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The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice - PubMed

. 2012 Nov;24(11):4748-62.

doi: 10.1105/tpc.112.105429. Epub 2012 Nov 30.

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The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice

Chan-Ho Park et al. Plant Cell. 2012 Nov.

Abstract

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

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Figures

Figure 1.
Figure 1.

Translocation of AvrPiz-t into Rice Cells during Rice Blast Infection. The fungal transformant KV129 expressing AvrPiz-t:mCherry:NLS and putative interfacial matrix protein BAS4:EGFP at 30 h after infection in the sheath cells of rice cultivar YT16 rice is shown as a projection of confocal optical sections over a depth of 4 μm. “Merge” shows bright-field, mCherry (red), and enhanced GFP (green). Arrows indicate BIC, arrowheads indicate rice nucleus, and yellow indicates overlapping mCherry and enhanced GFP fluorescence signals. Pinhole settings were 1 airy unit for enhanced GFP and 12.5 airy units for mCherry. mCherry fluorescence occurred in the BIC and in the nucleus of the invaded rice cell with brighter fluorescence in the presumed nucleolus. Bar = 5 μm.

Figure 2.
Figure 2.

Response of AvrPiz-t Transgenic Rice to Blast Infection, flg22, and Chitin Treatments. (A) Punch inoculation of the AvrPiz-t transgenic and the segregated wild-type (NPB) plants. Rice leaves of 6-week-old plants were inoculated with the virulent isolate RB22. Leaves were photographed 10 DAI. (B) Sporulation (left) and relative fungal growth [2 [CT(OsUBQ) − CT(MoPot2)] × 10,000] (right) on the inoculated leaves. Samples were taken for the assays 10 DAI. Values are the means of three replications, and error bars represent the

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(n = 8, *P value < 0.05). (C) Flg22-induced ROS burst in the AvrPiz-t and NPB plants. Rice leaf disks were treated with 100 nM flg22 and water. ROS were detected with a luminol-chemiluminescence assay. Error bars represent the

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(n = 3). (D) Chitin-induced ROS burst in the AvrPiz-t and NPB plants. Rice leaf disks were treated with 8 nM chitin (hexa-N-acetyl-chitohexaose) and water. ROS were detected with a luminol-chemiluminescence assay. Error bars represent the

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(n = 3). (E) Induction of the defense-related genes KS4 and PAL at 1 h after incubation in water, chitin, or flg22. Gray bars indicate the AvrPiz-t transgenic plants, and white bars indicate NPB control plants. qRT-PCR was performed with gene-specific primers. Values are the means of three replications, and error bars represent the

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(n = 3).

Figure 3.
Figure 3.

Interaction between AvrPiz-t and APIP6 in Vitro and in Vivo. (A) Yeast two-hybrid assay between BD-AvrPiz-t (without the signal peptide sequence) and AD-APIP6. Cells were plated on -Leu-Trp-His media containing 50 mM 3-amino-1,2,4-triazole (3AT), a competitive inhibitor of the His3p enzyme. (B) GST pull-down of MBP:APIP6 and MBP:APIP6 H58Y by GST:AvrPiz-t:HA. GST:HA was used as a negative control (C) Co-IP analysis of FLAG:APIP6 H58Y and GFP:AvrPiz-t:HA (without the signal peptide sequence) in vivo. The GFP:AvrPiz-t:HA and FLAG:APIP6 H58Y genes were expressed in N. benthamiana using agroinfection. The Co-IP experiment was performed with the anti-HA antibody, and the isolated protein was analyzed by immunoblot using anti-FLAG antibody to detect APIP6 and anti-HA antibody to detect AvrPiz-t. Asterisk indicates nonspecific bands (left panel) or IgG bands from anti-HA antibody used for Co-IP (right panel).

Figure 4.
Figure 4.

Ubiquitination of AvrPiz-t by APIP6 and Suppression of APIP6 E3 Ligase Activity by AvrPiz-t in Vitro. (A) In vitro ubiquitination assay of GST:AvrPiz-t:HA by the MBP:APIP6 fusion protein. Ubiquitination of AvrPiz-t by APIP6 was detected by immunoblot with the anti-HA antibody. GST:Avr-Pita:HA was used as a negative control for determining the specificity of AvrPiz-t ubiquitination by APIP6. (B) Suppression of APIP6 E3 ligase activity by AvrPiz-t. E3 ligase activity of APIP6 in the presence of AvrPiz-t or AvrPi-ta was determined by immunoblot with the antiubiquitin antibody. Relative E3 ligase activity was calculated by comparison to the control (lane 7) using ImageJ software. (C) Immunoblot with the anti-MBP antibody to quantify the MBP:APIP6 or MBP:APIP6 H58Y protein in each lane.

Figure 5.
Figure 5.

Degradation of AvrPiz-t in the Presence of APIP6 in N. benthamiana. FLAG:APIP6 or FLAG:APIP6 H58Y (H58Y) was coexpressed with GFP:AvrPiz-t:HA (without the signal peptide sequence) by agroinfection, and the tissues were harvested 2 DAI. MG132 (50 μM) was infiltrated with DMSO 18 h before sampling. The TAP tag gene was expressed as an internal control, and the protein was detected by immunoblot with the peroxidase antiperoxidase (PAP) antibody. The transcriptional level of each gene was determined by RT-PCR.

Figure 6.
Figure 6.

Degradation of APIP6 by AvrPiz-t in N. benthamiana. Agrobacterium carrying FLAG:APIP6 H58Y construct was coinfiltrated into N. benthamiana leaves with either an empty vector (ev), GFP:HA (G), or GFP:AvrPiz-t:HA (without the signal peptide sequence). The agroinfected tissues were harvested 3 d after treatment. MG132 (50 μM) was infiltrated with DMSO 18 h before sampling. The transcriptional level of each gene was determined by RT-PCR.

Figure 7.
Figure 7.

Responses of APIP6 RNAi Plants to Blast Infection and PAMP Elicitor Treatments. (A) Punch inoculation of the APIP6 RNAi and segregated wild-type TRD plants. Leaves of 6-week-old rice plants were inoculated with the virulent isolate RB22. The leaves were photographed 10 DAI. (B) Sporulation (left) and relative fungal growth [2 [CT(OsUBQ) − CT(MoPot2)] × 100] (right) were measured 10 d after punch inoculation. Values are the means of three replications, and error bars represent the

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(n = 8; *P value < 0.05). (C) Measurement of the flg22-induced ROS burst. Leaf disks from the APIP6 RNAi and the control plants were treated with 100 nM flg22 and water. ROS were detected with a luminol-chemiluminescence assay. Error bars represent the

se

(n = 3). (D) Measurement of chitin-induced ROS burst. Leaf disks from the APIP6 RNAi and the control plants were treated with 8 nM chitin (hexa-N-acetyl-chitohexaose) and water. ROS were detected with a luminol-chemiluminescence assay. Error bars represent the

se

(n = 3). (E) Induction of the defense-related genes NAC4 (1 h after incubation) and PAL (3 h after incubation) in water, chitin, or flg22. Gray bars indicate APIP6 RNAi plants, and white bars indicate the control plants. qPCR was performed with gene-specific primers. Values are the means of three replications, and error bars represent the

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(n = 3; *P value < 0.05).

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