Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum Immune receptor R2 to mediate disease resistance

Abstract

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.

Figure 1.

P. infestans AVR2 Specifically Associates with BSL1. (A) Immunoblots showing AVR2 specifically coimmunoprecipitates with tomato BSL1 (Sl-BSL1) in planta. FLAG:AVR2 was transiently expressed alone, with the empty vector pK7WGF2 or with GFP-tagged putative target proteins in N. benthamiana. Immunoprecipitates (IP) obtained with anti-FLAG or anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. The expected sizes of the GFP fusion proteins are indicated by red dots in the crude extracts and GFP co-IP probed with anti-GFP antibody. PS, Ponceau stain; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase. (B) Y2H analysis illustrating that the two allelic variants of AVR2, P. infestans AVR2^K31 and AVR2^N31, and the C-terminal effector domain of AVR2 (amino acids 66 to 116) interact with potato BSL1 (St-BSL1) in vivo. Both LacZ (blue) and His3 (providing growth on medium lacking His [-his]) reporter genes were activated. Empty vector (pDEST32) was used as a negative control. The RXLR effector PITG_08949, which is closely related to AVR2 across the N-terminal translocation domain but which differs across the C-terminal effector domain due to a likely recombination event (Gilroy et al., 2011), did not interact with St-BSL1 using Y2H analysis.

Figure 2.

Immunoblots Showing AVR2 Specifically Associates with the Putative Phosphatase Domain of Sl-BSL1 in Planta. (A) FLAG:AVR2 was transiently expressed alone, with SlBSL1^Kelch:GFP, SlBSL1^Phospho:GFP, or SlBSL1:GFP in N. benthamiana. Immunoprecipitates (IP) obtained with anti-FLAG or anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. The expected sizes of the GFP fusion proteins are indicated by red dots in the crude extracts and GFP co-IP probed with anti-GFP antibody. PS, Ponceau stain; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase. (B) Schematic illustrating the regions of Sl-BSL1 used to assess interaction with AVR2. Numbers indicate position of the amino acid residues.

Figure 3.

P. infestans AVR2 Accumulates around Haustoria and Colocalizes with the Perihaustorial-Localized Effector AVRblb2 in Planta. (A) P. infestans (red)–infected N. benthamiana cells preferentially accumulated transiently expressed GFP:AVR2 (green) around haustoria (arrowheads) when compared with the localization of GFP alone. (B) P. infestans–infected cells accumulated GFP:AVR2 and RFP:AVRblb2 at sites of haustorial penetration (arrowheads), illustrated by overlapping peaks of fluorescence intensity. Pictures were taken at 4 DAI. Bars + 5 µm.

Figure 4.

Sl-BSL1 and P. infestans AVR2 Colocalize and Accumulate around Haustoria in Planta. (A) Transient expression of SlBSL1:RFP in N. benthamiana reveals that Sl-BSL1 is excluded from the nucleus and localizes at the cell periphery and within the cytoplasm. (B) Transient expression of GFP:AVR2 and SlBSL1:RFP fusion proteins revealed similar patterns of localization for the two markers, accumulating at the cell periphery, as shown by overlapping peaks in fluorescence intensity. GFP:AVR2 was also detected in the nucleus. (C) GFP:AVR2 and SlBSL1:RFP localize strongly at sites of P. infestans haustorial penetration in N. benthamiana. Pictures were taken at 3 DAI. Bars + 10 µm.

Figure 5.

Immunoblots Showing That Five of the 13 AVR2 Family Members Associate with Sl-BSL1 and Activate R2-Mediated Recognition. FLAG-tagged protein fusions of AVR2 family members were transiently expressed with pK7WGF2 or SlBSL1:GFP in N. benthamiana. Immunoprecipitates obtained with anti-FLAG or anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. IP, immunoprecipitate; PS, Ponceau stain. [See online article for color version of this figure.]

Figure 6.

BSL1 Expression Is Required for R2-Mediated Recognition and Resistance. (A) and (B) VIGS of Nb-BSL1 specifically perturbed R2-mediated hypersensitivity (HR). AVR-R combinations were coexpressed (only the R proteins are indicated in the figure) in N. benthamiana using agroinfiltration. HR (%) indicates the percentage of infiltration sites showing a confluent zone of cell death. (C) and (D) VIGS of Nb-BSL1 inhibited R2-mediated resistance. Growth of P. infestans (also visualized by trypan blue staining) was significantly increased on BSL1-silenced plants transiently expressing R2 when compared with Rpi-sto1 expression. Letter “b” indicates a significant difference (P < 0.001) in the mean number of plants that show infection following R2 treatment, compared with Sto1 treatment (“a”) on TRV:3′NbBSL1 plants, using logistical regression analysis. Error bars represent ± se.

Figure 7.

Immunoblots Showing That AVR2 Expression Is Required for Interaction of Sl-BSL1 with R2. FLAG:AVR2, SlBSL1:myc, and GFP:SdR2 were transiently expressed in combination in N. benthamiana. Immunoprecipitates obtained with anti-FLAG or anti-GFP antiserum, and total protein extracts were immunoblotted with appropriate antisera. IP, immunoprecipitate; PS, Ponceau stain; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase. [See online article for color version of this figure.]

Figure 8.

A2L Associates with BSL1 but Does Not Promote an Interaction between BSL1 and R2. (A) Immunoblots showing A2L coimmunoprecipitates with tomato BSL1 in planta. FLAG:A2L was transiently expressed alone or with SlBSL1:GFP in N. benthamiana. Immunoprecipitates obtained with anti-FLAG or anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. IP, immunoprecipitate; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase. (B) Y2H analysis demonstrating that A2L (excluding the signal peptide) and the C-terminal effector domain of A2L (amino acids 66 to 116) interact with potato BSL1 (St-BSL1) in vivo. Both LacZ (blue color) and His3 (providing growth on medium lacking His [-his]) reporter genes were activated. Empty vector (pDEST32) and effector PITG_08949 (Gilroy et al., 2011) acted as negative controls (no reporter activity). (C) Immunoblots showing A2L does not promote interaction of Sl-BSL1 with R2. FLAG:A2L, SlBSL1:myc, and GFP:SdR2 were transiently expressed in combination in N. benthamiana. Immunoprecipitates obtained with anti-FLAG or anti-GFP antiserum and total protein extracts were immunoblotted with appropriate antisera. PS, Ponceau stain.

Figure 9.

Models Illustrating the Potential Link between Association of AVR2 Family Members with BSL1 and R2-Mediated Recognition and Resistance. Activation of R2 by AVR2 could be mediated by an AVR2-modifed BSL1, BSL1-modified AVR2, or AVR2-BSL1 complex (left to right). Circles, corresponding effectors; yellow diamonds, BSL1; green rectangles, R2. Potential modifications are indicated by red highlighting.