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

植物病原体导致疾病和作物减产，分泌针对宿主蛋白并抑制免疫的“效应器”蛋白。了解效应器功能和寄主靶标有助于培育抗病作物。这个项目涉及一种令人兴奋的新方法来理解效应器功能。邻近标记(PL)加速了发现与感兴趣的蛋白质相互作用的蛋白质，该蛋白质与生物素连接酶融合在一起，将生物素连接到附近的蛋白质上。用质谱仪(MS)鉴定了链霉亲和素偶联微球捕获的生物素化蛋白质。Branon等人。(2018)为PL-MS创造了具有活性的生物素连接酶TurboID。学生将与2名博士后和我们的蛋白质组学团队密切合作，以利用这一进展。目标1)使用针对WRKY转录因子的细菌效应器AvrRps4和PopP2发现效应器靶标。AvrRps4和PopP2靶向参与防御的WRKY转录因子蛋白。在RPS4/RRS1免疫受体复合体中，RRS1携带一个WRKY结构域，有助于检测AvrRps4和PopP2。这些效应物将与TurboID融合并在拟南芥中表达，以定义相互作用的蛋白。目的2)鉴定丝状病原体效应物的靶标白念珠菌可引起拟南芥白锈病并抑制免疫。为了了解Albugo是如何使宿主变得敏感的，学生将在拟南芥中表达用TurboID标记的防御抑制Albugo效应器，并识别和表征宿主蛋白靶标。目的3)识别在感染过程中与宿主靶标相互作用的效应器宿主“枢纽蛋白”，如转录因子TCP14，是病原体效应物的靶标。学生将创建TCP14-TurboID株，感染不同的病原体，并使用TurboID的敏感性来揭示感染期间传递的与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.