Fig 1.
PsGPA1 physically interacts with the N-terminal region of PsYPK1 in vitro and in vivo.
(A) The domain architecture of PsYPK1. The numbers under the STKc (serine/threonine protein kinases, catalytic) domain are the amino acid positions in the full-length protein. (B) Confirmation of the association of PsGPA1 and PsYPK1 in vitro. GST-PsGPA1- or GST-bound resins were incubated with E. coli supernatant containing His-PsYPK1, or the N-terminal or the C-terminal portions of PsYPK1. The presence of His-tagged proteins was detected by western blot analysis using a His antibody. (C) Validation of the association between PsYPK1 and PsGPA1 in vivo. Co-immunoprecipitations (Co-IP) were performed in extracts of Phytophthora sojae mycelium expressing PsGPA1-FLAG with PsYPK1-GFP or PsYPK1ΔN-GFP. The presence of FLAG-tagged proteins was detected by western blot analysis using a FLAG antibody. The bands detected with anti-GFP were quantified with the ODYSSEY infrared imaging system (application software version 2.1).
Fig 2.
PsYPK1 is important for growth in nutrient poor conditions and for sporangium and oospore production.
(A) Growth characteristics after 4 days on V8 medium and 7 days on Plich medium, and microscopic visualization of oospores and sporangia of the wild-type (P6497), PsYPK1-knockout (ΔPsYPK1), empty vector control line (EV), complemented transformants (ΔPsYPK1-C1, C2), and empty control line of ΔPsYPK1 (ΔPsYPK1-EV). (B) Growth rates in cm/day on V8 medium and (C) Plich medium. (D) The relative numbers of sporangia and (E) oospores with the number in wild-type strain P6497 set at 1. All experiments were repeated three times with similar results. Scale bar, 50 μm. Asterisk indicates significant difference at P<0.01 (**).
Fig 3.
The PsYPK1 knockout mutant is significantly impaired in virulence.
(A) Lesions on soybean (cultivar Hefeng 47) at 72 hours post inoculation (hpi) of 4-day-old etiolated hypocotyls with zoospores of the wild-type strain (P6497), PsYPK1-knockout (ΔPsYPK1), empty vector control line (EV), complemented transformants (ΔPsYPK1-C1, C2), and empty control line of ΔPsYPK1 (ΔPsYPK1-EV). Experiments were repeated three times with similar results. (B) Relative pathogen biomass in inoculated etiolated hypocotyls expressed as the ratio between the amounts of P. sojae DNA and soybean DNA detected at 72 hpi with the ratio P6497/soybean set at 1. Asterisks indicate significant differences at P<0.01 (**). (C) Microscopic observations of invasive hyphae in epidermis of soybean hypocotyls at 12 hpi and 24 hpi. Trypan Blue staining was performed on the epidermis of seedling hypocotyls. Red arrowheads, cysts; black arrowheads, invasive hyphae. Bar, 10 μm. (D) Percentage of abnormal invasive hyphae in epidermal cells of soybean hypocotyls at 24 hpi. In each sample 50 invasive hyphae were examined and the experiments were repeated three times.
Fig 4.
PsGPA1 negatively regulates sporangium formation in P. sojae.
(A) Expression of PsGPA1 in hyphae of PsGPA1 silenced and overexpression strains analysed by qRT-PCR). Relative expression levels of the empty control line (PsGPA1-EV), the PsGPA1-silenced mutant (PsGPA1-M27), and the PsGPA1 overexpression strain (PsGPA1-OE) were calculated using the expression level of the wild-type strain P6497 as reference. All qRT-PCR experiments were repeated three times using independent RNA isolations. (B) Microscopic visualization of oospores and sporangia, growth characteristics after 4 days on V8 medium and 7 days on Plich medium, and virulence on soybean hypocotyls of P6497, PsGPA1-EV, PsGPA1-M27, and PsGPA1-OE. (C–G) Quantification of oospore and sporangia production, growth rate and pathogen biomass. All experiments were repeated three times with similar results. Asterisks indicate significant differences at P<0.01 (**), P<0.05 (*).
Fig 5.
PsGPA1 overexpression inhibits the nuclear localization of PsYPK1.
(A) Microscopic analyses of P. sojae transformants expressing PsYPK1-GFP or PsYPK1ΔN–GFP in the wild-type recipient stain P6497 (marked as PsYPK1-GFP or PsYPK1ΔN–GFP, respectively) or in a PsGPA1 silenced or overexpression strain (marked as PsGPA1-M27/PsYPK1-GFP and PsGPA1-FLAG/PsYPK1-GFP, respectively). DAPI (4', 6-diamidino-2-phenylindole) staining was performed by adding DAPI to the cultures 5 min prior to the microscopic analysis. DIC: differential interference contrast; Merge: overlay of DIC, GFP fluorescence and DAPI staining. Bar, 20 μm. (B) Relative fluorescence intensity along the red arrows in A. Green line: PsYPK1-GFP or PsYPK1ΔN–GFP, blue line: nucleus (stained with DAPI).
Fig 6.
The N-terminal region of PsYPK1 is required for PsGPA1 to suppress sporangium formation.
(A) Virulence and sporangium production of wild-type P. sojae (P6497), and its transformants EV (empty vector control), ΔPsYPK1 (PsYPK1-knockout mutant) and PsYPK1ΔN (PsYPK1 N-terminal knockout), and of P. sojae transformants in a ΔPsYPK1 background expressing PsYPK1 (complemented strain), PsYPK1 with PsGPA1, or PsYPK1ΔN with PsGPA1. For the virulence assays 4-day-old etiolated soybean hypocotyls (cultivar Hefeng 47) were inoculated with zoospores and photographs of lesions were taken 72 hpi. (B) Relative pathogen biomass in inoculated etiolated hypocotyls expressed as the ratio between the amounts of P. sojae DNA and soybean DNA detected at 72 hpi with the ratio P6497/soybean set at 1. (C) Relative numbers of sporangia with the number produced in wild-type strain P6497 set at 1. All experiments were repeated three times with similar results. Asterisks indicate significant differences at P<0.01 (**), P<0.05 (*). (D) Western blot analysis for all the strains mentioned above. Total proteins were extracted from these strains and SDS-PAGE and immunoblots were performed with GFP or FLAG antibodies.
Fig 7.
Overlap in genes differently expressed in the PsGPA1 overexpression strain and PsYPK1 knockout mutant.
(A) Overview of differentially expressed genes (DEGs) in the PsYPK1 knockout and PsGPA1 overexpression strains, compared to wild-type (P6497) and (B) comparison of the consistency of DEGs between the PsYPK1 knockout mutant and wild-type (ΔPsYPK1 vs. P6497) and the PsGPA1 overexpression strain and wild-type (PsGPA1-OE vs. P6497). Red dots, upregulated genes; blue dots, downregulated genes. (C) Verification of expression levels of selected genes by qRT-PCR. Standard error bar (SD) represents relative expression level calculated by qPCR using the 2-ΔΔCT method. The level of gene expression in the wild-type was set equal to 1 and used to calculate the relative expression levels of the genes in the transformants. All experiments were repeated three times with similar results. Asterisks indicate significant differences at P<0.01 (**), P<0.05 (*). (D) Functional annotation and classification of co-differentially expressed genes in PsGPA1 overexpression and PsYPK1 knockout strains.