The Phytophthora sojae nuclear effector PsAvh110 targets a host transcriptional complex to modulate plant immunity

Abstract

Plants have evolved sophisticated immune networks to restrict pathogen colonization. In response, pathogens deploy numerous virulent effectors to circumvent plant immune responses. However, the molecular mechanisms by which pathogen-derived effectors suppress plant defenses remain elusive. Here, we report that the nucleus-localized RxLR effector PsAvh110 from the pathogen Phytophthora sojae, causing soybean (Glycine max) stem and root rot, modulates the activity of a transcriptional complex to suppress plant immunity. Soybean like-heterochromatin protein 1-2 (GmLHP1-2) and plant homeodomain finger protein 6 (GmPHD6) form a transcriptional complex with transcriptional activity that positively regulates plant immunity against Phytophthora infection. To suppress plant immunity, the nuclear effector PsAvh110 disrupts the assembly of the GmLHP1-2/GmPHD6 complex via specifically binding to GmLHP1-2, thus blocking its transcriptional activity. We further show that PsAvh110 represses the expression of a subset of immune-associated genes, including BRI1-associated receptor kinase 1-3 (GmBAK1-3) and pathogenesis-related protein 1 (GmPR1), via G-rich elements in gene promoters. Importantly, PsAvh110 is a conserved effector in different Phytophthora species, suggesting that the PsAvh110 regulatory mechanism might be widely utilized in the genus to manipulate plant immunity. Thus, our study reveals a regulatory mechanism by which pathogen effectors target a transcriptional complex to reprogram transcription.

Figure 1

PsAvh110 localizes to the plant nucleus and contributes to the virulence of P. sojae. A, PsAvh110 localizes to the nucleus. GFP, GFP-PsAvh110, or GFP-PsAvh110^mNLS were co-expressed with the nuclear marker (H2B-RFP) in N. benthamiana leaves for 36 h before imaging. Fluorescence intensity profiles of GFP and RFP were assessed in the nucleus and PM along the transects shown as white lines. y-axis, GFP intensity (arbitrary units [au]); x-axis, transect length (μm). Scale bar, 10 μm. B, Schematic diagram of PsAvh110 showing its two nuclear localization signals (NLS1 and NLS2) and the mNLS mutant. The lysine (K) and arginine (R) residues within NLS1 and NLS2 are highlighted in red and were mutated to alanine (A) labeled with blue. C, PsAvh110 is a conserved effector in the Phytophthora species. Phylogenetic analysis of PsAvh110 homologs from Phytophthora parasitica (PpPPTG_12652T0), P. infestans (PiPITG_22926), and P. capsici (Pc_22959) is shown. Blue bars indicate the percentage identity of PsAvh110 in different Phytophthora species. The conserved motifs of PsAvh110 and homologs are shown as different shapes with different colors. SP, signal peptide; RxLR, Arg–x–Leu–Arg; W, tryptophan (W) motif. The protein sequences were retrieved from JGI (https://genome.jgi.doe.gov/portal/) for MEGAX phylogenetic analysis using the neighbor-joining method with 1,000 bootstrap replicates. The phylogenetic tree was drawn in iTOL (https://itol.embl.de/). D and E, PsAvh110 promotes Phytophthora infection and nuclear localization is required for its virulence. Nicotiana benthamiana leaves individually expressing Flag-RFP, Flag-Avh110-RFP, or Flag-Avh110^mNLS-RFP were inoculated with P. capsici mycelial plugs at 24 h after Agrobacterium infiltration. D, Infected leaves were photographed under UV light and lesion areas are indicated by white circles at 36 hpi. E, Boxplot showing the average lesion diameter of the infected leaves in (D). The data are shown as an overlay of dot plots with mean ± sd, n=20. Different letters indicate significant differences (P < 0.01; one-way ANOVA). F, The expression of PsAvh110 is highly induced during the early infection stage. Total RNA extracted from P. sojae zoospores and soybean hairy roots infected with zoospores at the indicated time points was subjected to RT-qPCR analysis. Relative PsAvh110 expression levels were normalized to PsActin. Data are shown as means ± sd from three independent replicates, n = 3. G and H, Knockout of PsAvh110 reduces P. sojae virulence. Etiolated soybean hypocotyls were inoculated with zoospore suspensions of P. sojae WT strain P6497, two PsAvh110 knockout transformants (T52 and T119), or a control strain transformed with the EV (CK). The infected soybean hypocotyls were photographed at 48 hpi (G). Relative biomass of P. sojae was determined by qPCR of P. sojae genome DNA normalized to soybean genome DNA at 48 h post inoculation (hpi). The results are shown as an overlay of dot plots with means ± sd, n = 3. Asterisks indicate significant differences (****P < 0.0001; Student’s t test). The experiments were performed three times with similar results. I and J, The PsAvh110^mNLS complementation strains fail to facilitate P. sojae infection in soybean. PsAvh110 was replaced with PsAvh110^mNLS in P. sojae using a CRISPR/Cas9-mediated in situ complementation method. The experiments were performed as in (G and H). The data are shown as an overlay of dot plots with means ± sd from three independent replicates. Asterisks indicate significant differences (****P < 0.0001; Student’s t test).

