Bacterial effector HopF2 suppresses arabidopsis innate immunity at the plasma membrane

Abstract

Many bacterial pathogens inject a cocktail of effector proteins into host cells through type III secretion systems. These effectors act in concert to modulate host physiology and immune signaling, thereby promoting pathogenicity. In a search for additional Pseudomonas syringae effectors in suppressing plant innate immunity triggered by pathogen or microbe-associated molecular patterns (PAMPs or MAMPs), we identified P. syringae tomato DC3000 effector HopF2 as a potent suppressor of early immune-response gene transcription and mitogen-activated protein kinase (MAPK) signaling activated by multiple MAMPs, including bacterial flagellin, elongation factor Tu, peptidoglycan, lipopolysaccharide and HrpZ1 harpin, and fungal chitin. The conserved surface-exposed residues of HopF2 are essential for its MAMP suppression activity. HopF2 is targeted to the plant plasma membrane through a putative myristoylation site, and the membrane association appears to be required for its MAMP-suppression function. Expression of HopF2 in plants potently diminished the flagellin-induced phosphorylation of BIK1, a plasma membrane-associated cytoplasmic kinase that is rapidly phosphorylated within one minute upon flagellin perception. Thus, HopF2 likely intercepts MAMP signaling at the plasma membrane immediately of signal perception. Consistent with the potent suppression function of multiple MAMP signaling, expression of HopF2 in transgenic plants compromised plant nonhost immunity to bacteria P. syringae pv. Phaseolicola and plant immunity to the necrotrophic fungal pathogen Botrytis cinerea.

Fig. 1. HopF2 suppresses immune signaling triggered by multiple MAMPs

A. Real-time RT-PCR analysis of FRK1 and WRKY30 induction. Four-week-old Arabidopsis plants were inoculated with H₂O, DC3000, DC3000 ΔavrPto/avrPtoB, or DC3000hrcC at 1 × 10⁸ cfu/ml. The samples were collected 6 hr later for RNA isolation. The gene induction (fold change) by bacterial infiltration was compared with the expression level of H₂O infiltration. B. HopF2 suppresses flg22-induced FRK1-LUC activation. Protoplasts were co-transfected with GFP control or an effector and FRK1-LUC reporter. Three hours later, transfected protoplasts were treated with 10 nM flg22 for another 3 hr. The expression of effectors was detected by anti-GFP Western blot. C. HopF2 suppresses multiple MAMP-mediated FRK1-LUC activation. Protoplasts were transfected with FRK1-LUC with or without HopF2, AvrPto or AvrPtoB for different MAMP treatments. The concentrations for different MAMPs are flg22, 10 nM; elf18, 10 nM; HrpZ1, 100 nM; PGN, 50 µg/ml; chitin, 50 µg/ml; and LPS, 50 µg/ml.

Fig. 2. HopF2 attenuates MAP kinase signaling

A. HopF2 suppresses flg22-mediated MPK3, MPK4 and MPK6 activation. HA-tagged MPK3, MPK4 or MPK6 was co-expressed with GFP-tagged effectors. Transfected protoplasts were incubated for 6 hr before 1 µM flg22 treatment for 10 min. An anti-HA antibody was used for immunoprecipitation of MAPKs. Kinase activity was detected by an in-vitro kinase assay (top). Protein expression is shown for MAPKs (middle) and effectors (bottom). B. HopF2 suppresses elf18-mediated MPK3 and MPK6 activation. Transfected protoplasts were treated with 1 µM elf18 for 10 min.

Fig. 3. HopF2 suppresses flg22-induced BIK1 phosphorylation in vivo

A. HopF2 blocks flg22-induced BIK1 phosphorylation. Protoplasts were co-transfected with BIK1-HA and GFP-tagged HopF2, HopF2G2A or AvrPto for 6 hr and treated with 1 µM flg22 for 10 min. B. HopF2 did not affect BIK1 autophosphorylation and phosphorylation on BAK1. An in vitro kinase assay was performed by incubating GST-BIK1, GST-BAK1K with or without GST-HopF2. Proteins were separated with SDS-PAGE and analyzed by autoradiography (top panel). The top band is autophosphorylated GST-BIK1, and then phosphorylated GST-BAK1K. The protein loading control was shown by Coomassie blue staining (bottom panel).

Fig. 4. Myristoylation and conserved surface residues of HopF2 are required for its MAMP-suppression function

A. G2, R71 and D175 are required for HopF2 suppression of FRK1 induction. Protoplasts were co-transfected with HopF2 or its mutants and FRK1-LUC reporter, and incubated for 3 hr before treated with 10 nM flg22 for another 3 hr. B. R71 and D175 are required for HopF2 suppression of MAPK activation. HA-tagged MAPK was co-expressed with HA-tagged HopF2 or its mutants for 6 hr before 1 µM flg22 treatment for 10 min. Kinase activity was detected by an in-vitro kinase assay (top). Protein expression is shown for MAPKs and effectors (bottom).

Fig. 5. HopF2 suppresses plant nonhost resistance

A. HopF2 transgenic plants support non-adaptive bacteria growth. Four-week-old plants (Col-0 control, HopF2 or AvrPto transgenic plants) were sprayed with 10 µM DEX for 24 hr before bacterial inoculation. Arabidopsis leaves were inoculated with Pph NPS3121 (Pph) at 5 × 10⁵ cfu/ml. The bacterial counting was performed 3 days after inoculation. * indicates a significant difference with p<0.05 when compared with data from control plants based on the results of an unpaired Student’s t-test. B. RT-PCR analysis of MAMP marker gene induction. Ten-day-old seedlings were treated with 10 µM DEX for 24 hr, then treated with 10 nM flg22 for 0.5 and 1 hr. C. Susceptibility to botrytis. Four-week-old plants were sprayed with 10 µM DEX for 24 hr before Botrytis cinerea strain BO5 were sprayed at concentration of 10⁵ spores/ml. Disease symptom was recorded 2 days after infection. D. Lesion development of B. cinerea infection. The area of infection was measured 4 days postinoculation. * indicates a significant difference with p<0.05.