Search for a biological role of elicitins secreted by the soilborne pathogen of soybean Phytophthora sojae.

Lina Yousef, 210 Kottman Hall, Ohio State University - Columbus, Ohio State University, 2021 Coffey Road, Columbus, OH 43210, Richard Dick, 406C Kottman Hall, 2021 Coffey Road, Ohio State University - Columbus, Ohio State University, School of Environment and Natural Resources, Columbus, OH 43210-1085 and Warren Dick, 1680 Madison Ave., Ohio State University - OARDC, Ohio State University, School of Natural Resources, Wooster, OH 44691-4096

The genus Phytophthora comprises a large number of soilborne plant pathogens that completely lack sterol biosynthetic ability, and must acquire sterols from the environment to support their growth and development.The mechanism of sterol uptake by Phytophthora is unknown, but may involve a conserved group of small extracellular proteins named elicitins, because in vitro studies have shown elicitins to trap and transfer sterols between phospholipidic and plant plasma membrane preparations.This suggests elicitins might be sterol scavengers, but conclusive evidence for their exact role in Phytophthora is still lacking.Moreover, elicitins are the most abundant proteins present in spent filtrates of Phytophthora spp., indicating a significant role due to the high expenditure of energy to these organisms.We are using the model organism and soybean pathogen Phytophthora sojae to study the biological role of elicitins in Phytophthora.It is technically challenging to generate null mutants for studying gene function in Phytophthora due to the diploid nature and gene redundancy of members from this genus.We designed an experiment to circumvent this problem, and allow us to study the role of elicitins in Phytophthora.Elicitins of P. sojae were labeled with ¹⁵N, and then placed into the growth medium of P. sojae with or without added sterols.  Following this, tissue of P. sojae was harvested and analyzed on an IR-MS for shifts in the ¹⁵N signature over time.  It is hypothesized that signature shifts will be observed only when sterols are added to the growth medium of P. sojae, indicating the involvement of ¹⁵N-elicitins in sterol uptake by Phytophthora.   Labeling of elicitins was done by expressing them as six HIS-tagged recombinant proteins in Escherichia coli using a minimal M9 medium with ¹⁵NH₄Cl as the sole nitrogen source.