Towards Functional Characterization of a Novel ER-Associated Protein in Plant Immunity via CRISPR/Cas9 Technology
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To sabotage pathogen infection, plants have evolved a sophisticated immune system, in which ubiquitination has emerged in recent years as a key regulator of the interactions between plants and pathogens. To elucidate the role of endoplasmic reticulum (ER)-associated ubiquitin-conjugating enzymes (E2) in plant immunity, a novel Arabidopsis protein, ER-associated E2-interacting protein1 (EREI1) was previously identified through mining a gene co-expression network database. Preliminary studies revealed that EREI1 is localized to ER and interacts in vivo with the ER-localized Arabidopsis E2s UBC7, UBC13, and UBC14. In this study, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) gene editing technology was employed to develop Arabidopsis transgenic lines where the EREI1 gene is mutated. Out of two sites within the EREI1 gene that were identified to be putatively desirable for targeted mutation by the CRISPR/Cas9 technology, gRNA targeting a site within the first exon of the EREI1 gene was used for transforming Col-0 plants. Sequence analysis confirmed two independent mutation events in the T2 plant population where a ‘A’ and ‘T’, respectively was inserted after the 52nd nucleotide downstream the start codon of the EREI1 gene, resulting in a premature stop codon such that the mutated gene would produce a 31-amino-acid-only peptide. Arabidopsis plants that are Cas9-free and homozygous at the two mutations were further screened and obtained in the T3 generation. Bacterial growth assay using the homozygous mutant lines and wild type Col-0 plants indicated that the mutant plants displayed comparable growth of the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) strain DC3000 to that of the Col-0 plants three days after inoculation. Measurement of reactive oxygen species (ROS) production in the leaves after flg22 treatment revealed no significant difference between the control Col-0 plants and the mutant lines. Examination of the expression pattern for the EREI1 gene at 0-, 3-, 6-, and 12-hours post inoculation of Pst DC3000 by quantitative PCR was inconclusive potentially due to the extremely low abundance of the EREI1 transcript. Thus, these preliminary characterizations were unable to support the notion that EREI1 plays a key role in plant immunity.
Advisor: Lirong Zeng
A THESIS Presented to the Faulty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Plant Pathology, Under the Supervision of Professor Lirong Zeng. Lincoln, Nebraska: May 2023
Copyright © 2023 Mitchell A. Hockbein