Figure 2

PsAvh110 suppresses the expression of a subset of immune-associated genes. A, Heatmap representation of gene expression in P. sojae T52-infected and WT-infected soybean hairy roots. The original FPKM values were subjected to data adjustment against reference genes. Rows and hierarchical clustering were generated by the average linkage method using TBtools; red, high expression; blue, low expression. B, GO analysis of PsAvh110-regulated genes. The fold enrichment was calculated based on the transformed values of –Log10(P-value). A list of PsAvh110-regulated genes is shown in Supplemental Data Set S1. C, RT-qPCR analysis of PsAvh110-regulated immune-associated genes in soybean hairy roots infected by P. sojae WT or the T52 mutant at 6 hpi. Relative expression levels were normalized to the soybean control gene GmCYP2; the data are shown as means ± sd (n = 3) from three biological replicates. Asterisks indicate significant differences (***P < 0.001; ****P < 0.0001; Student’s t test). D, Schematic diagram of the effector and reporter, and internal control constructs used in the transfection assays in N. benthamiana protoplasts. proBAK1, plant BAK1 promoter driving the LUC reporter gene; effector constructs contain the 35S promoter (pro35S), RXLR effector genes, or GFP (negative control); the internal control contains the NbActin2 promoter and the RLUC reporter gene. E and F, PsAvh110, but not PsAvh23 or PsAvr3C, inhibits plant BAK1 promoter activity induced by XEG1 in N. benthamiana protoplasts. Nicotiana benthamiana mesophyll protoplasts co-transfected with effector, reporter, and control constructs were treated with or without 200 nM XEG1 for 2 h. The activity of the BAK1 promoter from N. benthamiana (E) or soybean (F) induced by XEG1 was calculated relative to untreated samples and was normalized to the internal control NbActin2. The data are shown as means ± sd from three biological replicates. Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; Student’s t test).

