了HopPtoK、HopPtoC和AvrPphE(PTO)被转移到植物细胞中。这些结果使DC3000中HRP系统分泌的蛋白质数量增加到40个。虽然大多数新发现的Hop蛋白具有与先前描述的Hop蛋白相同的N端特征，但HopPtoV没有这些特征。我们的结果表明，CyA应该是探索紫丁香HRP系统的多个方面的有用报告。
Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (lie, Len, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB,, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE(Pto) are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.