Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry

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dc.contributor.authorSun, Furongen
dc.contributor.committeechairCapelluto, Daniel G. S.en
dc.contributor.committeecochairSantos, Webster L.en
dc.contributor.committeememberWi, Sungsoolen
dc.contributor.committeememberLazar, Iuliana M.en
dc.contributor.committeememberFinkielstein, Carla V.en
dc.contributor.committeememberBevan, David R.en
dc.contributor.departmentChemistryen
dc.date.accessioned2014-03-14T21:10:34Zen
dc.date.adate2012-05-04en
dc.date.available2014-03-14T21:10:34Zen
dc.date.issued2012-04-09en
dc.date.rdate2012-05-04en
dc.date.sdate2012-04-23en
dc.description.abstractOomycetes, 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.en
dc.description.degreePh. D.en
dc.identifier.otheretd-04232012-191603en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-04232012-191603/en
dc.identifier.urihttp://hdl.handle.net/10919/37657en
dc.publisherVirginia Techen
dc.relation.haspartSun_F_D_2012.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectPhytophthora sojaeen
dc.subjectProtein-lipid interactionsen
dc.subjectPhosphatidylinositol 3-phosphateen
dc.subjectAvirulence homolog-5en
dc.titleStructural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entryen
dc.typeDissertationen
thesis.degree.disciplineChemistryen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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