Volume 12, Issue 4, Pages 552-564 (April 2019) Phytophthora sojae Effector PsAvh240 Inhibits Host Aspartic Protease Secretion to Promote Infection  Baodian Guo, Haonan Wang, Bo Yang, Wenjing Jiang, Maofeng Jing, Haiyang Li, Yeqiang Xia, Yuanpeng Xu, Qinli Hu, Fangfang Wang, Feng Yu, Yan Wang, Wenwu Ye, Suomeng Dong, Weiman Xing, Yuanchao Wang  Molecular Plant  Volume 12, Issue 4, Pages 552-564 (April 2019) DOI: 10.1016/j.molp.2019.01.017 Copyright © 2019 The Authors Terms and Conditions

Figure 1 PsAvh240 Promotes P. sojae Infection in Soybean and Is a Plasma Membrane-Localized Effector. (A) Overexpression of PsAvh240 in soybean hairy roots promotes P. sojae infection. Soybean hairy roots expressing the empty vector (EV) or GFP-PsAvh240 were inoculated with RFP-labeled P. sojae strain P6497 (WT-RFP). Scale bars, 0.2 mm. Relative biomass of P. sojae was determined by qPCR at 48 h post inoculation (hpi). The means and standard errors from six replicates are shown (**P < 0.01; one-way ANOVA). (Right) PsAvh240 recombinant protein is detected in soybean hairy roots. Western blot analysis of protein in soybean hairy roots expressing GFP or GFP-PsAvh240. EV, empty vector. (B) PsAvh240 localizes to the plasma membrane. Expression of GFP-PsAvh240 or PsAvh240-RFP fusion proteins in N. benthamiana through agroinfiltration revealed that PsAvh240 is localized at the cell periphery. Fluorescence from epidermal cells in the infiltrated tissues was observed by confocal microscopy at 36 hpi. Scale bars, 15 μm. (C) Fluorescence intensity profiles of GFP-PsAvh240 and Remorin-RFP in membrane transects (white arrowheads). y axis, GFP or RFP relative fluorescence intensity; x axis, transect length (μm). Molecular Plant 2019 12, 552-564DOI: (10.1016/j.molp.2019.01.017) Copyright © 2019 The Authors Terms and Conditions

Figure 2 Overall Structure of PsAvh240. (A) Schematic representation of the domain architecture of PsAvh240. PsAvh240 contains a signal peptide, an RxLR motif, and an effector domain, which are shown as different colors. (B) Ribbon diagram of the overall structure of the PsAvh240 effector domain. Six α helices in the effector domain include two WY domains. The helix α2 is distorted at Gln96, displayed as sticks. The helix α1 and the N-terminal segment of helix α2 are colored yellow. The WY1 and WY2 motifs are colored gray and red, respectively. The conserved residues (W104, Y140, W148, Y183) in the WY motif are shown as sticks. (C) Sequence alignment of the WY1 and WY2 motifs. The WY1 and WY2 motifs share 43% sequence identity. The identical residues are highlighted in blue. (D) Structure alignment of the WY1 and WY2 motifs. A 50° rotation allows the WY2 motif to be superimposed nicely on the WY1 motif, with a Cα RMSD of 1.36 Å. (E) Structure alignment of the WY1 motif, WY2 motif, and PexRD2. The WY1 and WY2 motifs adopt a similar structure as PexRD2, with Cα RMSD values of 1.85 Å and 2.36 Å, respectively. Molecular Plant 2019 12, 552-564DOI: (10.1016/j.molp.2019.01.017) Copyright © 2019 The Authors Terms and Conditions

Figure 3 PsAvh240 Functions as a Dimer. (A) Two PsAvh240 molecules in the crystal asymmetric unit are assembled as a dimer and they form extensive intermolecular interactions through the WY2 motif (colored by red). (B) The residues located in dimerization interface are shown by stick and colored by grey (from one molecule) and red (from the other molecule). D156 forms several hydrogen bonds with D156 from the other subunit and Y177, Y180, and Y181 contribute to formation of four intermolecular hydrogen bonds. Dashed lines represent hydrogen bonds. Y159, Y180, L184, and F188 form hydrophobic interactions. (C) Blue native gel electrophoresis analysis of purified untagged PsAvh240. The band of PsAvh240 in the gel corresponds to 30–40 kDa. (D) Dimerization of PsAvh240 in vitro. GST-PsAvh240 and His-PsAvh240 were expressed in E. coli. Coprecipitation of His-PsAvh240 with GST-PsAvh240 was examined by western blotting before (input) and after affinity purification (pull-down) using glutathione agarose beads. (E) Self-association of PsAvh240 in planta. Total proteins were extracted from N. benthamiana leaves expressing GFP-PsAvh240 and PsAvh240-HA. The immune complexes were pulled-down by GFP-Trap_A beads, and the coprecipitation of PsAvh240-HA was detected by western blotting. (F) PsAvh240 harboring a mutation disrupting dimerization of PsAvh240 lost its function in promoting P. sojae infection in soybean hairy roots. Soybean hairy roots expressing EV, GFP-PsAvh240, GFP-PsAvh240-Y180A were inoculated with RFP-labeled P. sojae strain P6497 (WT-RFP). Scale bars, 0.2 mm. (G) Relative biomass of P. sojae was determined by qPCR at 48 hpi. The means and standard errors from six replicates are shown (∗∗P < 0.01; one-way ANOVA). Molecular Plant 2019 12, 552-564DOI: (10.1016/j.molp.2019.01.017) Copyright © 2019 The Authors Terms and Conditions

