Screening Tomato Protein Kinases for interaction with Pseudomonas syringae effector AvrPto
The integral role of protein kinases in signal transduction and initiating immune responses in plants suggests that these signaling molecules could be a target for inactivation by plant pathogenic effectors. The goal of my project was to identify novel tomato protein kinases that interact with Pseudomonas syringae effector, AvrPto. P. syringae, the causal agent of bacterial speck disease, causes leaf wilting and black lesions on fruit, reducing nutrients available to the plant for growth and development and thereby reducing crop yield.
To detect kinase-AvrPto associations in tomato protoplast, I used split-luciferase complementation assays (SLCA). In the SLCA, the luciferase protein from Renilla reniformis (sea pansy) is divided into two fragments, each of which is fused to a protein of interest and transformed into tomato protoplasts (cells lacking a cell wall). Interaction between the proteins causes reconstitution of the complete luciferase protein, which in turn catalyzes its substrate and emits light that can be detected using a luminescence plate reader.
After screening 20 proteins from the Popescu Lab tomato kinase library, no obvious interactors were found. Additional trials are necessary to more consistently show that these 20 protein kinases are not interacting with AvrPto. Continued work on bacteria-plant protein-protein interactions is necessary to gain insight into novel proteins involved in plant defense against bacterial speck disease. By understanding the underlying molecular basis of immunity, scientists will be better suited to develop plants with increased disease resistance.
This internship afforded me a unique opportunity to work in a lab setting alongside Master’s and PhD students as well as a Postdoctoral researcher, exposing me to the demands of each position in academia and research. Being responsible for my own project helped cultivate my time management skills. Working with a mentor helped to improve my interpersonal communication skills and my ability to work as part of a team. The PGRP seminar series exposed me to interesting topics and research currently being carried out in plant biology, painting a broader picture of the invaluable place that plant science holds in our past, present, and future. After completing my undergraduate degree in Biology at the University of Georgia, I look forward to using what I have learned during my time at Boyce Thompson Institute as I pursue a master’s degree in plant breeding.