RXLR effector candidates for Avr3b

Briefly summarized, previous studies have described the following features of Avr effectors [12], [13], [14]. (1) The transcripts of Avr effectors are detectable in avirulent strains; (2) all of the Phytophthora Avr effectors cloned so far possess the RXLR motif; and (3) many Avr proteins are polymorphic. Previous genetic studies also indicated that Avr3b segregates as a single dominant gene [34]. Thus, we hypothesized that Avr3b was likely encoded by an infection-expressed RXLR effector that is polymorphic among P. sojae strains. To identify expressed RXLR effector candidates in avirulent strains, we examined transcription of potential RXLR effector genes in P. sojae using digital gene expression (DGE), Affymetrix arrays, and expressed sequence tags (ESTs). The DGE method resulted in the identification of 81 expressed RXLR effector genes [35]. A BLAST search in the P. sojae EST database identified 16 expressed RXLR effector genes. After the addition of 59 previously identified expressed RXLR effector genes from microarray analysis [12], [31], a total of 119 RXLR effectors were detected as expressed in the P. sojae Avr3b avirulent strain P6497 (Table S1).

Genomic and mitochondrial DNA RFLP results delineated P. sojae strains into four major genetic lineages [36]. Phytophthora sojae isolates P6497, P7064, P7074, and P7076 are representative isolates from each lineage. In addition to the published genome sequence of P6497, the re-sequencing of three P. sojae lineages (P7064, P7074, and P7076) has recently been completed [31]. Investigation of the 131 expressed effector sequences among these lineages revealed that six effectors (Avh20, Avh113, Avh238, Avh258, Avh288, and Avh307) showed a sequence polymorphism pattern consistent with the Avr3b phenotype; namely, identical in P6497 and P7074 (Avr3b avirulent strains) but variant in P7074 and P7076 (virulent strains) (Table 1). Furthermore, Avh6 showed a presence/absence polymorphism between avirulent and virulent strains (Table 1). Thus, these seven effectors were selected as Avr3b candidates for further investigation.

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Table 1. Seven expressed RXLR effectors showed Avr3b polymorphism.

https://doi.org/10.1371/journal.ppat.1002353.t001

Genetic co-segregation of the Avh307 genotype with the Avr3b phenotype

To determine whether candidate effectors might match Avr3b, we conducted genetic mapping to identify effectors that co-segregated with the Avr3b avirulence phenotype. First, we crossed P6497 and P7076, and identified 3 F₁ individuals. All the 3 F₁ individuals showed avirulence on Rps3b soybeans. Then, one F₁ individual was selected for self-crossing, resulting in a total of 71 F₂ individuals (P. sojae is diploid and homothallic, and wild-type strains show low levels of heterozygosity, hence segregation for most loci occurs in the F₂ generation). Avirulence assays of these progeny on soybean plants carrying resistance gene Rps3b distinguished 51 avirulent and 20 virulent F₂ individuals. The observed F₂ population segregation ratio for Avr3b fit a 3∶1 ratio (χ²  = 0.38, p >0.05), suggesting that the Avr3b avirulence phenotype is conferred by a single dominant allele, consistent with a previous report [34]. Cleaved amplified polymorphic (CAP) DNA markers were designed based on the sequence polymorphisms of the candidate effector genes (Table S2). The Avh307 genotype was found to perfectly match (0% recombination) the Avr3b avirulence phenotype among the 71 F₂ progeny (Figure 1A). In contrast, Avh238 (8.3% recombination), Avh288 (29%), Avh20 (33%), Avh6 (40%), Avh113 (35%), and Avh258 (31%) did not match.

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Figure 1. Avh307 corresponds to Avr3b based on genetic mapping, sequence polymorphisms, and bioassays.

(A) Co-segregation of Avh307 with the Avr3b virulence phenotype in F₂ progeny from a cross between P. sojae isolates P6497 and P7076. A virulence assay was performed with cultures scored as virulent (V) or avirulent (A) on Rps3b soybean plants. Cleaved amplified polymorphic (CAP) markers, co-dominant and specific for Avh307, were scored using genomic DNA from the avirulent (A) and virulent (V) parents and F₁ and F₂ progeny. Shown is a photograph of an ethidium bromide-stained agarose gel of the CAP marker results for Avh307. (B) Physical map of the Avh307 region. Predicted RXLR genes and CAP DNA markers for genetic mapping are shown. Genes (black color) and CAP markers (blue color) are shown on P. sojae P6497 genome sequence scaffold_3 (assembly version 5.0), not to scale. (C) Predicted amino acid sequences of Avh307^P6497 (from P6497 and other Rps3b-avirulent strains) and Avh307^P7076 (from P7076 and other Rps3b-virulent strains). Predicted signal peptide, RXLR-dEER motif, W-motif, and a Nudix hydrolase motif are shown in grey, blue, green and orange frames. (D) Transient expression assays of Avh307 alleles in Rps3b plant tissue. Soybean hypocotyls of cultivars Williams (rps), L88-1479 (Rps3b), and PRX146-36 (Rps3b) were transformed by co-bombardment with a plasmid mixture consisting of a glucuronidase reporter gene (GUS) expression vector and a test construct. Expression of the GUS reporter and Avh307 alleles were controlled by the 35S promoter. The test constructs used in combination with the GUS reporter are shown on the top of the panel.

https://doi.org/10.1371/journal.ppat.1002353.g001

Avh238 and Avh307 are genetically linked and are located on the same genome sequence scaffold, 713 kb apart (Avh238, scaffold_3: 2234002–2234580; Avh307, scaffold_3: 1520669–1521613) (Figure 1B). Examination of 1.06 Mb of DNA sequence surrounding Avh307 revealed another six RXLR effector genes (Avh309, Avh308, Avh9, Avh198, Avh340, and Avh302) in this region (Figure 1B; Table S3). Analysis by RT-PCR of all of these effector candidates revealed that only Avh309 and Avh340 were expressed in P6497 during infection (Table S3). However, neither Avh309 nor Avh340 displayed sequence polymorphisms. The predicted effector gene Avh308 displayed polymorphisms but was not expressed during infection. To generate a more detailed genetic map of the Avr3b locus, we identified another two markers (CAPSAvh307_ds6k and CAPSAvh9_us10k) in the vicinity of Avh307. The marker CAPSAvh307_ds6k (scaffold_3:1515512–1516432) is located 6 kb downstream of Avh307; this marker also co-segregated with the Avr3b avirulence phenotype without recombination in our 71 F₂ progeny. The marker CAPSAvh9_us10 k (scaffold_3: 1424151–1424781) is located 96 kb upstream of Avh307; this marker showed 4.4% recombination with the Avr3b avirulence phenotype (Figure 1B). Thus, of the eight RXLR genes examined in this region, Avh307 emerged as the best candidate for Avr3b (Table S3).

