Characterization of tomato lines with CRISPR/Cas9-mediated mutations in immunity-associated genes
Pseudomonas syringae pv. tomato (Pst), or bacterial speck disease, causes unsightly blemishes on tomato that make the fruits unsellable. Better understanding the molecular mechanisms underlying tomato – Pst interactions may lead to the development of tomato cultivars with enhanced Pst resistance, improving the yield and quality of tomato fruits. Thus far, the Martin Lab has used the CRISPR/Cas9 genome editing technique to generate tomato lines knocking out ~150 candidate immunity-associated genes. Each of these tomato mutant lines must be genotyped to identify homozygous or biallelic mutants and phenotyped via reactive oxygen species (ROS) assay and bacterial growth assay to examine the effects of disrupting a putative immunity-associated gene on tomato immunity. A total of 90 tomato T1 plants have been genotyped and 12 plants harboring homozygous or biallelic mutations but lacking T-DNA were identified and kept for seeds that will be phenotyped in the next generation. Additionally, 8 homozygous or biallelic mutant lines were phenotyped via ROS assay and 15 lines via disease assay using several Pst variants. One mutant line, knocking out the LykA gene, lost ROS response to flg22 and showed enhanced susceptibility to a Pst variant, while the pic1 knockout line displayed an increased resistance to Pst, implicating both LykA and Pic1 as important contributors to plant immunity that may function in different pathways. Taken together, this study will help us to better understand the underlying mechanisms of plant immunity as well as provide key information for improving tomato resistance to Pst.
This summer, my internship at the Boyce Thompson Institute has been an incredibly rewarding experience; I have learned practical and personal skills essential for pursuing a career in scientific research. Under the guidance of my mentor, I learned a host of new laboratory techniques, project management, and interpersonal skills. Even more importantly, however, was the time I spent surrounded by fellow people passionate about plant science. The weekly seminars and conversations with postdocs, graduate students, and my fellow interns allowed me to see how what fascinates me is important in the context of the greater world. Recognizing that plant science addresses critical issues like food security and global climate change, BTI has given me not only skills with which to be better in the lab but also the world beyond.