Figure 3

PsAvh110 interacts with soybean GmLHP1-2, which is required for PsAvh110 virulence activity. A, PsAvh110 interacts with GmLHP1-2 in yeast. Yeast transformants were spotted as 10-fold serial dilutions on SD medium without leucine and tryptophan (SD–2), or without histidine, leucine, adenine, and tryptophan (SD–4) and containing 0.2 mM X-α-gal. Yeast cells were photographed 3–4 days later. pLAW10 and pLAW11 are bait and prey EVs, respectively. Experiments were performed at least three times with similar results. B, PsAvh110 strongly associates with GmLHP1-2 in soybean protoplasts. Protoplasts from soybean seedlings were co-transfected with GmLHP1 homologs with FLAG-PsAvh110 or FLAG-RFP (control) for 12 h. Co-IP assays were carried out with GFP-Trap beads, followed by immunoblotting (IB) with anti-Flag or anti-GFP antibody (top two blots) with input proteins shown (bottom two blots). The molecular weight (kD) is shown to the left. Protein loading is shown by Ponceau S staining (Ponc.) of Rubisco large subunit (RBCL). C, Schematic diagram of PsAvh110 motifs and its variants. PsAvh110 contains two W domains (W1 and W2). The GEGE and GKSE residues present in the W1 and W2 domains are shown in yellow diamonds. The PsAvh110 variants PsAvh110^ΔW1 (lacking the W1 domain), PsAvh110^ΔW2 (lacking W2), and PsAvh110^mW2 (substitution of GKSE residues with AAAA, shown as green diamonds) are shown. D, PsAvh110, but not PsAvh110^mW2, interacts with GmLHP1-2 in an in vitro pull-down assay. Recombinant His-GmLHP1-2 protein immobilized on Ni-NTA resin was incubated with MBP, MBP-Avh110, MBP-Avh110^ΔW1, or MBP-Avh110^mW2. Washed beads were subjected to IB with anti-His or anti-MBP antibodies. E, Y2H assay showing the interaction between GmLHP1-2 and PsAvh110 derivatives. The Y2H assay was performed as described in (A). F, PsAvh110 associates with soybean GmLHP1-2 in planta. Total protein was extracted from N. benthamiana leaves co-expressing GFP-GmLHP1-2 with Flag-PsAvh110-RFP, Flag-PsAvh110^mW2-RFP, or Flag-RFP (control). Co-IP assays were performed as described in (B). G and H, Virulence activity of different PsAvh110 mutants. Nicotiana benthamiana leaves expressing Flag-RFP (control), or different Flag-PsAvh110-RFP derivatives were inoculated with P. capsici mycelial plugs at 36 h after Agrobacterium infiltration. Confocal images show subcellular localization of Flag-RFP (control) and Flag-Avh110-RFP derivatives in N. benthamiana at 36 h after Agrobacterium infiltration (top panel; scale bar, 5 μm). RFP-NLS was used as a nuclear marker. The infected leaves were imaged under UV light at 36 hpi and the diameter of the lesion areas, shown by white circles (bottom panel; scale bar, 1.5 cm), was measured. The data are shown as an overlay of dot plots with means ± sd (n = 22) in (G). Different letters indicate significant differences (P < 0.01; one-way ANOVA). Experiments were performed at least three times with similar results.

Figure 4

Both PsAvh110 and GmPHD6 interact with the C terminus of GmLHP1-2. A, Y2H assay showing the interaction between GmLHP1-2 and PsAvh110 or GmPHD6. Ten-fold serial dilutions of yeast cells harboring the indicated constructs were spotted on SD medium without leucine and tryptophan (SD–2), or without histidine, leucine, adenine, and tryptophan (SD–4) supplemented with 0.2 mM X-α-gal. B, PsAvh110 reduces the association between GmLHP1-2 and GmPHD6 in soybean protoplasts. Protoplasts were co-transfected with GFP-GmLHP1-2 and Flag-GmPHD6 with or without PsAvh110-HA for 12 h. Co-IP assays were performed with GFP-Trap beads, followed by IB with anti-Flag or anti-GFP antibody (top two blots) with input proteins shown (bottom three blots). Protein loading is shown by Ponceau S staining (Ponc.) of Rubisco large subunit (RBCL). C, Schematic diagram of GmLHP1-2 and its truncated variants. GmLHP1-2 truncation variants GmLHP1-2^ΔCSD (without the CSD motif), GmLHP1-2^CΔCD (C terminus without the CD motif), GmLHP1-2^C (C terminus with CD and CSD motifs), and GmLHP1-2^EHH (with residues I437,L444,L450 changed to E437,H444,H450) are shown. D, The C-terminal CSD of GmLHP1-2 is required for interacting with both PsAvh110 and GmPHD6 in yeast. The Y2H assay was performed as described in (A). E, Co-IP assay showing the interaction of GmLHP1-2 derivatives with PsAvh110 or GmPHD6. Total protein was extracted from N. benthamiana leaves co-expressing different GmLHP1-2 derivatives with EV (control), PsAvh110, or GmPHD6. Co-IP assays were performed as described in (B).

Figure 5

PsAvh110 competes with GmPHD6 for binding to GmLHP1-2 in vitro and in vivo. A, PsAvh110 disrupts the GmPHD6–GmLHP1-2 interaction in a co-IP assay. GFP-GmLHP1-2 and Flag-GmPHD6 were co-transfected with or without Flag-PsAvh110 or Flag-PsAvh110^mW2 in N. benthamiana leaves. Total protein was incubated with GFP-Trap beads for co-IP assays, and immunoprecipitated proteins were analyzed using anti-GFP, or anti-Flag antibodies (top two blots). Input proteins are shown in blots 4 and 5. The black arrows indicate the multiple bands of GFP-GmLHP1-2. Protein loading is indicated by Ponceau S staining of RBCL (bottom panel). B, PsAvh110 competes with GmPHD6 to bind to GmLHP1-2 in vitro in a dose-dependent manner. Recombinant GST-GmLHP1-2 immobilized on glutathione sepharose beads was incubated with His (control), Myc-GmPHD6, and increasing amounts of His-PsAvh110 protein (different gradient dilutions: 1×, 2×, 3×). Protein eluates from washed beads were used for immunoblot with anti-GST, anti-His, or anti-Myc antibodies. C, PsAvh110 displays a higher binding affinity to GmLHP1-2 than GmPHD6 in MST assays. Recombinant purified GmLHP1-2 was labeled with a fluorophore, and purified PsAvh110, GmPHD6, and MBP proteins were used as flow-through analytes for MST assays. Left: dose–response curves of PsAvh110 proteins at gradient concentrations flowing through immobilized and labeled GmLHP1-2; the calculated K_d is 7.03 µM. Middle: dose–response curves of PsAvh110 at gradient concentrations flowing through immobilized and labeled GmLHP1-2; the calculated K_d is 44.83 µM. Right: dose–response curves of PsAvh110 at gradient concentrations flowing through immobilized and labeled GmLHP1-2; no binding affinity was observed between GmLHP1-2 and MBP proteins. The above experiments were performed three times with similar results.

Figure 6

The GmLHP1-2/GmPHD6 complex positively regulates plant immunity. A, Knockdown efficiency of GmLHP1-2 or GmPHD6 in corresponding hairy roots as indicated by RT-qPCR. Total RNA was extracted from RNAi-GmLHP1-2, RNAi-GmPHD6, or RNAi-GmLHP1-2/GmPHD6 hairy roots. Gene expression was normalized to the soybean internal control GmCYP2. The data are shown as means ± sd from three replicates. Asterisks represent significant differences (***P < 0.001; ****P < 0.0001; Student’s t test). B and C, RNAi-GmLHP1-2, RNAi-GmPHD6, and RNAi-GmLHP1-2/GmPHD6 hairy roots enhance soybean susceptibility to P. sojae infection. Oospore production in RNAi-GmLHP1-2, RNAi-GmPHD6, and RNAi-GmLHP1-2/GmPHD6 hairy roots infected by WT P. sojae labeled with RFP (WT-RFP) was imaged under a microscope (B) and calculated at 48 hpi (C). The data are shown as an overlay of dot plots with means ± sd, n = 20. Different letters indicate significant differences (P < 0.01; one-way ANOVA). Scale bars, 0.2 mm. The experiments were performed at least three times with similar results. D, Relative biomass of P. sojae in RNAi-GmLHP1-2, RNAi-GmPHD6, and RNAi-GmLHP1-2/GmPHD6 hairy roots, as determined by qPCR at 48 hpi of the ratio of P. sojae genome DNA compared with soybean genome DNA. The data are shown as means ± sd, n = 3. Different letters indicate significant differences (P < 0.01; one-way ANOVA). E, Relative expression levels of immune-associated genes in RNAi-GmLHP1-2 and RNAi-GmPHD6 hairy roots infected with P. sojae, as determined by RT-qPCR at 6 hpi. Gene expression levels were normalized to the internal control gene GmCYP2. The data are shown as means ± sd from three biological repeats. Asterisks indicate significant differences (****P < 0.0001; Student’s t test). F, Sequence alignment of the cis-elements within the 1-kb promoter before the translation start codon of the selected immune-associated genes. The sequences were aligned using ClustalW and displayed in ESPript3. The conserved residues of GREs are highlighted in black, and the GREs are highlighted by the black box. The consensus of the GREs was analyzed by WebLogo 3. G, Occupancy of GmPHD6 at different promoter regions of selected immune genes, as assessed by ChIP-qPCR in 35S:GmPHD6-FLAG transgenic hairy roots. IgG was used as the negative control. The ChIP signals were normalized to input, and the data are means ± sem of three replicates. Asterisks indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test). P1–P3 represent the promoter regions examined by ChIP-qPCR. GREs in the promoter are shown as red triangles.