Oomycete Avr effectors often show significant sequence polymorphisms among field populations [12], [13], [14]. We examined Avh307 polymorphisms among 20 P. sojae field isolates from China. As shown in Figure 1C, Avh307 from each of the 16 avirulent strains encoded a 315 amino acid protein identical in sequence to Avh307^P6497. However, the four virulent isolates encoded a protein identical to Avh307^P7076 which is truncated to 230 amino acids by a premature stop codon (Figure 1C). Outside of the truncated region, an additional 46 amino acid substitutions and two deletions were present in Avh307^P7076 compared to Avh307^P6497.

In addition to a secretory signal peptide and RXLR and dEER motifs, the sequence of the Avh307^P6497 allele contains a W motif [30], and a nuclear diphosphate hydrolase (Nudix) motif, G5XE7XREUXEEXGU (Conserved Domain Database ID: cd04666, E-value  = 1.52^e-07) (Figure 1C). The Avh307^P7076 allele retained all of these motifs but with polymorphic sites in the dEER-, Nudix-, and W-motifs, as illustrated in Figure 1C.

Expression of Avh307 from avirulent strains triggers Rps3b-mediated cell death

The proteins encoded by P. sojae Avr genes trigger cell death when they are expressed in or enter into soybean cells containing their matching soybean Rps proteins. To test whether Avh307 alleles could trigger cell death in the presence of Rps3b, we conducted a soybean transient expression experiment by co-bombardment of soybean hypocotyls to measure cell death [37]. Plasmid constructs carrying Avh307 alleles encoding mature proteins without native signal peptides were delivered by particle bombardment into soybean hypocotyls, along with a beta-glucuronidase (GUS) reporter gene to measure cell survival. The Avh307^P6497 allele triggered cell death in the presence of Rps3b, but not in the absence of Rps3b, as evidenced by a strong reduction in GUS staining. This experiment was performed on two different soybean lines carrying the Rps3b gene and on control plants without Rps3b (Figure 1D). In contrast, expression of Avh307^P7076 did not reduce GUS staining in the presence of the Rps3b gene, nor in its absence (Figure 1D). To further confirm our results, we performed a double-barreled bombardment which could compare cell death in a more direct fashion [30]. These data confirmed that expression of the Avh307^P6497 allele strongly triggered cell death on Rps3b soybeans, but no reaction on near-isogenic soybean lines that do not contain Rps3b (Figure S1). Avh307^P7076, the virulence allele, did not induce significant cell death on Rps3b plants. Thus, both kinds of co-bombardment assays provided functional evidence that Avh307 is Avr3b, and that Avh307^P6497 and Avh307^P7076 are avirulence and virulence alleles, respectively, of Avr3b.

The Avr3b gene is expressed during infection and shows transcriptional polymorphisms between virulent and avirulent strains

To explore how Avr3b may be involved in P. sojae-soybean interactions, we examined the Avr3b transcriptional profile at different stages of infection and among different isolates. To define the transcriptional pattern of Avr3b, we isolated total RNA from mycelia, zoospores, cysts, germinating cysts, and from infected susceptible soybean leaves at 6 hours post-infection (hpi), 12 hpi, and 24 hpi. Real-time RT-PCR data indicated that Avr3b transcript levels are significantly elevated during infection (Figure 2A). Compared with in vitro grown mycelia, the Avr3b transcript level was elevated 20-fold, 150-fold, and 50-fold in the cyst, germinating cyst, and 24-hpi stages, respectively. These data indicate that Avr3b is strongly induced in pre-infection structures and during infection.

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Figure 2. Avr3b is induced during infection and shows transcriptional polymorphisms.

(A) Avr3b transcript levels in different developmental and infection stages measured by real-time RT-PCR. Total RNA was extracted from mycelia (MY), zoospores (ZO), cysts (CY), germinating cysts (GC), and infected soybean leaves at 6 hours post-infection (hpi), 12 hpi, and 24 hpi. All inoculations for RNA isolation were performed on susceptible soybean cultivar Williams. Real-time RT-PCR analysis employed primers specific for Avr3b and the actin gene. Transcrip level represent the P. sojae Avr3b mRNA levels compared with actin mRNA levels. Bars represent standard errors from 2 independent replicates each. (B) Avr3b transcriptional polymorphism between virulent (P7076) and avirulent (P6497) strains. Real-time RT-PCR analysis was conducted to compare Avr3b transcript levels at two infection-related stages, germinating cysts (GC) and infected leaves (24 hpi). Primers used in this assay amplified identical regions from Avr3b^P6497 and Avr3b^P7076. Bars represent standard errors from 2 independent replicates each.

https://doi.org/10.1371/journal.ppat.1002353.g002

P. sojae Avr effector genes Avr1b, Avr1a, and Avr3a all showed transcriptional polymorphisms among P. sojae strains [12], [13], [14]. To test for transcriptional polymorphisms of Avr3b, we compared Avr3b transcript levels in the germinating cyst and 24-hpi stages between P. sojae isolates P6497 (avirulent) and P7076 (virulent) using real-time RT-PCR. In P6497 compared to P7076, Avr3b transcript levels were five times higher at the germination cyst stage and three times higher at the 24-hpi stage (Figure 2B), indicating that Avr3b exhibits significant transcriptional polymorphism. However, despite the relatively lower expression level, transcripts of the Avr3b virulence allele remain detectable, indicating that it might be required during infection.

Avr3b enhances Phytophthora virulence on N. benthamiana and decreases ROS accumulation

Several oomycete avirulence effectors can suppress aspects of plant immunity [8], [32], [38]. To determine whether Avr3b interferes with plant immunity, we transiently expressed recombinant Avr3b^P6497 (without a signal peptide) fused with a FLAG tag in N. benthamiana using agroinflitration with a potato virus X (PVX) vector. Total protein samples were isolated from agroinfiltrated plant leaves and subjected to western blot analysis, employing an anti-FLAG monoclonal antibody as a probe. The results show expression of a protein of the expected size (34-kDa) in the tissue infiltrated with the Agrobacterium cells carrying the FLAG:Avr3b^P6497 construct (Figure 3A), consistent with expression of FLAG:Avr3b^P6497 in N. benthamiana.

