Volume 6, Issue 5, Pages 1592-1604 (September 2013) Intracellular and Extracellular Phosphatidylinositol 3-Phosphate Produced by Phytophthora Species Is Important for Infection  Shan Lu, Linlin Chen, Kai Tao, Nannan Sun, Yuren Wu, Xiaoxue Lu, Yuanchao Wang, Daolong Dou  Molecular Plant  Volume 6, Issue 5, Pages 1592-1604 (September 2013) DOI: 10.1093/mp/sst047 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Secreted PtdIns(3)P-Binding Proteins Expressed in N. benthamiana Are Targeted to Infecting P. parasitica Hyphae. (A, B) Observations of P. parasitica hyphae in N. benthamiana leaf tissues expressing PtdIns(3)P-binding proteins (A), and various mutants of Avr1b (B). (C) Confocal microscopic images of PI3K inhibitor LY294002 (20 μM) treatment of P. parasitica hyphae. FYVE1 and FYVE2 indicate fusions of GFP with duplicated PtdIns(3)P-binding FYVE domains from EEA1 and Hrs proteins, respectively. Avr1b proteins were full-length (FL), C-terminus (Ct; lacking residues 22–69), N-terminus (Nt; containing only residues 1–69, including sp), RxLR mutant (r–; RSLR->AAAA, RFLR->AAAA), dEER mutant (d–, EEDDAGER->AAAAAGAA), RxLR, dEER double mutant (r–,d–). sp indicates the presence of a secretory signal peptide from soybean PR1a (FYVE constructs) or from Avr1b (Avr1b and GFP constructs). The N. benthamiana leaves were infected with P. parasitica hyphae 3 d after infiltration, and the GFP signal of infectious hyphae was detected 36 h later. The invasive hyphae are marked by arrows. Bar = 100 μm. Molecular Plant 2013 6, 1592-1604DOI: (10.1093/mp/sst047) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Expression of Secreted or Non-Secreted PtdIns(3)P-Binding Proteins in P. sojae Reduces Virulence. (A) Verification of the presence of the transgene by genomic DNA PCR. spFYVE2 and FYVE2 are secreted and non-secreted Hrs 2xFYVE–GFP fusions cloned into P. sojae expression vector. + indicates the plasmid DNA positive control. P6497 indicates the non-transformed wild-type isolate. M indicates markers. (B) Relative expressional levels of GFP gene in the indicated transformants. GFP18 is a transgenic P. sojae line with GFP gene only. The P. sojae TEF1 gene was used as a reference. Bars represent standard errors from two independent RNA isolations and qRT–PCR replicates. (C) Localization of expressed GFP proteins in P. sojae transformants. (D) Symptoms produced by wild-type and transgenic P. sojae lines on etiolated seedling hypocotyls. The inoculated seedlings were photographed at 36 hpi. The two least virulent transformants (FYVE2-T16 and spFYVE2-T328) (see Figure 2E) are shown. (E) Virulence of wild-type and all transgenic P. sojae lines measured by Q–PCR assays of pathogen DNA levels in infected seedling hypocotyls relative to host DNA. The experiments were repeated four times in all transformants with similar results. Stars above bars indicate significant differences between the control and each transgenic line (Dunnett’s test: * p < 0.05; ** p < 0.01). (F) Microscopic observations of the defense response of soybean root epidermal cells at 10 hpi. The experiments were repeated four times in all transformants, with similar results. The two least virulent transformants are shown. Molecular Plant 2013 6, 1592-1604DOI: (10.1093/mp/sst047) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Transcriptional Profiles of P. sojae PI3K Genes. Transcript levels fold of the five PI3K genes was calculated relative to the level at 0 h. The P. sojae TEF1 gene was used as a reference. Error bars indicate SE = SD/sqrt(n) and SD consistent in three technical replicates. Lines were analyzed for significant differences in their expressional levels based on Duncan’s multiple range test among the means on the analysis of variance (P < 0.05). Lines designated with the same letter exhibit no significant difference in the tested time points. Molecular Plant 2013 6, 1592-1604DOI: (10.1093/mp/sst047) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Gene Silencing Indicates that P. sojae Genes PI3K1 and PI3K2 Are Required for Full Virulence. (A) Verification of the presence of the transgenes by genomic DNA PCR analysis. T32 and T37 were silenced using PsPI3K1 while T94 and T114 were silenced with PsPI3K2. + indicates the plasmid DNA positive control, WT indicates wild-type strain P6497, and M indicates size markers. (B) Relative