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

JONES_S20DTP

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.

Objective 1) Effector target discovery using bacterial effectors AvrRps4 and PopP2 that target WRKY transcription factors. AvrRps4 and PopP2 target WRKY transcription factor proteins involved in defence. 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.

Objective 2) 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.

The student will learn skills in proteomics, bioinformatics, molecular biology, GoldenGate cloning and plant pathology. Applicants should have a strong interest in plant/microbe interactions and cutting-edge biochemical methods.

Branon et al https://www.ncbi.nlm.nih.gov/pubmed/30125270