Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1

Abstract

Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3a(KI), which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3a(EM), which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3a(KI/Y147del), a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3a(KI) or Avr3a(EM) but not the Avr3a(KI/Y147del) mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.

Fig. 1.

AVR3a^KI and AVR3a^EM interact with and stabilize CMPG1. (A) AVR3a^KI and AVR3a^EM with (21-147) and without (60-147; shown only for AVR3a^KI) RXLR-encoding portions interact with CMPG1 in Y2H (LacZ and -His reporter genes activated). Whereas AVR3a^KI/Y147del (Y147 deletion) and AVR3a^KI/Y147S mutants fail to interact with CMPG1, the AVR3a^KI/Y147F mutant interacts. (B) Western blots probed with anti-myc and anti-HA antibodies following expression of mycCMPG1 in binding domain vector, with or without HA-AVR3a^KI in activation domain vector, in yeast. (C) Western blots probed with anti-myc and anti-FLAG antibodies, showing a time course of transient coexpression (by agroinfiltration) of FLAG-AVR3a^KI, FLAG-AVR3a^EM, FLAG- AVR3a^KI/Y147del, or a vector control with 4×-myc-ΔN-StCMPG1a at 2 and 3 dpi. (D) As in C, but at 5 dpi. (E) As in C but at 5 dpi with 4×-myc-StCMPG1b (full-length) and 4×-myc-ΔN-StCMPG1b (lacking the N-terminal 29 amino acids). Protein sizes are indicated in kDa. Protein loading is shown by Coomassie blue (CBB) or Ponceau S (PS) staining. (F) Fluorimeter measurements (in relative fluorescence units) following coexpression in N. benthamiana of the split YFP constructs CMPG1::N-YFP (YN), CMPG1::C-YFP (YC), C-YFP::AVR3a^KI, C-YFP::AVR3a^EM, and C-YFP::AVR3a^KI/Y147del, as indicated. (G) Confocal microscopy following coexpression in N. benthamiana of split YFP constructs CMPG1-YC with a vector expressing free N-YFP and CMPG1-YC with N-YFP::AVR3a^KI, N-YFP::AVR3a^EM, and N-YFP:: AVR3a^KI/Y147del constructs as indicated in the panels. (Scale bars, 200 μm.)

Fig. 2.

CMPG1 stabilization is consistent with its inactivation or modification. (A) Western blot probed with anti-myc and anti-FLAG antibodies following coexpression of 4×-myc-ΔN-StCMPG1a with FLAG-AVR3a^KI, with INF1, or with empty vector control. (B) Western blots probed with anti-myc antibody, following expression of ΔN-4×-myc-StCMPG1a with or without MG132 inhibitor treatment as indicated. (C) Western blot probed as in B following expression of ΔN-4×-myc-StCMPG1a with or without epoxomicin inhibitor treatment. Confocal images of CMPG1-YFP following water (D) or epoxomicin (E) treatment. (Scale bar, 200 μm.) (F) Western blot probed as in A following expression of 4×-myc-NbCMPG1a, 4×-myc-NbCMPG1aC34A, or 4×-myc-NbCMPG1aW64A with or without FLAG-AVR3a^KI. Sizes are indicated in kDa. Protein loading is shown by Coomassie blue (CBB) or Ponceau S (PS) staining.

Fig. 3.

CMPG1 is stabilized during infection. (A) Western blot probed with anti-myc antibody indicating stabilization of constitutively expressed ΔN-StCMPG1a-4×-myc across a time course of N. benthamiana infection with P. infestans isolates 88069 and CA65. (B) Disease development, measured as per cent infected leaf area, of isolates 88069 and CA65 on N. benthamiana at 4 and 5 dpi. (C) Typical infection of P. infestans isolate 88069 on TRV::GFP-inoculated N. benthamiana (Left) and TRV::CMPG1-inoculated N. benthamiana (Right) at 6 dpi. (D) Real-time RT-PCR showing relative expression of NbCMPG1 in N. benthamiana plants inoculated with TRV::GFP and TRV::CMPG1. (E) Percentage of P. infestans infection lesions that developed sporangia on TRV::GFP and TRV::CMPG1-inoculated N. benthamiana leaves at 6 dpi. (F) Diameter (mm) of P. infestans infection lesion in TRV::GFP and TRV::CMPG1-inoculated N. benthamiana leaves at 6 dpi. Vector-induced gene-silencing experiments were repeated on six occasions with six replicate plants for each construct, with reproducible results.

Fig. 4.

Avr3a is essential for virulence. (A) Infection of potato cultivar Bintje by wild-type P. infestans isolate 88069 (left leaf) and Avr3a-silenced line CS12 (right leaf) at 4 dpi. (B) Infection of N. benthamiana by wild-type P. infestans isolate 88069 at 6 dpi. (C) Infection of N. benthamiana by Avr3a-silenced line CS12 at 6 dpi. (D) Infection of N. benthamiana by Avr3a-silenced line CS12 at 6 dpi, following agroexpression of pGRAB::Avr3a^EM (left half of leaf) or empty pGRAB (right half of leaf). (E) As in D, but leaf is stained with trypan blue. (F) Infection of N. benthamiana by Avr3a-silenced line CS12 at 6 dpi, following agroexpression of pGRAB::Avr3a^KI (left half of leaf) or empty pGRAB (right half of leaf). (G) As in F, but leaf is stained with trypan blue. (H) Measurements at 6 dpi of lesion sizes (mm) following infection of isolate 88069 or silenced line CS12 on leaves that were uninfiltrated, infiltrated with agromix, agroinfiltrated to express AVR4a^KI, Avr3a^EM, or empty pGRAB vector. These complementation experiments were repeated on four occasions, with leaves from six plants for each treatment/construct.