University of Missouri-Columbia Cape Girardeau, Missouri
Body of Abstract: Because plants are a major food source for humans and life-stock, understanding how plants protect themselves against pathogenic infection is vital to protect the world’s food supply. With large percentages of yearly crop yield lost to pathogens, our lab’s research will provide new insights into how to engineer more resistant plants, reducing crop losses and providing food security for a rapidly growing world population.
The Heese lab focuses on the cellular machinery that shuttles the critical plant immune receptor Flagellin Sensing2 (FLS2) to the cell surface. FLS2 must be at the cell surface to detect extracellular bacteria and trigger defense responses. EPSIN1 (EPS1) is an adaptor protein which plays a role in the formation of vesicles trafficked to the cell surface. Our lab has published that Arabidopsis eps1 null mutants are more susceptible to infection by pathogenic bacteria due to decreased levels of FLS2 and other defense proteins at the cell surface. EPS1 has an Epsin N-terminal Homology (ENTH) domain which is highly conserved between plant orthologs; but role(s) of ENTH in physiological response is unknown for any plant EPSIN [3].
As a first step towards understanding roles of plant ENTH-domains, my project goal is to produce a polyclonal antibody against the EPS1 ENTH domain and confirm its specificity. I expressed a 6His-tagged ENTH domain into bacterial cells and purified it using nickel affinity chromatography. I then sent my purified ENTH protein to the company Eurogentec for polyclonal antibody production in rabbits. I received and tested the antibody bleeds to confirm by immunoblot analysis using Col-0 wild-type and eps1 mutants that these αENTH antibodies detect Arabidopsis EPS1 in vivo. My future work will assess whether this antibody also detects Zea mays EPS1 and Arabdiopsis EPS1 deletion forms in planta to gain insight into the physiological roles of EPS1 subdomains.