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. 2011 Jun 28;108(26):10774-9.
doi: 10.1073/pnas.1103338108. Epub 2011 Jun 13.

Effector-triggered immunity blocks pathogen degradation of an immunity-associated vesicle traffic regulator in Arabidopsis

Kinya Nomura  1 , Christy Mecey, Young-Nam Lee, Lori Alice Imboden, Jeff H Chang, Sheng Yang He
Affiliations

Effector-triggered immunity blocks pathogen degradation of an immunity-associated vesicle traffic regulator in Arabidopsis

Kinya Nomura et al. Proc Natl Acad Sci U S A. .

Abstract

Innate immunity in plants can be triggered by microbe- and pathogen-associated molecular patterns. The pathogen-associated molecular pattern-triggered immunity (PTI) is often suppressed by pathogen effectors delivered into the host cell. Plants can overcome pathogen suppression of PTI and reestablish pathogen resistance through effector-triggered immunity (ETI). An unanswered question is how plants might overcome pathogen-suppression of PTI during ETI. Findings described in this paper suggest a possible mechanism. During Pseudomonas syringae pathovar tomato (Pst) DC3000 infection of Arabidopsis, a host ADP ribosylation factor guanine nucleotide exchange factor, AtMIN7, is destabilized by the pathogen effector HopM1 through the host 26S proteasome. In this study, we discovered that AtMIN7 is required for not only PTI, consistent with the notion that Pst DC3000 degrades AtMIN7 to suppress PTI, but also ETI. The AtMIN7 level in healthy plants is low, but increases posttranscriptionally in response to activation of PTI. Whereas DC3000 infection led to degradation of AtMIN7, activation of ETI by three different effectors, AvrRpt2, AvrPphB, and HopA1, in Col-0 plants blocks the ability of Pst DC3000 to destabilize AtMIN7. Further analyses of bacterial translocation of HopM1 and AtMIN7 stability in HopM1 transgenic plants show that ETI prevents HopM1-mediated degradation of AtMIN7 inside the plant cell. Both AtMIN7 and HopM1 are localized to the trans-Golgi network/early endosome, a subcellular compartment that is not previously known to be associated with bacterial pathogenesis in plants. Thus, blocking pathogen degradation of trans-Golgi network/early endosome-associated AtMIN7 is a critical part of the ETI mechanism to counter bacterial suppression of PTI.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HopM1 is localized to the trans-Golgi network/early endosome (TGN/EE). (Top) HopM1-GFP is localized in intracellular punctate structures 8 h postinduction with 30 μM DEX in DEX::hopM1-GFP transgenic Arabidopsis. These structures could be stained with FM4-64, indicating that they are membrane derived. (Middle) Transiently expressed HopM1-GFP colocalizes with the TGN/EE marker VHA-a1-RFP in leaf cells of Nicotiana benthamiana. (Bottom) Transiently expressed HopM1-GFP colocalizes with AtMIN7-DsRed2 in leaf cells of Nicotiana benthamiana. (Scale bars: 10 μm.) Dashed lines in bright field images indicate borders between leaf cells.
Fig. 2.
Fig. 2.
Role of AtMIN7 in BTH- and flg22-triggered immunity. Arabidopsis Col-0 plants were sprayed with H2O, 2 μM flg22, or 30 μM BTH. Plants were dip-inoculated with Pst DC3000 bacteria (1 × 108 cfu/mL) 24 h later. (A) Disease symptoms (chlorosis and necrosis) at day 4 and (B) bacterial populations in infected leaves were recorded. *P < 0.05 and **P < 0.01 between Col-0 and atmin7 plants. (C) AtMIN7 protein amounts in Arabidopsis Col-0leaves 9 h after infiltration with H2O, 30 μM BTH, or 2 μM flg22. One to three closely migrating bands of molecular weights between 200 and 250 kDa could be detected in immunoblotting by the AtMIN7 antibody (Upper). A portion of a Coomassie Blue-stained gel loaded with the same protein samples used in immunoblotting is shown to indicate equal loading (Lower).
Fig. 3.
Fig. 3.
The role of AtMIN7 in effector-triggered immunity. (A) Multiplication of Pst DC3000(avrRpt2) in Col-0 and atmin7 plants. Arabidopsis plants were dip inoculated with bacteria at 1 × 108 cfu/mL. Bacterial populations were determined at day 4. *P < 0.05 and **P < 0.01 between Col-0 and atmin7 plants. (B) Callose deposition in leaves of Col-0 and atmin7 plants. Arabidopsis leaves were hand infiltrated with 1 × 108 cfu/mL bacteria. Leaves were stained for callose deposition 9 h after treatment. Average numbers of callose depositions per field of view (0.9 mm2) are presented with SDs displayed as errors. *P < 0.05 and **P < 0.01 between Col-0 and atmin7 plants. (C) Responses of Col-0 and atmin7 plants to nonhost P. syringae strains. Plants were dip inoculated with Pst DC3000 or a nonhost pathogen, P. syringae tabaci 11528 or P. syringae pv. phaseolicola NPS3132, at 1 × 108 cfu/mL. Bacterial populations were determined at day 4. *P < 0.05 between Col-0 and atmin7 plants.
Fig. 4.
Fig. 4.
(A) RT-PCR analysis of AtMIN7 and ACTIN1 gene expression. Arabidopsis Col-0 leaves were hand infiltrated with 1 × 108 cfu/mL bacteria, 30 μM BTH, or 2 μM flg22. Total RNA samples were purified 9 h after treatment and subjected to RT-PCR assays (25 cycles). (B–D) Western blot analysis of the AtMIN7 protein. Arabidopsis Col-0 leaves were hand infiltrated with H2O, 1 × 108 cfu/mL bacteria, 30 μM BTH, or 2 μM flg22. atmin7 leaves infiltrated with distilled water were used as a negative control. Total protein samples were extracted from leaves 9 h after treatment and loaded onto SDS/PAGE gel. AtMIN7 was detected with AtMIN7 antibody (α-AtMIN7).
Fig. 5.
Fig. 5.
(A) Detection of bacterial translocation of HopM1-CyaA during bacterial infection. Bacterial strains harboring a shcM-hopM1-cyaA reporter plasmid were hand infiltrated into Arabidopsis Col-0 leaves at 1 × 108 cfu/mL. Samples were collected at 0 and 9 h postinfiltration. **P < 0.01 indicates significant difference in cAMP levels between Pst DC3000-infected leaves and hrcC mutant-infected leaves. (B–E) Western blot analysis of the AtMIN7 protein in plants. (B) Col-0 gl1 and 6×His-HopM1 Arabidopsis leaves were coinfiltrated with 1 × 108 cfu/mL bacteria plus DEX (final concentration of 10 nM). (C) Col-0 gl1 and 6×His-HopM1 Arabidopsis leaves were coinfiltrated with 1 × 108 cfu/mL bacteria plus DEX (final concentration of 200 nM) or with H2O plus DEX (final concentration of 200 nM). (D) 10 nM DEX was infiltrated into leaves of 6×His-HopM1 plants 9 h before infiltration with H2O or 1 × 108 cfu/mL bacteria. (E) H2O or 1 × 108 cfu/mL bacteria were infiltrated into leaves of 6×His-HopM1 plants 3 h before infiltration of 10 nM DEX. Total protein samples were extracted from leaves 9 h after the last leaf infiltration. AtMIN7 (indicated by arrow) was detected using an AtMIN7 antibody (α-AtMIN7).
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