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. 2013 Jun;162(2):1018-29.
doi: 10.1104/pp.113.219659. Epub 2013 Apr 30.

The Pseudomonas syringae type III effector AvrRpt2 promotes pathogen virulence via stimulating Arabidopsis auxin/indole acetic acid protein turnover

Fuhao Cui  1 , Shujing Wu, Wenxian Sun, Gitta Coaker, Barbara Kunkel, Ping He, Libo Shan
Affiliations

The Pseudomonas syringae type III effector AvrRpt2 promotes pathogen virulence via stimulating Arabidopsis auxin/indole acetic acid protein turnover

Fuhao Cui et al. Plant Physiol. 2013 Jun.

Abstract

To accomplish successful infection, pathogens deploy complex strategies to interfere with host defense systems and subvert host physiology to favor pathogen survival and multiplication. Modulation of plant auxin physiology and signaling is emerging as a common virulence strategy for phytobacteria to cause diseases. However, the underlying mechanisms remain largely elusive. We have previously shown that the Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis (Arabidopsis thaliana) auxin physiology. Here, we report that AvrRpt2 promotes auxin response by stimulating the turnover of auxin/indole acetic acid (Aux/IAA) proteins, the key negative regulators in auxin signaling. AvrRpt2 acts additively with auxin to stimulate Aux/IAA turnover, suggesting distinct, yet proteasome-dependent, mechanisms operated by AvrRpt2 and auxin to control Aux/IAA stability. Cysteine protease activity is required for AvrRpt2-stimulated auxin signaling and Aux/IAA degradation. Importantly, transgenic plants expressing the dominant axr2-1 mutation recalcitrant to AvrRpt2-mediated degradation ameliorated the virulence functions of AvrRpt2 but did not alter the avirulent function mediated by the corresponding RPS2 resistance protein. Thus, promoting auxin response via modulating the stability of the key transcription repressors Aux/IAA is a mechanism used by the bacterial type III effector AvrRpt2 to promote pathogenicity.

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Figures

Figure 1.
Figure 1.
AvrRpt2 promotes the turnover of AXR2 and AXR3 but not TIR1. A, Both FLAG and HA epitope-tagged AvrRpt2 stimulate AXR2-HA protein turnover in wild-type Col-0 protoplasts. The control plasmid (Ctrl), AvrRpt2-FLAG, or AvrRpt2-HA was cotransfected with AXR2-HA into wild-type protoplasts. The protoplasts were incubated for 4 h before treatment with1 µm NAA for an additional 2 h, and western-blot analysis was carried out using an α-HA or α-FLAG antibody. The band intensity of AXR2 was quantified by ImageJ software and is labeled on the bottom. B, AvrRpt2 promotes AXR2-GFP but not GFP protein turnover in wild-type protoplasts. C, AvrRpt2-FLAG promotes AXR3-HA protein turnover in wild-type protoplasts. D, AvrRpt2-FLAG promotes AXR2-HA protein turnover in rps2 protoplasts. E, AvrRpt2 does not affect TIR1 protein expression in wild-type protoplasts. All the above experiments were repeated three to five times, and representative results are shown.
Figure 2.
Figure 2.
AvrRpt2 promotes AXR2 turnover during bacterial infection. A, Inducible expression of AvrRpt2 in transgenic plants leads to AXR2-FLAG protein turnover. Leaves from two independent lines of 4-week-old 35S::AXR2-FLAG/Dex::AvrRpt2/rps2 transgenic plants were hand inoculated with either 5 µm Dex or water control and harvested 24 h after inoculation. The AXR2-FLAG protein was detected by western blot with an α-FLAG antibody. A nonspecific band (NSB) on the same blot is shown as the protein-loading control. AvrRpt2 expression was detected by RT-PCR analysis, and the UBQ10 gene was used as a control. The band intensity of AXR2 was quantified by ImageJ software and is labeled on the bottom. B, Bacterium-delivered AvrRpt2 stimulates AXR2 protein turnover. Four-week-old 35S::AXR2-FLAG/Dex::AvrRpt2/rps2 transgenic plants were hand inoculated with the indicated bacteria at a concentration of 1 × 108 cfu mL−1 or water as a mock control. Samples were harvested 30 h after inoculation for AXR2-FLAG protein detection with an α-FLAG antibody. The above experiments were repeated three times, and representative results are shown. WT, Wild type.
Figure 3.
Figure 3.
The AvrRpt2-mediated auxin response depends on its Cys protease activity. A, The Cys protease catalytic mutants H208A and C122A of AvrRpt2 are no longer able to activate auxin reporter pIAA5::LUC expression. Protoplasts were cotransfected with a control vector (Ctrl), wild-type AvrRpt2 or its mutants, and the pIAA5::LUC reporter. pUBQ::GUS was included in the transfection as an internal control. The luciferase and GUS activities were detected 6 h after transfection. The promoter activity is shown as the LUC-GUS ratio. Data are shown as means ± sd, and the asterisks indicate significant differences between wild-type AvrRpt2 and its mutants (P < 0.05). B, The H208A mutant blocks AvrRpt2-mediated AXR2 turnover. The control plasmid, AvrRpt2-HA, or AvrRpt2 H208A-HA was cotransfected with AXR2-HA into wild-type protoplasts. The protoplasts were incubated for 4 h before treatment with 1 µm NAA for an additional 2 h. Note that the H208A mutant accumulated as unprocessed protein because of its proteolytic deficiency. The band intensity of AXR2 was quantified by ImageJ software and is labeled on the bottom. C, The C122A mutant blocks AvrRpt2-mediated AXR2 turnover. D, The wild type but not the H208A mutant of AvrRpt2 diminishes the AXR2-GFP and AXR3-GFP fluorescence signals in the nucleus. The control plasmid, AvrRpt2-HA, or AvrRpt2H208A-HA was cotransfected with AXR2-GFP or AXR3-GFP into wild-type protoplasts. An NLS-RFP construct was included in the transfection for nuclear localization control. The samples were collected 10 h after transfection for microscope observation. All the above experiments were repeated three times, and representative results are shown.
Figure 4.
Figure 4.
AvrRpt2-mediated AXR2 turnover is proteasome dependent. A, MG132 suppresses auxin-mediated AXR2 turnover. Four-week-old 35S::AXR2-FLAG/Dex-AvrRpt2/rps2 transgenic plants were hand inoculated with 4 µm NAA with or without 5 µm MG132, and the samples were collected 24 h after inoculation for western blot with an α-FLAG antibody. A nonspecific band (NSB) on the same blot is shown as the protein-loading control. B, MG132 suppresses AvrRpt2-mediated AXR2 turnover. Four-week-old 35S::AXR2-FLAG/Dex-AvrRpt2/rps2 transgenic plants were hand inoculated with 5 µm Dex with or without 5 µm MG132, and samples were collected 24 h after inoculation for western blot. C, AvrRpt2 possesses an additive effect with auxin to stimulate AXR2 turnover. Wild-type protoplasts were transfected with a control plasmid or AvrRpt2-FLAG together with AXR2-HA. The protoplasts were incubated for 4 h before treatment with different concentrations of NAA for an additional 2 h. The band intensity of AXR2 was quantified by ImageJ software and is labeled underneath the gel. The numbers in the top row represent the relative value of AXR2 protein level when the sample without NAA treatment or AvrRpt2 transfection was set as 1. The numbers in the bottom row show the relative percentage of AXR2 protein compared with samples without NAA treatment in the absence (control [Ctrl]) or presence of AvrRpt2. All the above experiments were repeated three times, and representative results are shown.
Figure 5.
Figure 5.
axr2-1 is resistant to AvrRpt2-mediated degradation. A, The axr2-1 (AXR2P87S) mutant is insensitive to auxin- and AvrRpt2-mediated degradation in protoplasts. Ctrl, Control. B, The axr2-1 protein is insensitive to AvrRpt2-mediated degradation in transgenic plants. Leaves from three independent lines of 4-week-old axr2-1-FLAG/Dex::AvrRpt2/rps2 transgenic plants were hand inoculated with either 5 µm Dex or water control and harvested 24 h after inoculation. The axr2-1 proteins were detected by western blot with an α-FLAG antibody. A nonspecific band (NSB) on the same blot is shown as the protein-loading control. The expression of AvrRpt2 after Dex treatment is shown by RT-PCR, with UBQ10 as a control. The band intensity of axr2-1 was quantified by ImageJ software and is labeled on the bottom. The above experiments were repeated three times, and representative results are shown.
Figure 6.
Figure 6.
The axr2-1 mutant suppresses AvrRpt2 virulence. A, The AvrRpt2-mediated suppression of Pst-induced defense gene expression is abolished in axr2-1 transgenic plants. Leaves from 4-week-old two independent lines (#14 and #21) of axr2-1-FLAG/Dex::AvrRpt2/rps2, Dex::AvrRpt2/rps2, or rps2 plants were syringe inoculated with Pst at a concentration of 1 × 108 cfu mL−1. Samples were harvested at 6 hpi for ICS1, PAD4, and PR1 and at 3 dpi for PR2 and PR5 gene expression with quantitative RT-PCR analysis. UBQ10 was used as an internal control. The data are shown as means ± se from three independent biological replicates. Asterisks indicate significant differences at P < 0.05 when compared with data from rps2 plants with Pst inoculation. B, AvrRpt2-mediated suppression of AvrRpm1 disease resistance is blocked in axr2-1 transgenic plants. Leaves from 4-week-old three independent lines of axr2-1-FLAG/Dex::AvrRpt2/rps2, Dex::AvrRpt2/rps2, or rps2 plants were hand inoculated with Pst avrRpm1 at a concentration of 5 × 105 cfu mL−1. Bacterial growth was measured 0, 2, and 4 dpi. The data are shown as means ± se of three repeats, and asterisks indicate significant differences at P < 0.05 when compared with data from rps2 plants. Disease symptoms at 5 dpi are shown at the bottom. C, AvrRpt2-mediated suppression of AvrRpm1 HR is blocked in axr2-1 transgenic plants. Leaves from 4-week-old two independent lines of axr2-1-FLAG/Dex::AvrRpt2/rps2, Dex::AvrRpt2/rps2, or rps2 plants were hand inoculated with Pst avrRpm1 at a concentration of 1 × 108 cfu mL−1. The HR was observed at the indicated time points and calculated as the percentage of leaves exhibiting the wilting phenotype of total inoculated leaves (n > 25). All the above experiments were repeated three times, and representative results are shown. [See online article for color version of this figure.]
Figure 7.
Figure 7.
The axr2-1 mutant does not affect AvrRpt2 avirulence. A, The axr2-1 mutant does not affect AvrRpt2-mediated HR. Leaves from 4-week-old three independent lines of axr2-1-FLAG/WT or wild-type (WT) plants were hand inoculated with Pst avrRpt2 at a concentration of 1 × 108 cfu mL−1. The HR was observed at the indicated time points and calculated as the percentage of leaves exhibiting the wilting phenotype of total inoculated leaves (n > 25). B, The axr2-1 mutant does not affect AvrRpt2-mediated disease resistance. Leaves from 4-week-old three independent lines of axr2-1-FLAG/WT or wild-type plants were hand inoculated with Pst avrRpt2 at a concentration of 5 × 105 cfu mL−1. Bacterial growth was measured 0, 2, and 4 dpi. The data are shown as means ± se of three repeats. C, The axr2-1 mutant does not affect AvrRpt2-mediated WRKY46 activation. Protoplasts were cotransfected with the control plasmid, AvrRpt2, AXR2, or the axr2-1 mutant together with the pWRKY46::LUC reporter. pUBQ::GUS was included in the transfection as an internal control. The luciferase and GUS activities were detected 6 h after transfection. The promoter activity is shown as the LUC-GUS ratio with means ± sd. D, A model of the AvrRpt2 virulence mechanism via stimulating Arabidopsis Aux/IAA protein turnover. All the above experiments were repeated three times, and representative results are shown. [See online article for color version of this figure.]
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References

    1. Abel S, Nguyen MD, Theologis A. (1995) The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana. J Mol Biol 251: 533–549 - PubMed
    1. Albrecht C, Boutrot F, Segonzac C, Schwessinger B, Gimenez-Ibanez S, Chinchilla D, Rathjen JP, de Vries SC, Zipfel C. (2012) Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1. Proc Natl Acad Sci USA 109: 303–308 - PMC - PubMed
    1. Axtell MJ, Chisholm ST, Dahlbeck D, Staskawicz BJ. (2003) Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease. Mol Microbiol 49: 1537–1546 - PubMed
    1. Axtell MJ, Staskawicz BJ. (2003) Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112: 369–377 - PubMed
    1. Belkhadir Y, Jaillais Y, Epple P, Balsemão-Pires E, Dangl JL, Chory J. (2012) Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns. Proc Natl Acad Sci USA 109: 297–302 - PMC - PubMed

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