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Figure 3. Expression of Avr3b^P6497 in N. benthamiana increased Phytophthora susceptibility and suppressed ROS accumulation.

(A) Western blot assay of Avr3b^P6497 and GFP protein levels in N. benthamiana leaves following Agrobacterium-mediated transient expression. Anti-FLAG monoclonal antibody was used to detect levels of FLAG-tagged Avr3b^P6497 protein. Anti-GFP monoclonal antibody was used to detect GFP protein levels. (B) Real-time PCR measurement of pathogen and plant DNA ratios was used to determine P. capsici biomass in infected plant tissues following Agrobacterium-mediated transient expression of FLAG-Avr3b or GFP. Nicotiana benthamiana leaves infiltrated with Agrobacterium carrying different constructs were grown in a greenhouse for 48 hours then inoculated with agar plugs containing P. capsici mycelium. DNA from P. capsici infected regions was isolated at 36 hpi. Real-time PCR employed primers specific for the N. benthamiana and P. capsici actin genes. Statistical analysis was performed by the Wilcoxon rank sum test (asterisk indicates p<0.01). Bars represent standard errors from 6 replicates (two technical replicates each from three biological replicates). (C) Photomicrographs from P. capsici infected regions. Leaf regions transiently expressing Avr3b^P6497 or GFP, and inoculated with P. capsici for 16 hours, were stained with trypan blue. Bar  = 2.5 µm. IS: inoculation sites, SP: sporangia. (D) ROS generation during P. capsici infection following Avr3b^P6497 or GFP transient expression in N. benthamiana leaves at 12 hpi (upper panel) and 36 hpi (lower panel). Leaves were stained with DAB. Bar  = 5 mm. Typical results are shown from 4 replicates.

https://doi.org/10.1371/journal.ppat.1002353.g003

Two Phytophthora pathogens of N. benthamiana, P. capsici and P. parasitica, were used to the challenge the infiltrated tissue. After 48 hours following Agrobacterium infiltration, we inoculated the infiltrated regions with an agar plug (5×5 mm) containing freshly grown P. capsici and P. parasitica mycelia, then evaluated disease development. On leaves infiltrated with strains carrying a control gene (GFP), the diameter of the disease lesions was approximately 0.7 to 0.8 cm at 36 hpi; however, on leaves infiltrated with strains carrying Avr3b^P6497 the lesion diameter expanded to 1.3 to 1.4 cm, as shown in Figure S2. To more precisely measure P. capsici infection, we measured the ratio of P. capsici DNA to N. benthamiana DNA using real-time PCR to determine the Phytophthora biomass in the infected plant tissues (Figure 3B). The data show that the P. capsici/N. benthamiana biomass ratio was significantly higher in Avr3b^P6497-infiltrated leaves (38%) than in GFP leaves (24%) (P value < 0.01). Both the lesion size data and the biomass data suggest that Avr3b expression in planta enhanced the susceptibility of N. benthamiana to Phytophthora.

To explore possible mechanisms behind the increased Phytophthora susceptibility, trypan blue and diaminobenzidine (DAB) staining were performed to examine infected plant tissues for the distribution of hyphae and for ROS production, respectively. At 16 hpi, a higher density of infected mycelium was observed in Avr3b^P6497-transformed tissues (Figure 3C), consistent with the real-time PCR data showing greater P. capsici biomass in Avr3b^P6497-expressing tissues. Furthermore sporangia had formed within the infected regions of Avr3b^P6497-expressing leaves, whereas none were found within the infected regions of GFP-expressing leaves (Figure 3C). In contrast, there was more extensive trypan blue staining of plant cells in infected tissues expressing the GFP control than in those expressing Avr3b^P6497, suggesting that more defense-related cell death may have occurred in those tissues. Substantially less DAB staining of 16-hpi plant tissues was observed in infected regions of Avr3b^P6497-expressing leaves compared to the GFP control, suggesting that ROS accumulation in response to infection was significantly reduced in the Avr3b^P6497-expressing tissue (Figure 3D). Overall, transient expression of Avr3b^P6497 in N. benthamiana leaf tissue resulted in a greater degree of Phytophthora infection with less ROS accumulation and less cell death, compared to the GFP control.

Avr3b-like RXLR-Nudix proteins are conserved in Phytophthora species, but not in other oomycetes

To examine the distribution of Avr3b homologs in other oomycete species, we performed a BLAST similarity search in several oomycete genome databases. A search of the P. sojae genome identified another 12 predicted genes that had the Nudix motif. A total of 15 Nudix hydrolases were found in the P. infestans genome; four of these hydrolases (PITG05846, PITG06308, PITG15679, and PITG15732) had a signal peptide and RXLR motif. The P. ramorum genome encoded a total of 14 Nudix hydrolases; three (PrAvh165, PrAvh268, and PrAvh281) were RXLR effectors. Only one RXLR Nudix hydrolase (ID: 102433) was defined in the JGI P. capsici genome. We also searched other plant and animal oomycete databases, including H. arabidopsidis, Pythium ultimum, and Saprolegnia parasitica without finding any predicted RXLR Nudix hydrolase effectors, suggesting that this gene family might be present only in the Phytophthora genus. Thus, an Avr3b family composed of nine members from four Phytophthora species was identified (Table S4).

Sequence alignment of Avr3b family members plus two characterized Nudix hydrolases (AtNUDT7 from Arabidopsis and ScYSA1 from Saccharomyces cerevisiae) revealed that all of these sequences contain the conserved residues of the Nudix hydrolase motif (GX5EX7REUXEEXGU) (Figure 4A). The Nudix motif is located at the C-terminus of each of the oomycete effectors. The motif is located at the extreme C-terminus in many family members including P. capsici Pc102433, P. infestans PITG05846, PITG06308, PITG15679, and the three P. ramorum Nudix hydrolase effectors, a pattern similar to that of Avr3b^P7076 (Figure S3). However, in Avr3b^P6497 and PITG15732 the motif is closer to the middle of the protein, which is more similar to AtNUDT7 and ScYSA1.

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Figure 4. Avr3b is an ADP-ribose/NADH pyrophosphorylase.

(A) Sequence alignment of the Nudix motif region from Avr3b homologs and characterized nudix hydrolases. Pc102433 is a predicted P. capsici RXLR effector. PITG05846, PITG06308, PITG15679, and PITG15732 are predicted P. infestans RXLR effectors. PrAvh165, PrAvh268, and PrAvh281 are P. ramorum RXLR effectors. AtNUDT7 is an ADP-ribose/NADH pyrophosphorylase from Arabidopsis. ScYSA1 is an ADP-ribose pyrophosphorylase from Saccharomyces cerevisiae. The black line refers to the Nudix hydrolase motif “G5XE7XREUXEEXGU.” The threshold for amino acid shading in this alignment is 70%. Avr3b^QQQQ is an Avr3b^P6497 non-functional mutant; the glutamine (Q) substitutions in the nudix hydrolase motif are marked. (B) Western blot of purified Avr3b and control proteins. Immuno-precipitated recombinant proteins FLAG:AtNUDT7, FLAG:GFP, FLAG:Avr3b^P6497 and predicted non-functional Avr3b mutant FLAG:Avr3b^QQQQ were detected with anti-FLAG antibody. (C) Enzyme assays of purified Avr3b and control proteins. The hydrolase activities of FLAG:AtNUDT7, FLAG:Avr3b^P6497 and FLAG:Avr3b^QQQQ were measured separately with FLAG:GFP as a control for each assay. The relative hydrolase activity was calculated as a ratio of Avr3b/AtNUDT7 over GFP. ADPR: adenosine diphosphate ribose, NADH: nicotinamide adenine dinucleotide (reduced form). Bars represent standard errors from 6 independent replicates each. Student's t-test was used to compare whether testing sample activities are significantly different from the GFP control. The double asterisk and single asterisk indicates statistical significance p <0.01 and p <0.05, respectively. The letters represent statistical significance (P <0.01) as measured by Duncan's multiple range test. (D) Western blot of the total plant extract from the Agrobacterium infiltrated region. The supernatants from FLAG:AtNUDT7, FLAG:GFP, FLAG:Avr3b^P6497 and FLAG:Avr3b^QQQQ transgenic N. benthamiana leaves were electrophoresed and detected with anti-FLAG antibody. (E) Enzyme assays of the total protein extracts from the Agrobacterium infiltrated region. The relative hydrolase activities were measured as a ratio to extracts from a GFP-expression control. Bars represent standard errors from 4 independent replicates each. Student's t-test was used to compare whether testing sample activities are significantly different from the GFP control. The double asterisk and single asterisk indicates statistical significance p <0.01 and p <0.05, respectively. The letters represent statistical significance (P <0.01) as measured by Duncan's multiple range test.

https://doi.org/10.1371/journal.ppat.1002353.g004

Avr3b has ADP-ribose/NADH pyrophosphorylase activity

To examine whether Avr3b has Nudix hydrolase activity, and to identify possible substrates for it, we expressed FLAG:Avr3b^P6497 (with the FLAG tag fused to the Avr3b N-terminus) in N. benthamiana (attempts to produce active protein in E. coli were unsuccessful). Western blot data showed a clear 34∼35 kDa band which suggested the recombinant protein was expressed. As a positive control, we fused AtNUDT7 from Arabidopsis with a FLAG tag. We then conducted immuno-precipitation using anti-FLAG M2 affinity gel. After immuno-precipitation, western blotting of the elution samples showed clear signals, indicating that, Avr3b protein had been recovered (Figure 4B). To test the hydrolase activity of the precipitated FLAG:Avr3b^P6497 protein and its possible substrates, relative hydrolase activity was measured as a ratio of FLAG:Avr3b^P6497 samples over immuno-precipitated GFP, using a series of nucleotide derivatives as substrates. The results showed that FLAG:AtNUDT7 and FLAG:Avr3b^P6497 immunoprecipitates could both hydrolyze ADPR and NADH, as shown in Figure 4C and Table S5. The AtNUDT7 showed higher activity than Avr3b^P6497. To confirm Avr3b^P6497 that is a nudix hydrolase, and to validate that the hydrolase activity in the immunoprecipitates was due to Avr3b, we mutated Avr3b^P6497 at four conserved residues (R220Q, E221Q, E225Q, E226Q simultaneously, Figure 4A) to generate a non-functional Nudix mutant [39], called Avr3b^QQQQ. The hydrolase activity of immunoprecipitated Avr3b^QQQQ was significantly weaker than Avr3b^P6497, on either ADPR or NADH, and not significantly greater than the GFP control (Figure 4C). Besides ADPR and NADH, other nucleotide derivatives including NADPH, NAD, Ap4A, FAD and Coenzyme A were also examined as possible substrates for Avr3b^P6497. The Avr3b^P6497 immunoprecipitate caused weak but significant hydrolysis of all these derivatives except NAD, whereas there was no significant hydrolysis by the Avr3b^QQQQ immunoprecipitate, except possibly of NADPH (Table S5). In our assay, the hydrolase data from AtNUDT7 (Figure 4C), a positive control, is consistent with previous reports [40]. Together, these data are consistent with the hypothesis that Avr3b^P6497 contains ADP-ribose and NADH pyrophosphorylase activity.

To measure the elevation of total nudix hydrolase activity in the plant tissue resulting from Avr3b^P6497 expression, we examined the hydrolase activity of total plant extracts. Avr3b^P6497, Avr3b^QQQQ , GFP and AtNUDT7 were transiently expressed in N. benthamiana by using Agrobacterium infiltration. Western blotting of total plant extracts confirmed expression of the proteins (Figure 4D). Consistent with the results from the immunoprecipitated protein, the extracts from Avr3b^P6497-expressing plant tissue showed significantly elevated hydrolase activity with NADH and ADPR than the control whereas expression of Avr3b^QQQQ did not significantly elevate the hydrolase activity (Figure 4E). No significant elevation of hydrolase activity against NADPH, NAD, Ap4A, FAD or Coenzyme A could be detected in total extracts from Avr3b^P6497- and Avr3b^QQQQ-expressing tissue (Table S5). Thus, in planta expression of Avr3b^P6497 elevated the ADP-ribose/NADH pyrophosphorylase activity in the plant tissue by approximately two-fold.

Avr3b virulence functions depend on its nudix motif, but its avirulence activity does not

To examine the relationship between Avr3b's Nudix motif and its virulence and avirulence activities, we fused a FLAG tag to the N-terminus of Avr3b^P6497, Avr3b^QQQQ, Avr3b^P7076 (virulence allele), Avr3b^16-174 (N-terminus of Avr3b^P6497), and Avr3b^170-315 (C-terminal of Avr3b^P6497) (Figure 5A). These proteins were then transiently expressed in N. benthamiana leaves using agroinfiltration. Western blots confirmed that all of the proteins were expressed in planta with the expected sizes (Figure 5B).

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Figure 5. Functional assays of Avr3b alleles and mutants.

(A) Schematic view of pGR107-FLAG:Avr3b constructs. Avr3b^16-174, Avr3b^170-315 and Avr3b^QQQQ are derived from the Avr3b^P6497 avirulence allele. (B) Western blot confirmation of in planta expression of Avr3b alleles and mutants (as in Figure 4A). (C) ROS accumulation assay. DAB staining of inoculation sites around P. capsici infection sites on Agrobacterium-infiltrated N. benthamiana leaf tissue expressing Avr3b alleles and mutants at 36 hpi, as in Figure 3D. The quantification of the DAB staining based on three replicates is shown on the right. The quantification data was shown in a pattern of mean ± standard deviation. (D, E) ADP-ribose (D) and NADH (E) hydrolase activity of total protein extracts from Agrobacterium-infiltrated N. benthamiana tissue transiently expressing Avr3b alleles and mutants, as in Figure 4C. The letters represent statistical significance (P <0.01) determined by Duncan's multiple range test. Bars represent standard errors from 5 independent replicates each. Student's t-test was used to compare whether testing sample activities are significantly different from the GFP control. The double asterisk indicates statistical significance p <0.01 and p <0.05, respectively. (F) Virulence assay. The P. capsici biomass index in N. benthamiana infected tissues was determined by PCR of plant and pathogen DNA as in Figure 3B. The letters represent statistical significance (P <0.01). (G) Avirulence assay. Avr3b alleles and mutants were transiently expressed in leaves of soybean cultivar Williams (rps) and PRX146–36 (Rps3b) by double-barreled bombardment, as described in the Methods. Statistical analysis was performed using Wilcoxon rank sum test for each pair of Williams and PRX146–36 measurements. The asterisks indicate a statistically significant difference between Williams and PRX146–36 (P <0.01). A significantly reduced cell survival ratio indicates avirulence activity.

https://doi.org/10.1371/journal.ppat.1002353.g005

Next, N. benthamiana leaves expressing each protein were challenged with P. capsici and then stained with DAB to test for suppression of ROS production. Expression of Avr3b^P6497, Avr3b^P7076, and Avr3b^170-315 all noticeably reduced ROS generation, whereas, expression of Avr3b^16-174 and Avr3b^QQQQ failed to do so, similar to expression of the GFP control (Figure 5C).

To measure the elevation in hydrolase activity caused by expression of the Avr3b proteins, total proteins from leaves expressing each construct were assayed with ADP-ribose and NADH as substrates. Protein extracts from leaf tissue expressing Avr3b^P6497, Avr3b^P7076, and Avr3b^170-315 exhibited significantly elevated ADP-ribose and NADH pyrophosphorylase activity whereas leaves expressing Avr3b^16-174 did not (Figure 5D, 5E). Avr3b^QQQQ expression resulted in no significant elevation in ADP-ribose pyrophosphorylase activity, and a weak but not statistically significant elevation in NADH pyrophosphorylase activity (Figure 5D, 5E).

To determine the effect of each mutant on susceptibility to P. capsici infection, real-time PCR was used to quantify the pathogen biomass in infected plant tissues expressing each construct. The pathogen biomass was significantly higher in tissues expressing Avr3b^P6497, Avr3b^P7076, and Avr3b^170-315 (p <0.01) than in tissues expressing the GFP control, but was not significantly higher in tissues expressing Avr3b^16-174 or Avr3b^QQQQ (p >0.05) (Figure 5F).

To quantitate the avirulence activity of each Avr3b allele and mutant, the double-barreled version of the co-bombardment assay was used. This assay allows precise quantitation of cell death triggered by a construct by comparing the number of blue spots on one side of a leaf with the number produced by a parallel empty vector bombardment on the other side of the leaf [29]. Consistent with the qualitative assay results shown in Figures 1D and S1, Avr3b^P6497, but not Avr3b^P7076, significantly reduced the cell survival ratio to around 40% on leaves containing Rps3b, whereas no significant effector-triggered cell death was observed in control leaves lacking Rps3b (Figure 5G). Transient expression of Avr3b^QQQQ and Avr3b^170-315 also reduced the cell survival ratio to around 40% in leaves with Rps3b but not in those without Rps3b. On the other hand, Avr3b^16-174 did not trigger significant Rps3b-dependent cell death (Figure 5G). Therefore the avirulence activity of Avr3b (i.e. triggering of Rps3b-dependent cell death) required the C-terminal half of the protein, containing the Nudix motif, but did not require the Nudix motif to be intact.

Transient silencing of Avr3b^P6497 impaired P. sojae virulence on susceptible soybean

To more directly test the contribution of Avr3b, to P. sojae virulence, transient silencing of Avr3b expression in P. sojae was carried out. In vitro synthesized Avr3b^P6497 dsRNA was introduced into P. sojae P6497, and 16 lines were recovered. Avr3b transcript levels were determined in germinating cysts from two biological replicates of each line using Real-Time PCR. In five lines (T5, T7, T9, T10, T13) levels of the Avr3b^P6497 transcript were significantly reduced (14%∼29%) compared to P6497 (Figure 6A). In five non-silenced lines (T1, T2, T3, T12, T16) levels of the Avr3b^P6497 transcript were consistently similar to P6497. In six lines, Avr3b transcript levels were not consistent between the two biological replicates; these lines were not examined further.

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Figure 6. Transient silencing of Avr3b impaired P. sojae virulence on soybean.

In vitro synthesized Avr3b dsRNA was introduced into P. sojae P6497, generating 16 lines. Avr3b transient transformants and recipient strain P6497 were examined for Avr3b transcript levels and virulence. RNA samples were extracted at the germinating cyst stage. (A) Avr3b transcript levels were determined by Real-time PCR using P. sojae actin transcripts as a reference and then normalized to the wildtype (P6497). Bars represent standard errors from 2 independent RNA isolation and Real-time PCR replicates. Letters indicated above each column represent statistical significance based on Duncan's multiple range test (p <0.01). (B) The disease lesion lengths in etiolated soybean hypocotyls (susceptible cultivar Williams) infected with zoospores of Avr3b transient transformants and P6497 at 36 hpi. Means and standard errors from at least 10 measurements are shown. Two statistical tests was used. The letters indicated above each column represent statistical significance based on Duncan's multiple range test (p <0.01). The asterisk indicates statistical significance based on student's t-test (p <0.01).

https://doi.org/10.1371/journal.ppat.1002353.g006

Zoospore inoculation of etiolated soybean hypocotyls was performed on susceptible soybean cultivar Williams. The disease lesion length was measured to quantify the virulence of Avr3b-silenced lines at 36 hpi, as shown in Figure 6B. The lesion lengths generated by the Avr3b-silenced lines were significantly smaller than those of the non-silenced lines and of P6497 (P <0.01). Thus, the silencing of Avr3b expression in P6497 significantly impaired virulence.