transcript levels of five PI3K genes in the silenced transformants, measured by qRT–PCR. Levels were standardized to the wild-type (WT; P6497) using TEF1 transcripts as the reference gene. Bars represent standard errors from three independent RNA isolations and quantitative RT–PCR replicates. Stars above bars indicate significant differences between the control and each transgenic line (Dunnett’s test, p < 0.05). (C) Symptoms produced by wild-type and silenced P. sojae lines on etiolated seedling hypocotyls. Susceptible soybean cultivar Williams was used. The photos were taken at 36 hpi. Two representative silenced lines (T37 and T114) are shown as examples. (D) Virulence of wild-type and all silenced P. sojae lines measured by Q–PCR assays of pathogen DNA levels in infected seedling hypocotyls relative to host DNA. The infection level of the wild-type was set as 1. Samples were taken at 36 hpi. The means and SEs were calculated from five seedlings. Stars above bars indicate significant differences between the control and each transgenic line (Dunnett’s test, p < 0.01). (E) Labeling of hyphae of P. sojae wild-type and PI3K-silenced lines by PtdIns(3)P biosensor proteins following inoculation onto N. benthamiana leaves expressing the biosensors. Pictures were taken at 4 dpi. The arrows indicate invasive hyphae. Bar = 100 μm. Left panels, fluorescence images; right panel, bright field images; center panels, merged images. Molecular Plant 2013 6, 1592-1604DOI: (10.1093/mp/sst047) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Treatment with PI3K Inhibitor LY294002 Decreases the Virulence of P. sojae Hyphae. (A) Etiolated seedlings of the susceptible soybean cultivar Williams were inoculated with P. sojae hyphae that had been treated with ddH2O or 30 μM LY294002. The photographs were taken 36 h after inoculation (36 hpi). (B) Dose-dependent effects of LY294002 on P. sojae virulence. The lesion lengths on the inoculated seedlings were measured 12 h, 24 h, or 36 h following inoculation. The hyphae were treated with indicated concentration of LY294002 or water (CK). The lengths shown were averaged from five seedlings each from two independent experiments. The bars indicate standard errors. Stars above bars indicate significant differences between the control and each transgenic line (Dunnett’s test: * p < 0.05; ** p < 0.01). Molecular Plant 2013 6, 1592-1604DOI: (10.1093/mp/sst047) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Secretion of PtdIns(3)P-Binding Proteins or AtPIP5K1 Increases Resistance of N. benthamiana to Phytophthora Infection. (A) Average lesion area of N. benthamiana leaves transiently expressing the indicated genes, relative to leaves expressing the control gene (GFP). Averages were calculated from 10 lesions per construct. Error bars represent standard errors. * and ** indicate significance at p < 0.05 or < 0.01, respectively (Dunnett’s test). (B) Comparison of P. parasitica lesions on N. benthamiana leaves expressing GFP or the secreted PtdIns(3)P-binding proteins. The upper panel shows a decolorized infected leaf photographed under UV illumination at 36 hpi. The lower panels show trypan blue staining of lesions, revealing a lower hyphal density in the presence of spFYVE1. The experiments were repeated four times and shown with a representative image. (C) P. capsici colonization of N. benthamiana leaves transiently expressing the indicated genes, measured by Q–PCR assays of pathogen DNA levels relative to host DNA. The infection level in leaves expressing GFP was set as 1. Samples were taken at 36 hpi. The means and SEs were calculated from five leaves. * and ** indicate significance at p < 0.05 or < 0.01, respectively (Dunnett’s test). (D) P. capsici colonization of N. benthamiana leaves observed by trypan blue staining. The leaves were inoculated with P. capsici zoospores and stained 16 hpi. Leaves transiently expressing spFYVE1 exhibit a lower hyphal density than with GFP. The experiments were repeated four times with spFYVE1; the figure shows representative images. (E) RT–PCR assays of expressional levels of the genes in N. benthamiana. C, + and – indicate amplification products from the target gene, plasmid DNA control, and no transgenic leaves, respectively. Molecular Plant 2013 6, 1592-1604DOI: (10.1093/mp/sst047) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions