Insight into the Structural Basis and Intermolecular Interactions of the Phytophthora Sojae Avirulence Homolog-5 with Phosphatidylinositol-3-Phosphate

Abstract

Listen

Translate article

Oomycetes, such as P.sojae, are plant pathogens that employ protein effectors (Avirulence or Avr proteins) to facilitate infection. Plants are able to overcome this infection by their ability to recognize pathogen effectors by the plant defense system. The entry of Avr proteins to plant cells is mediated by external membrane-bound phosphoinositides such as phosphatidylinositol-3-phosphate (PtdIns(3)P). Furthermore, the N-terminal RXLR motif of several Avr proteins is critical for phosphoinositide recognition. However, a recent report indicates that the C-terminal basic domain of related effectors, Avr3a and Avr1b, is relevant for phosphoinositide recognition. Because there is a need to clarify these differences, we have structurally and functionally characterized P.sojae Avirulence homolog-5 (Avh5). Using NMR spectroscopy, we demonstrate that Avh5 is helical in nature with a long N-terminal disordered region. HSQC titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate (Ins(1,3)P2), allowed us to map two lipid-binding regions that comprise the basic-rich second helix and the charged fourth helix. The first residue of the RXLR motif, Arg24, was also perturbed by Ins(1,3)P2. Using both liposome-binding and lipid-protein overlay assays, we demonstrate that whereas mutations in RXLR slightly affect PtdIns(3)P binding; mutations in the basic second helix (residues Lys62, Lys64, and Lys65) almost abolished it. Avh5 exhibited moderate affinity for PtdIns(3)P (KD=2.3 μM) as determined using surface plasmon resonance. Consistent with our lipid-binding assays, mutations in the RXLR and the basic patch reduced the affinity for the phosphoinositide 1.5- and 44-fold, respectively. Thus, our findings suggest that the central Avh5 basic-rich region has a major role in PtdIns(3)P recognition. Therefore, our identification of PtdIns(3)P-binding site of Avh5 will provide the structural basis to understand the role of the lipid in facilitating entry of Avr proteins into plant cells.