TurboID-charging discovery of plant pathogen virulence and host defense mechanisms

植物病原体引起疾病并降低作物产量，分泌针对宿主蛋白质并抑制免疫力的“效应”蛋白。了解效应物的功能和寄主目标有助于培育抗性作物。这个项目涉及一种令人兴奋的理解效应函数的新方法。近距离标记（PL）加速发现与感兴趣的蛋白质相互作用的蛋白质，这些蛋白质融合到生物素连接酶上，将生物素附着到附近的蛋白质上。用质谱法（MS）鉴定了链霉亲和素偶联珠捕获的生物素化蛋白。Branon等人（2018）为pls - ms创建了一种活性生物素连接酶TurboID。该学生将与2名博士后和我们的蛋白质组学团队密切合作，利用这一进步。目的1)利用细菌效应物AvrRps4和PopP2靶向WRKY转录因子发现效应靶点。AvrRps4和PopP2靶向WRKY转录因子蛋白参与防御。在RPS4/RRS1免疫受体复合体中，RRS1携带一个WRKY结构域，帮助检测AvrRps4和PopP2。这些效应物将融合到TurboID中并在拟南芥中表达以确定相互作用蛋白。目的2)鉴定丝状病原菌效应靶点念珠白蛋白卵菌引起拟南芥白锈病并抑制免疫。为了了解Albugo如何使宿主易感，该学生将在拟南芥中表达带有TurboID标记的防御抑制Albugo效应物，并识别和表征宿主蛋白靶点。目的3)鉴定在感染过程中与宿主靶点相互作用的效应物。宿主“枢纽蛋白”，如转录因子TCP14，是病原体效应物的靶点。该学生将创建TCP14-TurboID细胞系，感染不同的病原体，并使用TurboID的敏感性来揭示在感染过程中与TCP14相互作用的病原体效应蛋白。

Plant pathogens cause disease and reduce crop yields, secreting "effector" proteins that target host proteins and suppress immunity. Understanding effector function and host targets could help develop resistant crops. This project involves an exciting new approach to understanding effector functions. Proximity labeling (PL) accelerates discovery of proteins that interact with a protein of interest, that is fused to a biotin ligase that attaches biotin to nearby proteins. Biotinylated proteins trapped by streptavidin-coupled beads are identified by mass spectrometry (MS). Branon et al. (2018) created an active biotin ligase TurboID for PL-MS. The student will work closely with 2 postdocs and our proteomics team to exploit this advance.Objective1) Effector target discovery using bacterial effectors AvrRps4 and PopP2 that target WRKY transcription factors. AvrRps4 and PopP2 target WRKY transcription factor proteins involved in defense. In the RPS4/RRS1 immune receptor complex, RRS1 carries a WRKY domain that helps detect AvrRps4 and PopP2. These effectors will be fused to TurboID and expressed in Arabidopsis to define interacting proteins.Objective2) Identifying targets of filamentous pathogen effectors The oomycete Albugo candida causes white rust in Arabidopsis and suppresses immunity. To understand how Albugo makes hosts susceptible, the student will express in Arabidopsis, defense-suppressing Albugo effectors tagged with TurboID and identify and characterize host protein targets.Objective 3) Identifying effectors that interact with host targets during infection Host "hub proteins", such as the transcription factor TCP14, are targets of pathogen effectors. The student will create TCP14-TurboID lines, infect with diverse pathogens, and use the sensitivity of TurboID to reveal pathogen effector proteins delivered during infection that interact with TCP14.