The nudix motif is required for Avr3b^P6497 to suppress ETI

To further elucidate the virulence mechanism of Avr3b, we tested if Avr3b could suppress ETI in soybean using a double-barreled bombardment protocol [31]. This experiment involves measuring ETI-associated cell death triggered by P. sojae effector Avr1b in the presence of soybean R gene Rps1b, in the presence or absence of Avr3b. The side-by-side comparison of cell death triggered by co-bombardment of Avr1b alone compared to Avr1b+Avr3b^P6497 on Rps1b soybean indicated that Avr1b-triggered cell death was strongly suppressed in the present of Avr3b^P6497 as shown in Figure 7A.

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Figure 7. Avr3b suppresses ETI triggered by P. sojae effector Avr1b.

Suppression of Avr1b-triggered PCD in soybean cultivar L77–1863 (Rps1b) by Avr3b^P6497 and Avr3b^QQQQ measured by indirect and direct double barrel particle bombardment assays. (A) Side-by-side comparison of cell death triggered by co-bombardment of Avr1b alone compared to Avr1b+Avr3b^P6497 on Rps1b soybean. Numbers in parenthesis indicate total GUS positive spots counted for each treatment on the leaf shown. Co-bombardments occur horizontally and are separated by dashed lines. Replicate bombardments are separated by the solid black line. (B) Quantitation of suppression by indirect and direct co-bomardment assays. For the indirect assay the number of GUS-expressing spots surviving in the presence of Avr1b on Rps1b soybean, was measured relative to a parallel GUS-only control in the presence (light grey bars) or absence (white bars) of Avr3b^P6497 or Avr3b^QQQQ. For the direct assay (dark gray bars) one barrel delivered DNA encoding Avr1b + Avr3b^P6497 or Avr3b^QQQQ and the other barrel delivered Avr1b DNA only. The direct ratio was then multiplied by the cell survival in the presence of Avr1b alone on Rps1b Soybean (white bars) to enable comparison to the results of the indirect assays. Based on the Wilcoxon signed ranks test (direct assays) or the Wilcoxon rank sum test (indirect assays and comparisons of two direct assays), outcomes that were significantly different p <0.001 are marked with different letters.

https://doi.org/10.1371/journal.ppat.1002353.g007

To quantify suppression, and to determine whether the Nudix motif is required for suppression, an indirect and a direct assay were carried out (Figure 7B). For the indirect assay, the number of GUS-expressing spots surviving in the presence of Avr1b on Rps1b soybean was measured relative to a parallel GUS-only control in the presence or absence of Avr3b^P6497 or Avr3b^QQQQ. For the direct assay one barrel delivered DNA encoding Avr1b + Avr3b (Avr3b^P6497 or Avr3b^QQQQ) and the other barrel delivered DNA encoding Avr1b alone. The results of both the direct and the indirect assays showed that the survival of GUS-spots from Avr1b + Avr3b^P6497 was significantly higher than from Avr1b alone, confirming that Avr3b could suppress Avr1b-triggered cell death. The survival of GUS-spots from Avr1b + Avr3b^QQQQ was significantly less than Avr1b + Avr3b^P6497 and not significantly higher than from Avr1b alone indicating that Avr3b^P6497 suppression of Avr1b mediated cell death is dependent on the nudix motif activity.

Plant and microbial culture, and virulence scoring

Detailed information about the P. sojae strains used in this study is listed in Table S6. The P. capsici (Pc263) and P. parasitica (P24-3) strains were obtained from the Phytophthora species collection at Nanjing Agricultural University. All of these isolates and the P6497× P7076 F₁ progeny and F₂ progeny used in this paper were routinely maintained on 2.5% vegetable (V8) juice medium at 25°C in the dark [58]. Phytophthora sojae mycelia, zoospores, and cysts were prepared as previously described [59]. Cysts germinating at 25°C for 6 hours were used for this study. For RNA samples, infection assays with P. sojae were performed by sandwiching P. sojae mycelium between pairs of soybean leaves [60]. The mycelium was removed at 6 hpi, and the infected soybean leaves were collected at 12 hpi and 24 hpi. All of the collected samples were immediately frozen in liquid nitrogen and stored at −80°C until used for RNA isolation.

Soybean (Glycine max) cultivar Williams (rps), Williams isoline L88–1479 (Rps3b), and PRX146–36 (Rps3b) from the collections at Nanjing Agricultural University and Northeast Agricultural University (Harbin, China) were used to score the virulence of P. sojae cultures. Williams isoline L77–1863 (Rps1b) was from the collection at the Virginia Bioinformatics Institute. Etiolated soybean seedlings were grown in vermiculite soaked with water at 25°C without light for 4 days before harvest for co-bombardment. For light-grown soybeans, ten soybean seeds were sown in 10 cm pots (a minimum of three pots per isolate) containing a soil (70%) and vermiculite (30%) mix soaked with water. Soybeans were grown in a greenhouse with a 16-hour photoperiod at 25°C. Soybeans were grown for 7 days for virulence assays, or for 13 to 14 days for use in co-bombardment. Phytophthora sojae cultures were grown on 0.9% (v/v) V8 agar plates 5 to 7 days prior to light-grown plant inoculations. The virulence of P. sojae cultures was scored in exactly the same manner as that previously described [14]. A minimum of three independent replicates of the disease assay were performed for each P. sojae culture tested. N. benthamiana plants were grown at 25°C with a 16 hr photoperiod in a greenhouse in styrofoam cups containing disinfected soil. Plants of 5 to 6 weeks old were used for agroinfiltration.