Figure 4 The First Two α Helices of PsAvh240 Are Required for Localization in the Plasma Membrane and Virulence Function. (A) Schematic view of the PsAvh240 deletion mutants. (B) Subcellular localization of PsAvh240 deletion mutants in N. benthamiana. GFP-PsAvh240 or GFP-tagged PsAvh240 deletion mutants were expressed in N. benthamiana through agroinfiltration. Fluorescence from epidermal cells in the infiltrated tissues was detected by confocal microscopy at 36 h post agroinfiltration. Scale bars, 20 μm. (C) Virulence assay of PsAvh240 deletion mutants in soybean hairy roots. Soybean hairy roots expressing EV, GFP-PsAvh240, or GFP-PsAvh240-M3 were inoculated with RFP-labeled P. sojae strain P6497 (WT-RFP). Relative biomass of P. sojae was determined by qPCR at 48 hpi. The means and standard errors from six replicates are shown (**P < 0.01; one-way ANOVA). Scale bars, 0.2 mm. Molecular Plant 2019 12, 552-564DOI: (10.1016/j.molp.2019.01.017) Copyright © 2019 The Authors Terms and Conditions

Figure 5 PsAvh240 Interacts with Soybean Aspartic Protease GmAP1. (A) PsAvh240 interacts with GmAP1 in planta. Total proteins were extracted from N. benthamiana leaves expressing GmAP1-HA and GFP-PsAvh240 or the deletion mutant GFP-PsAvh240-M3. The immune complexes were pulled down by using GFP-Trap_A beads, and the coprecipitation of GmAP1 was detected by western blotting. (B) PsAvh240 colocalizes with GmAP1 in the plasma membrane. GFP or the GFP-PsAvh240 fusion protein was coexpressed with GmAP1-RFP in N. benthamiana through agroinfiltration. Fluorescence from epidermal cells in the infiltrated tissues was detected by confocal microscopy at 36 h post agroinfiltration. Scale bars, 10 μm. (C) Fluorescence intensity profiles of GFP-PsAvh240 and GmAP1-RFP in membrane transects (white arrowheads). y axis, GFP or RFP relative fluorescence intensity; x axis, transect length (μm). (D) PsAvh240 specifically interacts with GmAP1 and GmAP2 in planta. Total proteins were extracted from N. benthamiana leaves expressing GFP-PsAvh240 and GmAP1-HA or its one of its homologs. The immune complexes were pulled down by using GFP-Trap_A beads, and the coprecipitation of GmAP1 or its homologs was detected by western blotting. Molecular Plant 2019 12, 552-564DOI: (10.1016/j.molp.2019.01.017) Copyright © 2019 The Authors Terms and Conditions

Figure 6 GmAP1 Contributes to Soybean Resistance against Phytophthora sojae. (A) Soybean hairy roots expressing EV or GmAP1-GFP were inoculated with RFP-labeled P. sojae strain P6497 (WT-RFP). Scale bars, 0.2 mm. (B) Relative biomass of P. sojae was determined by qPCR at 48 hpi, and means and standard errors from six replicates are shown (*P < 0.05; one-way ANOVA). (C) GmAP1 recombinant protein was detected in the soybean hairy roots by western blot. (D) Silencing of GmAP1/GmAP2 in soybean hairy roots followed by inoculation with RFP-labeled P. sojae strain P6497 (WT-RFP). Scale bars, 0.2 mm. (E) Relative expression levels of GmAP1/GmAP2 and GmAP3/GmAP4 determined by qPCR. The means and standard errors from six replicates are shown (***P < 0.001; one-way ANOVA). (F) Relative biomass of P. sojae in GmAP1-silenced soybean hairy roots determined by qPCR at 48 hpi. The means and standard errors from six replicates are shown (**P < 0.01; one-way ANOVA). Molecular Plant 2019 12, 552-564DOI: (10.1016/j.molp.2019.01.017) Copyright © 2019 The Authors Terms and Conditions

Figure 7 PsAvh240 Inhibits the Secretion of GmAP1. (A) PsAvh240 inhibits the secretion of GmAP1 in planta. GFP, GFP-PsAvh240, or the deletion mutant GFP-PsAvh240-M3 was co-expressed with GmAP1-HA in N. benthamiana. Apoplastic fluid and intracellular leaf extracts were separated and detected by western blot. The gel stained with Coomassie brilliant blue shows the overall protein level of apoplastic fluid. (B) PsAvh240-Y180A cannot inhibit the secretion of GmAP1 in planta. The experimental procedures same as (A). (C) PsAvh240 specifically inhibits the secretion of GmAP1 and GmAP2 in planta.The experimental procedures same as (A). (D) A proposed model illustrating the virulence mechanism of PsAvh240. Molecular Plant 2019 12, 552-564DOI: (10.1016/j.molp.2019.01.017) Copyright © 2019 The Authors Terms and Conditions