In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2

Abstract

The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD36(45-1), suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34-amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2.

Figure 1.

Overview of the Effectoromics Pipeline for Allele Mining, Cloning, and in Planta Expression of RXLR Effectors. The various steps in the pipeline are as follows: (1) PCR-based allele mining using primers designed to amplify sequences corresponding to the mature RXLR proteins and including an in-frame ATG start codon. (2) Sequencing of amplicons and prioritization for cloning. (3) Cloning of amplicons in the PVX-based expression vector pGR106. (4) Transformation of constructs into A. tumefaciens GV3101 and sequencing of inserts to yield a library of nonredundant clones. (5) Testing mutants of interest for suppression and promotion of cell death, as well as for specific activation of R genes, by agroinfiltration and wound inoculation in N. benthamiana.

Figure 2.

Functional Validation of the Signal Peptides of RXLR Effectors. Functional validation of the signal peptides of PexRD6/IpiO, PexRD8, PexRD39, and PexRD40 was performed using the yeast invertase secretion assay. Yeast YTK12 strains carrying the PexRD signal peptide fragments fused in frame to the invertase gene in the pSUC2 vector are able to grow in both the CMD-W media (with sucrose, yeast growth even in the absence of invertase secretion) and YPRAA media (with raffinose instead of sucrose, growth only when invertase is secreted), as well as reduce TTC to red formazan, indicating secretion of invertase. The controls include the untransformed YTK12 strain and YTK12 carrying the pSUC2 vector.

Figure 3.

PexRD8 and PexRD36_45-1 Suppress the HR Induced by P. infestans INF1 Elicitin. (A) and (B) Agroinfiltration sites in N. benthamiana leaves expressing either PexRD8 (A) or PexRD36_45-1 (B) were challenged with A. tumefaciens expressing the INF1 elicitin. The INF1-induced cell death was scored at 3 and 4 DAI. Two independent pGR106-derived clones of PexRD8 and PexRD36_45-1 were used (bottom panels; clone #1 on the bottom left side and #2 on the bottom right). A. tumefaciens strain carrying pGR106-ΔGFP (dGFP) was used as a negative control, and pGR106-AVR3a (AVR3a) was used as a positive control. (C) and (D) Quantification of suppression of INF1 cell death by PexRD8 and PexRD36_45-1 relative to AVR3a. The mean percentages of sites showing cell death and the standard errors were scored from 20 infiltration sites based on three independent experiments using N. benthamiana leaves expressing either PexRD8 (C) or PexRD36_45-1 (D). Two independent pGR106-derived clones of PexRD8 and PexRD36_45-1 were used (#1 and #2) as shown in (A) and (B).

Figure 4.

PexRD2 Promotes Cell Death in N. benthamiana. (A) Symptoms observed in N. benthamiana after wound inoculation with A. tumefaciens carrying pGR106 vector derivatives expressing a subset of the 62 RXLR effectors of P. infestans. The negative and positive controls were A. tumefaciens strains carrying pGR106-ΔGFP (dGFP) and pGR106-INF1, respectively. Note the small ring of dead cells triggered by the pGR106-PexRD2 strain relative to the more expanded cell death triggered by pGR106-INF1. All strains were inoculated in triplicate. The photo was taken 12 DAI. (B) The PexRD2-associated cell death is enhanced in the presence of gene silencing suppressor p19. A. tumefaciens carrying pGR106-PexRD2 was mixed with (+) p19 or without (−) an A. tumefaciens p19 strain and infiltrated into N. benthamiana leaves. The experiment was repeated three times with similar results. After 6 d, the PexRD2-associated cell death symptoms were observed in both cases but were enhanced in the presence of p19. All strains were inoculated in triplicate. (C) SGT1 is required for the cell death response induced by PexRD2. Leaves of N. benthamiana vector control (TRV2-dGFP) and SGT1-silenced (TRV2-NbSGT1) plants were challenged by agroinfiltration of A. tumefaciens carrying pGR106-ΔGFP (dGFP, negative control) or pGR106-PexRD2. Control-silenced plants showed symptoms of the cell death induced by the PexRD2 starting at 3 to 5 DAI, and this response was enhanced in the presence of gene silencing suppressor p19 (left panel). In the TRV2-NbSGT1 plants, the PexRD2-associated cell death was suppressed (right panel). (D) RT-PCR analysis of SGT1 expression in control (TRV2-dGFP) and SGT1-silenced (TRV2-NbSGT1) N. benthamiana. Total RNA was extracted from the silenced plants and subjected to RT-PCR analysis with SGT1 primers to detect SGT1 transcripts. The Actin gene was used to confirm equal total RNA amounts among samples. Similar results were obtained at least two times independent experiments.

Figure 5.

