AVR2 Targets BSL Family Members, Which Act as Susceptibility Factors to Suppress Host Immunity

Abstract

To be successful plant pathogens, microbes use "effector proteins" to manipulate host functions to their benefit. Identifying host targets of effector proteins and characterizing their role in the infection process allow us to better understand plant-pathogen interactions and the plant immune system. Yeast two-hybrid analysis and coimmunoprecipitation were used to demonstrate that the Phytophthora infestans effector AVIRULENCE 2 (PiAVR2) interacts with all three BRI1-SUPPRESSOR1-like (BSL) family members from potato (Solanum tuberosum). Transient expression of BSL1, BSL2, and BSL3 enhanced P. infestans leaf infection. BSL1 and BSL3 suppressed INFESTIN 1 elicitin-triggered cell death, showing that they negatively regulate immunity. Virus-induced gene silencing studies revealed that BSL2 and BSL3 are required for BSL1 stability and show that basal levels of immunity are increased in BSL-silenced plants. Immune suppression by BSL family members is dependent on the brassinosteroid-responsive host transcription factor CIB1/HBI1-like 1. The P. infestans effector PiAVR2 targets all three BSL family members in the crop plant S. tuberosum These phosphatases, known for their role in growth-promoting brassinosteroid signaling, all support P. infestans virulence and thus can be regarded as susceptibility factors in late blight infection.

Figure 1.

PiAVR2 interacts with StBSL1, StBSL2, and StBSL3. A, Yeast coexpressing BSL1, BSL2, or BSL3 with PiAVR2 grew on -His (LTH) medium and yielded β-galactosidase (LacZ) activity, while those coexpressed with the control effector Pi08949 did not. The +HIS (LT) control shows that all yeast were able to grow in the presence of His. B, IP of protein extracts from agroinfiltrated leaves using GFP-Trap confirmed that cMYC-StBSL1, cMYC-StBSL2, and cMYC-StBSL3 associated in N. benthamiana with GFP-tagged PiAVR2, but no association was seen with the GFP-PiAVR3a control. Expression of constructs in the leaves is indicated by +. Protein size markers are indicated in kilodaltons, and protein loading is indicated by Ponceau stain (PS).

Figure 2.

Interaction with the BSL family is essential for PiAVR2 virulence function. A, IP of protein extracts from agroinfiltrated leaves using GFP-Trap confirmed that cMYC-StBSL1, cMYC-StBSL2, and cMYC-StBSL3 associated in N. benthamiana with GFP-tagged PiAVR2 and PiAVR2-66_116, but no association was seen with GFP- PiAVR2-66_100. Expression of constructs in the leaves is indicated by +. Protein size markers are indicated in kilodaltons, and protein loading is indicated by Ponceau stain (PS). B, Transient coexpression of GFP-PiAVR2 or truncated forms of PiAVR2 with INF1 indicated that PiAVR2 and PiAVR2-66_116 can suppress ICD in N. benthamiana, similar to the GFP-AVR3a control, whereas PiAVR2-66_100 did not. Cell death sites were counted at 4 d post infiltration. Results combine data from three independent experimental replicates, each consisting of eight individual plants (biological replicates), with three leaves (technical replicates) infiltrated per plant. C, P. infestans lesion sizes at 8 d post inoculation of sporangia suspension (diameter in millimeters) following expression of GFP-tagged PiAVR2, PiAVR2-66_116, PiAVR2-66_100, and control GFP. Data shown combine 4 independent experimental replicates, each consisting of 18 leaves taken from 6 individual plants, with 4 inoculations per leaf. D, Transient coexpression of GFP-PiAVR2 or truncated forms of PiAVR2 with R2 indicated that PiAVR2 and PiAVR2-66_116 are recognized by R2 in N. benthamiana, whereas PiAVR2-66_100 is not. Cell death sites accounted at 3 d post infiltration. Results combine data from three independent experimental replicates, each consisting of 8 individual plants, with 3 leaves infiltrated per plant. Error bars indicate SEM. Significant difference in (B to D) is denoted by lowercase letters (P < 0.001 in one-way ANOVA, using the Student-Newman-Keuls method).

Figure 3.