Bioinformatics

In total, 395 predicted RXLR effectors in P. sojae P6497 were previously predicted [42]. For DGE expression data, RNA samples were collected at Nanjing Agricultural University, and sequencing and analysis were performed at Beijing Genomics Institute. Affymetrix microarray data were previously reported [12], [31]. All RXLR sequences were used as queries for BLAST searches of the P. sojae EST unigene database (http://vmd.vbi.vt.edu) [61] , with an E-value cutoff at e⁻²⁰. We identified a total of 131 RXLR effectors that are expressed in P. sojae P6497. P. sojae 454 genome sequencing data (P7064, P7074, and P7076) [31] were accessed from Virginia Microbial Database (http://vmd.vbi.vt.edu). Nudix homologs were identified in two ways: genome annotation searching and BLAST searching. The P. sojae, P. capsici and P. ramorum homolog searches were performed at the Joint Genome Institute database (http://genome.jgi-psf.org), the H. arabidopsidis search was performed at Virginia Microbial Database, and the P. infestans search was conducted at the Broad Institute P. infestans database (http://www.broadinstitute.org). For Pythium ultimum genome searching, the genome sequence was downloaded from the Pythium genome database available at http://pythium.plantbiology.msu.edu and a local BLAST search was conducted. SignalPv3.0 (http://www.cbs.dtu.dk/services/SignalP) was used for secretion signal peptide prediction. Protein domain and motif analyses were conducted using the NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Sequence alignment was performed using BioEdit2005 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The Avr3b-like family member sequences have been deposited into NCBI GenBank with accession numbers (Table S7).

Generation of F₂ mapping population and scoring of genotypes

F₁ hybrids were derived from P6497 (Avr3b) × P7064 (avr3b) crosses. Oospores from F₁ hybrids were produced as described [14]. Single germinating oospores were harvested for DNA isolation. DNA isolation was performed using a fast DNA isolation kit (Axygen Biotech, China). Of 79 germinating oospores, three F₁ hybrids were confirmed using two different CAP DNA markers corresponding to polymorphisms within the predicted genes Avh20 and Avh238). One of the F₁ hybrids was cultured on 2.5% (v/v) V8-medium plates for F₂ progeny oospore generation. A total of 71 F₂ progeny were produced to establish an expanded mapping population to test Avr3b candidate effectors. For each F₂ individual, virulence was scored on Williams (rps) and L88–1479 (Rps3b) plants, and DNA samples from mycelia were also isolated for genotyping. CAP markers for seven Avh genes (Avh238, Avh307, Avh113, Avh258, Avh20, Avh288, and Avh6) were designed based on polymorphisms between P6497 and P7076; details are provided in Table S2. The genotype of the candidate gene and the virulence type of the F₂ progeny were compared. For Avr3b allele sequencing in different strains, the specific primers Avh307 MF, Avh307 MR, Avh307 MFa, and Avh307 MFb were used for PCR amplification and sequencing. Primer sequences are presented in Table S2.

Soybean transient expression assays

The Avr3b^P6497 and Avr3b^P7076 genes, excluding regions encoding the predicted signal peptide sequences, were amplified using specific primers and cloned into a 35S promoter-derived plant expression vector pFF19 using BamH I and Sph I restriction sites (primers sequences are shown in Table S8). Similarly, constructs for bombardment of Avr3b^P6497-derived mutants (Avr3b^16-174, Avr3b^170-315 and Avr3b^QQQQ) were also generated. Co-bombardment and transient expression assays for Avr3b^P6497 and Avr3b^P7076 on etiolated soybean hypocotyls were performed as described [37]. Leaves were photographed using a digital camera system (V12, Zeiss Germany). For Avr3b-derived mutant avirulence assay and Avr3b suppression of Avr1b ETI assay, double-barreled particle bombardment assays on soybean leaves were performed as previously described [30]. Both hypocotyl co-bombardment and double-barreled particle bombardment were conducted using a Bio-Rad (USA) He/1000 particle delivery system. For each paired shot, the logarithm of the ratio of the spot numbers of the test construct to that of the control was calculated. The log ratios obtained from the Rps3b and non-Rps3b leaves were then compared using the Wilcoxon rank-sum test.

RNA isolation and real-time RT-PCR

To monitor Avr3b transcript profiling in P. sojae P6497 by real-time RT-PCR, total RNA samples from mycelia, zoospores, cysts, germinating cysts, and infected plant tissue samples were extracted using a PureLink RNA Mini Kit (Invitrogen USA). For Avr3b transformants, germinating cysts was prepared at 9 and 11 days after transformation. The RNA samples were isolated from these germinating cysts (two biological replicates for each transformants). The first-strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) following the manufacturer's directions. For Avr3b transcript profiling analysis, SYBR green real-time RT-PCR assays were carried out. Primer pairs (Table S8) were designed at the identical region between Avr3b^P6497 and Avr3b^P7076. Two P. sojae housekeeping genes were selected as endogenous controls, namely actin (JGI Gene ID: 109046) and the molecular chaperone HSP70 superfamily gene (JGI Gene ID: 144810). PCR reactions (20 µL) included 20 ng cDNA, 0.2 µM of each primer, and 10 µL SYBR Premix ExTaq (TaKaRa Inc., Dalian, China). Reactions were performed on an ABI PRISM 7300 fast real-time PCR system (Applied Biosystems, USA) under the following conditions: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 31 s; followed by 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s to obtain melt curves. The expression of each gene relative to average Ct values of the two housekeeping genes (Ct  =  Ct_gene - Ct_HKaverage) was determined and analyzed using ABI 7300 System Sequence Detection Software Version 1.4 [62].

Construction of recombinant Agrobacterium binary PVX vectors

Sequences corresponding to Avr3b^P6497 and Avr3b^P7076 with secretion signal peptides replaced by a FLAG tag sequence were amplified from P6497 and P7076 genomic DNA using high-fidelity DNA polymerase (TaKaRa, Inc.) with primers described in Table S8. The PVX vector pGR107 for N. benthamiana transient expression assay was isolated using a plasmid spin column small isolation kit (Axygen Biotech. China.). For the Avr3b^P6497 mutant, a method similar to that used for vector construction was used. The Avr3b^QQQQ mutant was generated with additional primers (Table S8) by overlapping PCR. High-fidelity PCR products were sub-cloned into pGR107 pre-digested by the restriction enzyme Sma I. Recombinant binary vectors were maintained and propagated in the Escherichia coli strain JM109, grown in the presence of 50 µg/mL kanamycin. The recombinant binary vectors were transformed into the Agrobacterium tumefaciens strain GV3101 by electroporation. After growing at 28°C on LB agar plates supplemented with 50 µg/mL kanamycin as selective agents for 2 days, individual Agrobacterium colonies were verified with PCR using vector primers. Agrobacterium tumefaciens was grown in LB broth cultures supplemented with 25 µg/mL kanamycin for 2 day at 28°C with constant shaking. The cultures were centrifuged at 4,000 rpm for 4 min in a tabletop centrifuge. The pellet was resuspended in 1 mL of induction medium (10 mM MES, 10 mM MgCl₂, and 150 mM acetosyringone, pH  = 5.6). The final concentration of Agrobacterium cells was adjusted to OD₆₀₀  = 0.4. Leaves of N. benthamiana were infiltrated with Agrobacterium cultures using a blunt syringe. The infiltrated plants were then kept in a greenhouse for 72 hours prior to Western blot, immuno-precipitation, or virulence assays.