Figure 7

PsAvh110 suppresses the transcriptional activity of the GmLHP1-2/GmPHD6 complex. A, The GmLHP1-2–GmPHD6 complex regulates the promoter activity of GmBAK1-3, GmPR1, GmGCN5-like, GmWRKY71-2, and GmGF14 in NbLHP1s-silenced plants. NbLHP1s-silenced N. benthamiana leaves were infiltrated with Agrobacterium cultures carrying the indicated constructs; relative LUC activity was measured at 48 hpi using a chemiluminescent imaging system. B, Schematic diagram of GREs of the GmBAK1-3 and GmPR1 promoters and their mutants. The DNA probes (GmBAK1-3^GRE and GmPR1^GRE) containing GREs from the GmBAK1-3 and GmPR1 promoters are shown. The GRE motifs of GmBAK1-3^GRE and GmPR1^GRE mutated to red-labeled adenine (A) are labeled GmBAK1-3^mGRE and GmPR1^mGRE. C and D, GmLHP1-2/GmPHD6 activates GmBAK1-3 transcription via the GREs. A 1-kb fragment of the GmBAK1-3 promoter before the start codon containing the GREs (proGmBAK1-3) or the GRE mutation (proGmBAK1-3m) shown in (B) was cloned upstream of the LUC gene to generate proGmBAK1-3/ProGmBAK1-3m:LUC-Nos constructs. C, N. benthamiana leaves were infiltrated with Agrobacterium strains carrying the indicated constructs as shown in the bottom panel, and LUC activity was quantified at 48 hpi (D). Ten individual leaf discs were collected for LUC activity measurement; data are shown as means ± sd (n = 10). Different letters indicate significant differences (P < 0.01, one-way ANOVA). E and F, GmLHP1-2/GmPHD6 activates GmPR1 transcription via the GREs. A 1-kb fragment of the GmPR1 promoter before the start codon containing the GREs (proGmPR1) or the GRE mutation (ProGmPR1m) shown in (B) was cloned upstream of the LUC gene to generate proGmPR1/ProGmPR1m:LUC-Nos. The experiments were performed as in (C and D). Different letters indicate significant differences (P < 0.01, one-way ANOVA). G, GmPHD6 binds to the GREs of the GmBAK1-3 and GmPR1 promoters in a dose-dependent manner. Biotin-labeled probes were incubated with increasing amounts of recombinant purified MBP-GmPHD6 in the absence or presence of 125-fold excess of the corresponding unlabeled probes. The bands corresponding to the DNA–protein complexes (shifted) or free probes are indicated by arrows. Protein loading was determined by Coomassie Brilliant Blue (CBB) staining (bottom panel). H and I, The transcription of GmBAK1-3 activated by GmLHP1-2/GmPHD6 is suppressed by PsAvh110. The experiments were performed as in (C and D). Different letters indicate significant differences (P < 0.01, one-way ANOVA). J and K, The transcription of GmPR1 activated by GmLHP1-2/GmPHD6 is suppressed by PsAvh110. The experiments were performed as described in (C and D). Different letters indicate significant differences (P < 0.01, one-way ANOVA).

Figure 8

A simplified working model of PsAvh110 functions in modulating plant transcriptional reprogramming during P. sojae infection. In the absence of PsAvh110, GmPHD6 binds to the GRE in the promoter of immunity genes to recruit GmLHP1-2 and form a transactivation complex during P. sojae Psavh110 mutant infection, thereby activating the expression of immune-associated genes and mounting robust plant immunity. In the presence of PsAvh110, PsAvh110 has a high binding affinity toward GmLHP1-2, which disrupts the formation of the GmLHP1-2/GmPHD6 complex, thereby suppressing its transcriptional activity, resulting in the suppression of the expression of immune genes and of plant immunity.