Functional Characterizations Of Phosphatidylinositol 3-Phosphate In Interactions Of Nicotiana Benthamiana And Phytophthora Pathogens

The genus Phytophthora contains many devastating plant pathogens, among which P. infestans and P. sojae are two of the most notorious species. The genome of each Phytophthora species encodes hundreds of host cytoplasmic effectors to alter host physiology for successful colonization. One major type is RxLR effectors, which are defined as modular secreted proteins that contain the characteristic amino-terminal motif RxLR-dEER with rapidly evolving C-terminal effector domains. RxLR effectors have been proposed to bind to PtdIns(3)P to mediate their translocation into host cells, and/or to increase their stability in planta. However it remains unclear whether Phytophthora pathogens exclusively utilize host PtdIns(3)P or whether they produce additional PtdIns(3)P themselves. In this study, we showed that Phytophthora pathogens may produce PtdIns(3)P to promote their infection by binding with RxLR effectors. Secretion by plants of proteins that sequester or metabolize PtdIns(3)P could reduce Phytophthora infection, suggesting a novel strategy for disease management. Additionly, we found that phosphoinositide 3-kinase complexes related genes might participate N. benthamiana resistance to Phytophthora pathogens.Localization and source analysis of PtdIns(3)P during the interaction between Phytophthora species and Nicotiana benthamiana. We found that GFP flurecent signals predominatly accumulated on Phytophthora hyphae during infection of N. benthamiana leaves transiently secreting PtdIns(3)P-specific GFP biosensors (FYVE-GFP). The levels of the biosensor accumulating on the hyphae were significantly reduced following pre-treatment of the P. parasitica hyphae with PI3K inhibitor LY294002. The accumulation levels of the biosensor were also significantly reduced on the PI3K-silenced transformants of P. sojae. These results suggest that the PtdIns(3)P on the surface of the hyphae may be produced by the pathogen itself. In addition, we used various Avrlb derivatives as PtdIns(3)P biosensors also provides valuable information about which domains of this effector are involved in binding PtdIns(3)P in the context of an actual infection, and we found that the C-terminal domain of Avrlb plays a major role in PtdIns(3)P binding while the RxLR domain plays a more subtle role. These results suggest that Phytophthora pathogens may produce PtdIns(3)P to facilitate infection by binding with RxLR effectors.A novel strategy for disease control:expression secreted proteins to dicrease the PtdIns(3)P levels in planta. PtdIns(3)P have been proposed to bind with effectors during the interaction of plant and Phytophthora pathogens. And Phytophthora may produce PtdIns(3)P to facilitate itself infection, pointing to a strategy for disease control by blocking or metabolizing PtdIns(3)P through expressing secreted PtdIns(3)P-binding proteins or a PtdIns(3)P-5-kinase. We observed that the lesion areas and the density of hyphae infection with P. parasitica were significantly smaller in N. benthamiana leaves transiently expressing spFYVE or spAtPIP5Kl. Then we examined the resistance levels of leaves transiently expressing the constructs to P. capsici, we found that the hyphal accumulation in planta by microscopic observation and by real-time PCR measurement of the ratio of pathogen-to-host DNA levels were significantly reduced. For further investigate, we obtained stable transgenic N. benthamiana plants and tested their resistance to P. capsici and P. parasitica. The results showed that the lesion diameters and the density of hyphae infected with the two Phytophthora were significantly reduced in transgenic plants expressing spFYVE or spAtPIP5K1, the AOS accumulation and callose deposition at the infection sites were increased distinctly. These results suggest that the plant resistance could be improved by expressing secreted fusion proteins to decrease the PtdIns(3)P contents.Functional characterizations of the PI3K complexes related genes in the resistance of Nicotiana benthamiana. To further investigate the function of plant PtdIns(3)P during plant resistance, we focus on the crucial components of phosphoinositide 3-kinase complexes (VPS 15, VPS30 and VPS34). Here we observed that the expression level of NbVPS15 and NbVPS34 genes were differently up-regulated during the infection stages of two Phytophthora pathogens (Pc35 or P6497), indicating that they may involve in N. benthaminana resistance to Phytophthora. Silencing NbVPS15 or NbVPS34 via Virus-induced gene silencing increases resisitance of N. benthamiana to Phytophthora infection, and the AOS accumulation and callose deposition were also increased. In contrast, silencing of the three indicated NbVPS genes did not result in resistance to the necrotrophic fungus Botrytis cinerea. We also found that silencing NbVPS34 reduced the stability of P. sojae RxLR effectors Avrlb and Avh18 in N. benthamiana leaves, and NbVPS15, NbVPS30 and NbVPS34 is differently required to limit spread of HR PCD induced during elicitin, effectors or R gene-mediated resistance. These results suggest that NbVPS15 or NbVPS34 might participate N. benthamiana resistance to Phytophthora pathogens by regulation PtdIns(3)P contents and PCD in plant cells.