Silencing of the BSL family increases INF1-triggered cell death in Nicotiana benthamiana. Silencing BSL1 slightly increases ICD, and BSL2/3 significantly accelerates ICD at 4 d post infiltration of Agrobacterium allowing transient expression of INF1. Silencing was achieved using VIGS. Significant difference is denoted by lowercase letters (P < 0.001 in one-way ANOVA, using the Student-Newman-Keuls method). Results shown are a combination of data from three independent experimental replicates, each consisting of seven individual plants, with three leaves infiltrated per plant. Error bars indicate SEM. Representative ICD lesions are shown.

Figure 4.

VIGS of BSL2 and BSL3 in Nicotiana benthamiana resulted in decreased susceptibility to P. infestans. BSL2/3-silenced plants were inoculated with a P. infestans zoospore suspension and a variety of measurements made at 7 d post inoculation: percentage of inoculation sites forming lesions (A), lesion diameter (B), and number of sporangia per milliliter (C). Results were combined from four independent experimental replicates, each involving 8 individual plants, with three leaves inoculated per plant. Error bars indicate SEM. Significant difference is represented by asteriks (***P < 0.01 in one way ANOVA, using the Student-Newman-Keuls method).

Figure 5.

The BSL family suppresses immunity and enhances P. infestans leaf colonization. A, INF1-triggered cell death following coexpression of INF1 with GFP-StBSL1, GFP-StBSL2, and GFP-StBSL3 transiently expressed in N. benthamiana. Data are a combination of 3 independent experimental replicates, consisting of eight individual plants, with three leaves infiltrated per plant. Representative ICD lesions are shown. B, P. infestans lesion sizes following transient expression of GFP-StBSL1, GFP-StBSL2, and GFP-StBSL3 in N. benthamiana at 8 d post inoculation of sporangia. Data are a combination of 3 independent experimental replicates, each involving 15 leaves from 7 individual plants. Significant differences in (A) and (B) are represented by lowercase letters (P < 0.05 in one-way ANOVA, using the Student-Newman-Keuls method). Error bars indicate SEM. Representative leaf images show the full extent of the lesion under UV light, converted to grayscale.

Figure 6.

Silencing of BSL2/3 compromises the suppression of INF1-triggered cell death by PiAVR2 in Nicotiana benthamiana. Coinfiltration INF1 with GFP-PiAVR2, GFP-PiAVR3a, and GFP control in TRV:BSL1, TRV:BSL2/3, and TRV:GFP plants. Cell death sites were counted at 5 d post infiltration. Significant difference was represented by letters (P < 0.001 in one-way ANOVA, using the Student-Newman-Keuls method). Results are combined data across three independent experimental replicates, consisting of seven individual plants, with three leaves infiltrated per plant. Error bars indicate SEM.

Figure 7.

Silencing of CHL1 attenuated the suppression of INF1-triggered cell death by GFP-BSL1 and GFP-BSL3 in Nicotiana benthamiana. INF1 was coinfiltrated with GFP-StBSL1, GFP-StBSL3, GFP-AVR3a, and GFP control in TRV:CHL1 and TRV:GFP control plants. Cell death sites were counted at 5 d post infiltration. Significant difference is represented by letters (P < 0.001 in one-way ANOVA, using the Student-Newman-Keuls method). Results shown are combined data across three independent biological replicates, each consisting of seven individual plants, with three leaves infiltrated per plant. Error bars indicate SEM.

Figure 8.

Schematic diagram illustrating main findings of this work. AVR2 is an RXLR effector secreted into the plant by P. infestans, where it interacts with the kelch-repeat phosphatases StBSL1, 2, and 3. A 16-amino acid region at the C terminus (C-term) of the effector is shown to be essential for BSL interaction. All three BSL family members can be considered to be susceptibility S factors, with overexpression increasing P. infestans leaf infection. Overexpression of StBSL1 and BSL3 can suppress ICD, with no effect seen for StBSL2. Silencing StBSL1 results in no change to P. infestans leaf infection or to ICD. Silencing StBSL2 and 3 in combination significantly reduces P. infestans leaf infection and significantly increases ICD. Notably, silencing StBSL2 and 3 reduces protein level of StBSL1, suggesting that one or both of these are required for StBSL1 stability. Finally, suppression of ICD by StBSL1 and StBSL3 has been shown to require the transcription factor StCHL1, a suppressor of immunity in Solanaceous plants.