Functional Identification of Avrblb1 and Avrblb2. (A) Wound inoculation screening of the pGR106-PexRD library on N. benthamiana leaves expressing the S. bulbocastanum R genes Rpi-blb1 (left panel) and Rpi-blb2 (right panel). The two HR-inducing PexRD6/IpiO clones (PexRD6_41-3/IpiO1-K143N and PexRD6_41-10/IpiO2) and two of the positive PexRD39 and PexRD40 clones (PexRD39_169-6 and PexRD40_170-1) are shown. Additional PexRD clones that yielded negative responses are also shown. All tested clones are labeled RD# for the corresponding PexRD clone number. The negative and positive controls were A. tumefaciens strains carrying pGR106-ΔGFP (dGFP) and pGR106-PiNPP1 (NPP1), respectively. (B) to (D) Confirmation of Avrblb cloning using agroinfiltration. Agroinfiltration of the positive A. tumefaciens pGR106 strains carrying Avrblb1 (PexRD6_41-3/IpiO1-K143N and PexRD6_41-10/IpiO2, top and bottom right panels, respectively) and Avrblb2 (PexRD39 and PexRD40, top and bottom panels, respectively) was performed in N. benthamiana corresponding to control plants (B) or leaves expressing Rpi-blb1 (C) or Rpi-blb2 (D). A. tumefaciens strain carrying pGR106-ΔGFP (dGFP) was used as a negative control (top and bottom left panels of leaves). Coinfiltration was performed with A. tumefaciens solutions mixed in 1:2 ratio (Avr:R gene). Hypersensitive cell death was observed starting at 4 DAI, and the photograph was taken at 7 DAI. The experiment was repeated three times with similar results.

Figure 6.

The AVRblb2 Family Is Highly Polymorphic and under Diversifying Selection in P. infestans. (A) Multiple sequence alignment of 24 AVRblb2 amino acid sequences from P. infestans. Single-letter amino acid codes were used. Residue numbers are denoted above the sequences. The predicted signal peptide, RSLR motif, and 34–amino acid functional domains are indicated above the alignment. (B) Posterior probabilities along the AVRblb2 protein sequence for site classes estimated under the discrete model M8 in the PAML software. The analysis was based on the 24 identified AVRblb2 sequences described in Figure 6A. Amino acid sites 42P, 47I, 69A, 70Q, 84G, 88E, and 95A marked in red have high posterior probabilities (P > 0.95 and ω > 8.9) and are potentially under positive selection. (C) Posterior probabilities along the AVRblb2 protein sequence obtained with a subset of four paralogous sequences from P. infestans T30-4 strain. In this analysis, only residue 69A (ω= 69.434) is under positive selection. The position of the signal peptide, RSLR motif, and the 34–amino acid domain are indicated below the graphs.

Figure 7.

Deletion Analysis of AVRblb2 Reveals a 34–Amino Acid Region Sufficient for Induction of Rpi-blb2–Mediated Cell Death. RXLR and deletion mutants of PexRD40_170-7 were coexpressed with Rpi-blb2 by agroinfiltration in N. benthamiana to determine the AVRblb2 domains required for induction of the Rpi-blb2–mediated HR. A schematic view of the different mutant and deletion constructs is shown on the left. Symptoms of infiltration sites coexpressing the AVRblb2 construct with Rpi-blb2 are shown on the right. HR cell death index with plus and minus signs indicate the presence and absence of effector activity, respectively. The assays were repeated at least three times with similar results. Photograph of symptoms were taken 5 to 7 DAI. SP, signal peptide.

Figure 8.

The Positively Selected Amino Acid 69 of AVRblb2 Is Critical for Activation of Rpi-blb2 Hypersensitivity. (A) Schematic view of pGR106-PexRD40_170-7 (AVRblb2) site-directed mutant constructs. FLAG refers to the FLAG epitope tag. V (Val), A (Ala); I (Ile), and F (Phe) refer to the amino acids at position 69 with the top construct (V69) corresponding to PexRD40_170-7. The numbers refer to the amino acid positions based on the full-length protein. (B) Symptoms observed in N. benthamiana infiltration sites coexpressing the PexRD40_170-7 constructs with (+) or without (−) Rpi-blb2. Photographs were taken 6 DAI. A. tumefaciens solutions were mixed in a 1:1 ratio before infiltration into N. benthamiana leaves. V69, A69, I69, and F69 refer to the constructs described in (A). The negative control was A. tumefaciens strains carrying pGR106-ΔGFP (GFP). (C) In planta accumulation of PexRD40 proteins. A FLAG immunoblot was performed on total protein extracts of leaves of N. benthamiana following agroinfiltration with the constructs described in (A). An ∼10-kDa protein band representing recombinant PexRD40 was detected in total extracts of plant tissues expressing all PexRD40 constructs but not the ΔGFP negative control. Equal loading was checked by PonceauS staining. (D) Percentages of infiltration sites with Rpi-blb2–mediated hypersensitive cell death based on two independent experiments scored at 4 DAI. Error bars indicate se.