Virulence assay of Phytophthora on N. benthamiana

Agrobacterium-infiltrated N. benthamiana plants were grown in a greenhouse for 48 hours, and transformed leaves were then detached and maintained on half-strength MS medium in a petri dish. Next, 2.5% V8 juice agar plugs (0.5×0.5 cm) infested with fresh P. capsici or P. parasitica mycelia were inoculated onto the infiltrated regions. The diameter of the disease lesion was photographed and measured at 36 hpi. Total DNA isolated from P. capsici-infected regions (2×2 cm) was isolated at 36 hpi. Real-time PCR was used to quantify the ratio of host to pathogen ratio DNA sequences, employing primers specific for the N. benthamiana and P. capsici housekeeping actin genes (Table S8). Three independent biological replicates were conducted. Diseased plant tissues at 16 hpi were stained by trypan blue and DAB according as described [63]. Quantification DAB was performed by analyzing DAB staining image by a combination of Photoshop and Quantity One. GFP was considered to be 100% standard.

Western blot and immuno-precipitation assays

Total plant protein was isolated as described [63] for Western blot and immuno-precipitation assays. The protein samples were quantified using a BioPhotometer (Eppendorf, Germany). A standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protocol was used for protein separation. The amount of total protein loaded per gel lane varied from 80 µg to 120 µg depending on each experiment. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using a semi-wet apparatus (Bio-Rad) according to the product instructions. Western blotting was performed as a standard protocol. Anti-FLAG monoclonal antibody (Sigma-Aldrich) and anti-mouse IgG-peroxidase conjugate (Beyotime Biotech, China) were used as the primary and secondary antibodies. The membrane was treated with Chemiluminescent Peroxidase Substrate-1 (Thermo Scientific Pierce, No. 34080, USA) for 5 min. The membrane was briefly drained and exposed to BioMax (Kodak, USA) light film several times (depending on results) for exposure signal development.

For immuno-precipitation, anti-FLAG M2 affinity gel (Sigma-Aldrich) was used. The gel suspension (40 µL) was transferred into a fresh 1.5 mL test tube and centrifuged in a pre-cooled rotor at 6000 g for 30 s. The resin was washed twice with 1 mL TBS (50 mM Tris HCL, 150 mM NaCl, pH = 7.4), and all washing buffer was removed. Total protein lysate was clarified by centrifugation (10000 g, 2 min) then 1000 µL of the supernatant was added to the washed resin, and the mixture was incubated using a roller shaker for 4 hours at 4°C. The suspension was centrifuged at 6000 g and the supernatant was removed. The remaining resin was washed three times with 1 mL of TBS. A total of 100 µL elution buffer (0.5 M Tris-HCl buffer, 150 ng/µL 3xFLAG, pH = 7.2) was then added to the resin, and the samples were incubated using a roller shaker for 10 min at room temperature. The resin was centrifuged for 30 s at 6000 g, and the supernatants were transferred to fresh tubes previously loaded with 10 µL of 0.5 M Tris HCl and 1.5 M NaCl, pH 7.4. The purified protein samples were used for gel electrophoresis and enzyme activity assay.

Nudix hydrolase activity assays

A general method for the Nudix hydrolase activity assay was performed to test for Avr3b enzymatic activity [40], [53]; a 50-µL reaction mixture contained 50 mM Tris-HCl, pH 8.5, 5 mM MgCl₂, 1 mM dithiothreitol, 2 units of calf alkaline phosphatase, 2 mM substrate (ADP-ribose, NADH, FAD, or Ap4A; Sigma-Aldrich), and certain amount of protein samples. Equal amount of protein (5 µg for purified protein, 2 mg for total plant protein extract) was loaded for hydrolase activity assay in each reaction. After incubation for 1 hr at 37°C, the reaction was stopped by adding 150 µL of 1 N H₂SO₄. For color development, 100 µL of water and 700 µL of a freshly made mixture containing 600 µL of 3.4 mM ammonium molybdate and 100 µL of 570 mM ascorbic acid was used. The reaction tubes were incubated in a 45°C dry bath for 10 min for color development and then cooled on ice. The solutions were measured using the Du-640 spectrophotometer (Beckman, USA) at A₈₂₀ The reaction without protein was used as a blank control for each substrate. For direct assay of purified proteins, immuno-precipitation eluate was directly added into enzymatic assay reaction mixture.

For hydrolase activity assay of total plant protein extract, two regions per leaf transiently expressing FLAG:GFP and FLAG:Avr3b^P6497 were created in N. benthamiana leaves using Agrobacterium infiltration. A total of 0.1 g of GFP- or Avr3b-infiltrated leaf tissue was collected from each leaf. For both kinds of protein preparations, the relative hydrolase enzyme activity was calculated as a ratio of Avr3b extract (A₈₂₀ reading) over GFP extract (A₈₂₀ reading). For NADH and ADPR, 17 and 21 biological replicates were performed, respectively. For FAD, Ap4A, NAD and NADPH, 4 biological replicates were conducted.

In vitro dsRNA synthesis and P. sojae transformation

Avr3b in vitro dsRNA synthesis was carried out as described [64]. Primers AVH307-T7F/R with T7 promoter sequence added to the 5′ end of both primers (see Table S8) were used to amplify a C-terminal specific Avr3b DNA fragment from the Avr3b^P6497 allele. The PCR product was cloned into pMD19 vector (TaKaRa) and sequenced. AVH307-T7F/R primers were also used to generate Avr3b in vitro dsRNA by using the Megascript RNAi kit (Ambion AM1626). A total of around 200 µg dsRNA was obtained as measured by spectrophotometry. P. sojae transient transformation was performed as described [30], [59] with a few modifications [32]. About 100 µg of Avr3b dsRNA was added into 1 mL MMg solution (0.4 M mannitol, 15 mM MgCl₂, 4 mM MES, pH 5.7) containing 5000–10000 P. sojae P6497 protoplasts. After transformation, the regenerated protoplasts were suspended in liquid pea agar (40 °C ) containing 0.5 M mannitol. The visible colonies could be observed after 36 h incubation at 25 °C. A total of 16 single colonies were randomly selected and propagated on V8 agar plates for RNA extraction and virulence assays.