Functional Analysis Of Phoshatidylinositol3-Phsphate Produced By Phytophthora Sojae During Infection

Phytophthora sojae is an important oomycete pathogen, and causes "damping off" in seedlings and root rot of soybean, resulting in an annual loss of more than1billion dollar in the whole world. P. sojae is a soilbone pathogen and almost obligate to soybean (Glycine max). It has an initial phase of biotrophic growth requiring living host cells to support their obligate parasitism, followed by a transition to necrotrophy that may actively kill its host during the disease process. Thus the manipulation of cell death may be complicated. This pathogen delivers a variety of extracellular effectors into the plant apoplast to maniplulate cell death. It is known that these effectors, such as Avrlb, can bind with phosphatidylinositol-3-phosphate (PI3P) to enter into host cells. However, it is still unclear whether efffector use PI3P produced by pathogen or host in the interaction. Here, we selected FYVE domain of Hrs gene in mice to study the roles of PI3P in P. sojae because FYVE domain can specifically bind with PI3P to sequester its content in the cell. The domain is also called as PI3P biosensor.We first constructed the vectors containing two duplicated FYVE domains fusion with GFP gene for P. sojae transformation with signal peptide or without signal peptetide, to express FYVE inside or outside cells. Then, we obtained the transgenic P. sojae lines based on PEG mediated protoplast transformation method. The phenotype analysis showed that the growth characterization is smilar to the wild type whether singal peptide is present or not. However, the FYVE domain is mainly distributed at the endomembrane vesicles inside the cells when the singal peptide os present. In contrast, the signal peptide will drive GFP outside of the cells. Virulent assays showed that both transformants exhibited lower pathogencity, comparing to the wild type and trasnformants with GFP gene only. Thus, we conclude that PI3P produced by the pathogen is important for the infection, which is first report of PI3P functions in virulence of oomycete pathogens. Our results provides a reference for understanding of the roles of PI3P and molecular mechanism in the process of infection.