Title page for ETD etd-04232012-191603


Type of Document Dissertation
Author Sun, Furong
URN etd-04232012-191603
Title Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Capelluto, Daniel G. S. Committee Chair
Santos, Webster L. Committee Co-Chair
Bevan, David R. Committee Member
Finkielstein, Carla V. Committee Member
Lazar, Iuliana M. Committee Member
Wi, Sungsool Committee Member
Keywords
  • Phytophthora sojae
  • Protein-lipid interactions
  • Phosphatidylinositol 3-phosphate
  • Avirulence homolog-5
Date of Defense 2012-04-09
Availability restricted
Abstract
Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using NMR spectroscopy, I demonstrate that Avh5 is helical in nature with a long N-terminal disordered region. Heteronuclear single quantum coherence titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate (Ins(1,3)P2), allowed us to identify a C-terminal lysine-rich helical region (helix 2) as the principal lipid-binding site in the protein, with the N-terminal RxLR (RFLR) motif playing a more minor role. Furthermore, mutations in the RFLR motif slightly affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Avh5 exhibited moderate affinity for PtdIns(3)P, which increased the thermal stability of the protein. Mutations in the RFLR motif or in the basic region of Avh5 both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. My findings support a model in which Avh5 transiently interacts with PtdIns(3)P by electrostatic interactions mainly through its positively charged helix 2 region, providing stability to the protein during RFLR-mediated host entry.
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