Petre Benjamin, Saunders Diane G O, Sklenar Jan, Lorrain Cécile, Win Joe, Duplessis Sébastien, Kamoun Sophien
1 The Sainsbury Laboratory, Norwich Research Park, NR4 7UH Norwich, U.K.;
2 INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280 Champenoux, France;
Mol Plant Microbe Interact. 2015 Jun;28(6):689-700. doi: 10.1094/MPMI-01-15-0003-R.
Rust fungi are devastating crop pathogens that deliver effector proteins into infected tissues to modulate plant functions and promote parasitic growth. The genome of the poplar leaf rust fungus Melampsora larici-populina revealed a large catalog of secreted proteins, some of which have been considered candidate effectors. Unraveling how these proteins function in host cells is a key to understanding pathogenicity mechanisms and developing resistant plants. In this study, we used an effectoromics pipeline to select, clone, and express 20 candidate effectors in Nicotiana benthamiana leaf cells to determine their subcellular localization and identify the plant proteins they interact with. Confocal microscopy revealed that six candidate effectors target the nucleus, nucleoli, chloroplasts, mitochondria, and discrete cellular bodies. We also used coimmunoprecipitation (coIP) and mass spectrometry to identify 606 N. benthamiana proteins that associate with the candidate effectors. Five candidate effectors specifically associated with a small set of plant proteins that may represent biologically relevant interactors. We confirmed the interaction between the candidate effector MLP124017 and TOPLESS-related protein 4 from poplar by in planta coIP. Altogether, our data enable us to validate effector proteins from M. larici-populina and reveal that these proteins may target multiple compartments and processes in plant cells. It also shows that N. benthamiana can be a powerful heterologous system to study effectors of obligate biotrophic pathogens.
锈菌是极具破坏力的作物病原体，可将效应蛋白输送到受感染组织中，以调节植物功能并促进寄生生长。杨树叶片锈菌（Melampsora larici-populina）的基因组揭示了大量分泌蛋白，其中一些被认为是候选效应蛋白。弄清楚这些蛋白在宿主细胞中的功能是理解致病机制和培育抗性植物的关键。在本研究中，我们使用效应蛋白组学流程在本氏烟草叶片细胞中筛选、克隆并表达了20种候选效应蛋白，以确定它们的亚细胞定位，并鉴定与它们相互作用的植物蛋白。共聚焦显微镜显示，六种候选效应蛋白靶向细胞核、核仁、叶绿体、线粒体和离散的细胞体。我们还使用了免疫共沉淀（coIP）和质谱技术来鉴定与候选效应蛋白相关的606种本氏烟草蛋白。五种候选效应蛋白与一小部分可能代表生物学相关相互作用蛋白的植物蛋白特异性相关。我们通过植物体内免疫共沉淀证实了候选效应蛋白MLP124017与杨树中TOPLESS相关蛋白4之间的相互作用。总之，我们的数据使我们能够验证来自M. larici-populina的效应蛋白，并揭示这些蛋白可能靶向植物细胞中的多个区室和过程。这也表明本氏烟草可以成为研究专性活体营养型病原体效应蛋白的强大异源系统。
