Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry.
Oomycetes such as Phytophthora sojae employ effector proteins that enter plant cells to facilitate infection. Entry of some effector proteins is mediated by RxLR motifs in the effectors and phosphoinositides (PIP) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effectors involved in PIP recognition. We have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that Avh5 is helical in nature, with a long N-terminal disordered region. NMR titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate, directly identified the ligand-binding residues. A C-terminal lysine-rich helical region (helix 2) was the principal lipid-binding site, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Mutations in the RFLR motif or in the basic region both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. Based on these findings, we propose a model where Avh5 interacts with PtdIns(3)P through its C terminus, and by binding of the RFLR motif, which promotes host cell entry.
Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA.
Furong
F
Sun
Shiv D
SD
Kale
Hugo F
HF
Azurmendi
Dan
D
Li
Brett M
BM
Tyler
Daniel G S
DG
Capelluto
D020816:Amino Acid Motifs; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D006790:Host-Parasite Interactions; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010838:Phytophthora / Q000166:cytology / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D010935:Plant Diseases / Q000469:parasitology*; D018517:Plant Roots / Q000469:parasitology; D011485:Protein Binding; D055550:Protein Stability; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013025:Glycine max / Q000469:parasitology*; D020349:Surface Plasmon Resonance; D013696:Temperature
D018129:Phosphatidylinositol Phosphates; D011506:Proteins; D011993:Recombinant Fusion Proteins; C055525:phosphatidylinositol 3-phosphate