Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.
Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.
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DOI：
10.1371/journal.ppat.1005874
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发表时间：
2016-09
期刊：
影响因子：
6.7
通讯作者：
van der Hoorn RA
中科院分区：
文献类型：
--
作者：
Shindo T;Kaschani F;Yang F;Kovács J;Tian F;Kourelis J;Hong TN;Colby T;Shabab M;Chawla R;Kumari S;Ilyas M;Hörger AC;Alfano JR;van der Hoorn RA
丁香假单胞菌番茄DC 3000（PtoDC 3000）是一种胞外模式植物病原体，但其产生操纵质外体的分泌效应物的潜力一直在研究中。在这里，我们确定了131个候选的小，分泌，非注释的蛋白质从PtoDC 3000基因组，其中大部分是常见的假单胞菌属物种，并可能在质外体定殖表达。我们通过定制的细胞外细菌蛋白的网关兼容表达系统产生了43种这些蛋白质，并使用基于竞争活性的蛋白质谱分析筛选它们抑制番茄分泌的免疫蛋白酶C14的能力。该筛选揭示了C14抑制蛋白-1（Cip 1），其含有chagasin样蛋白酶抑制剂的基序。Cip 1突变体对番茄的毒性较低，证明了这种效应子在质外体免疫中的重要性。Cip 1还抑制免疫蛋白酶Pip 1，已知Pip 1可抑制PtoDC 3000感染，但与其同源物Rcr 3的亲和力较低，这解释了为什么这种蛋白质在携带Cf-2抗性基因的番茄植物中不被识别，Cf-2抗性基因使用Rcr 3作为辅助受体来检测病原体衍生的蛋白酶抑制剂。因此，该方法揭示了番茄疫霉的蛋白酶抑制剂，表明番茄疫霉也分泌选择性靶向番茄的质外体宿主蛋白酶的效应物，类似于番茄病原真菌、卵菌和线虫。叶片的细胞外空间（质外体）被多种微生物定殖，这些微生物必须处理宿主分泌的水解酶，其中许多在防御反应期间积累。我们推测，除了真菌和卵菌病原体，细菌模式植物病原体假单胞菌也保护自己在质外体分泌针对这些质外体水解酶的抑制剂。注射假单胞菌的基因组含有超过131个编码推定的小的、未注释的分泌蛋白的基因，这些蛋白先前尚未被表征。在这里，我们生产并纯化了43个这些小蛋白，并测试了它们抑制番茄分泌的免疫蛋白酶C14的能力。我们发现了一种C14蛋白酶抑制剂，称为Cip 1，它携带chagasin样基序，并有助于毒力。Cip 1还有效地抑制Pip 1，这是番茄的另一种免疫蛋白酶，已知可抑制P. lingae感染。有趣的是，Cip 1对免疫蛋白酶Rcr 3的亲和力较低，这解释了为什么这种蛋白质和产生Cip 1的PtoDC 3000在携带Cf-2抗性基因的番茄植物中不被识别，该基因使用Rcr 3作为辅助受体来检测病原体入侵。
Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. The extracellular space in the leaf (the apoplast) is colonized by a diversity of microbes that will have to deal with host-secreted hydrolytic enzymes, many of which accumulate during defence responses. We hypothesize that in addition to fungal and oomycete pathogens, the bacterial model plant pathogen Pseudomonas syringae also protects itself in the apoplast by secreting inhibitors targeting these apoplastic hydrolases. The genome of P. syringe harbours over 131 genes encoding putative small, non-annotated secreted proteins that have not been characterized previously. Here, we produced and purified 43 of these small proteins and tested them for their ability to inhibit the secreted immune protease C14 of tomato. We discovered a C14 protease inhibitor, coined Cip1, which carries chagasin-like motifs and contributes to virulence. Cip1 also effectively inhibits Pip1, another immune protease of tomato, known to suppress P. syringae infection. Interestingly, Cip1 has a lower affinity for the immune protease Rcr3, explaining why this protein, and PtoDC3000 producing Cip1, is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen invasion.
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影响因子：
1.5
作者：
Huang, Robert;Que, Xuchu;Hirata, Ken;Brinen, Linda S.;Lee, Ji Hyun;Hansell, Elizabeth;Engel, Juan;Sajid, Mohammed;Reed, Sharon
通讯作者：
Reed, Sharon
DOI：
10.1073/pnas.1202867109
发表时间：
2012-06-19
影响因子：
11.1
作者：
Lozano-Torres, Jose L.;Wilbers, Ruud H. P.;Smant, Geert
通讯作者：
Smant, Geert
DOI：
10.1073/pnas.1731982100
发表时间：
2003-09-02
影响因子：
11.1
作者：
Buell, CR;Joardar, V;Collmer, A
通讯作者：
Collmer, A
影响因子：
48
作者：
Choi, KH;Gaynor, JB;Schweizer, HP
通讯作者：
Schweizer, HP
影响因子：
56.9
作者：
de Jonge, Ronnie;van Esse, H. Peter;Thomma, Bart P. H. J.
通讯作者：
Thomma, Bart P. H. J.