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An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus.

McLellan H, Boevink PC, Armstrong MR, Pritchard L, Gomez S, Morales J, Whisson SC, Beynon JL, Birch PR - PLoS Pathog. (2013)

Bottom Line: This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192).Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity.Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

View Article: PubMed Central - PubMed

Affiliation: The Division of Plant Sciences, College of Life Science, University of Dundee at the James Hutton Institute, Invergowrie, Dundee, United Kingdom ; Dundee Effector Consortium, James Hutton Institute, Invergowrie, Dundee, United Kingdom.

ABSTRACT
The potato late blight pathogen Phytophthora infestans secretes an array of effector proteins thought to act in its hosts by disarming defences and promoting pathogen colonisation. However, little is known about the host targets of these effectors and how they are manipulated by the pathogen. This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192). The effector interacts with NAC Targeted by Phytophthora (NTP) 1 and NTP2 at the endoplasmic reticulum (ER) membrane, where these proteins are localised. Transcripts of NTP1 and NTP2 rapidly accumulate following treatment with culture filtrate (CF) from in vitro grown P. infestans, which acts as a mixture of Phytophthora PAMPs and elicitors, but significantly decrease during P. infestans infection, indicating that pathogen activity may prevent their up-regulation. Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity. Transient expression of Pi03192 in planta restores pathogenicity of the Pi03192-silenced line. Moreover, colonisation by the Pi03192-silenced line is significantly enhanced on N. benthamiana plants in which either NTP1 or NTP2 have been silenced. StNTP1 and StNTP2 proteins are released from the ER membrane following treatment with P. infestans CF and accumulate in the nucleus, after which they are rapidly turned over by the 26S proteasome. In contrast, treatment with the defined PAMP flg22 fails to up-regulate NTP1 and NTP2, or promote re-localisation of their protein products to the nucleus, indicating that these events follow perception of a component of CF that appears to be independent of the FLS2/flg22 pathway. Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

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StNTP1, StNTP2 and Pi03192 are localised to the ER membrane in planta.(A) GFP-Pi03192 co-localises to the ER membrane with an RFP tagged ER marker. Scale bars indicate 10 µm, and insert-images show ER around the nucleus. (B) GFP-StNTP1 and 2 localise to the ER and are found in this membrane surrounding the nucleus (insert-images). Scale bars indicate 10 µm. (C) Immunoblots of GFP-StNTP1, GFP-StNTP2 and GFP-Pi03192 showing the stability of the full length constructs when probed with a specific GFP antibody. PS is Ponceau stain.
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ppat-1003670-g003: StNTP1, StNTP2 and Pi03192 are localised to the ER membrane in planta.(A) GFP-Pi03192 co-localises to the ER membrane with an RFP tagged ER marker. Scale bars indicate 10 µm, and insert-images show ER around the nucleus. (B) GFP-StNTP1 and 2 localise to the ER and are found in this membrane surrounding the nucleus (insert-images). Scale bars indicate 10 µm. (C) Immunoblots of GFP-StNTP1, GFP-StNTP2 and GFP-Pi03192 showing the stability of the full length constructs when probed with a specific GFP antibody. PS is Ponceau stain.

Mentions: The subcellular localisations of StNTP1, StNTP2 and Pi03192 were examined by Agrobacterium-mediated transient expression of each with N-terminal GFP fusions in N. benthamiana and imaging using confocal microscopy. Images of cells expressing each of the constructs revealed a network of fluorescence suggestive of localisation to the endoplasmic reticulum (ER) membrane in planta (Figure 3A and B). To confirm the ER localisation of the RxLR effector, GFP-Pi03192 was co-expressed with an RFP-ER-tagged construct and imaged by confocal microscopy. Both were observed to co-localise to the ER network as shown by the merge of the green and red channels (Figure 3A). The ER membrane localisation is consistent with the observation that both StNTPs possess predicted C-terminal TM domains. However, GFP-Pi03192 is also ER localised despite the absence of a predicted TM domain. Western blots hybridised with an antibody specific to GFP demonstrated that the GFP-StNTP1, GFP-StNTP2 and GFP-Pi03192 fusion proteins were stable, showing bands of the predicted sizes, respectively 111 kDa, 106 kDa and 39 kDa (Figure 3C).


An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus.

McLellan H, Boevink PC, Armstrong MR, Pritchard L, Gomez S, Morales J, Whisson SC, Beynon JL, Birch PR - PLoS Pathog. (2013)

StNTP1, StNTP2 and Pi03192 are localised to the ER membrane in planta.(A) GFP-Pi03192 co-localises to the ER membrane with an RFP tagged ER marker. Scale bars indicate 10 µm, and insert-images show ER around the nucleus. (B) GFP-StNTP1 and 2 localise to the ER and are found in this membrane surrounding the nucleus (insert-images). Scale bars indicate 10 µm. (C) Immunoblots of GFP-StNTP1, GFP-StNTP2 and GFP-Pi03192 showing the stability of the full length constructs when probed with a specific GFP antibody. PS is Ponceau stain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795001&req=5

ppat-1003670-g003: StNTP1, StNTP2 and Pi03192 are localised to the ER membrane in planta.(A) GFP-Pi03192 co-localises to the ER membrane with an RFP tagged ER marker. Scale bars indicate 10 µm, and insert-images show ER around the nucleus. (B) GFP-StNTP1 and 2 localise to the ER and are found in this membrane surrounding the nucleus (insert-images). Scale bars indicate 10 µm. (C) Immunoblots of GFP-StNTP1, GFP-StNTP2 and GFP-Pi03192 showing the stability of the full length constructs when probed with a specific GFP antibody. PS is Ponceau stain.
Mentions: The subcellular localisations of StNTP1, StNTP2 and Pi03192 were examined by Agrobacterium-mediated transient expression of each with N-terminal GFP fusions in N. benthamiana and imaging using confocal microscopy. Images of cells expressing each of the constructs revealed a network of fluorescence suggestive of localisation to the endoplasmic reticulum (ER) membrane in planta (Figure 3A and B). To confirm the ER localisation of the RxLR effector, GFP-Pi03192 was co-expressed with an RFP-ER-tagged construct and imaged by confocal microscopy. Both were observed to co-localise to the ER network as shown by the merge of the green and red channels (Figure 3A). The ER membrane localisation is consistent with the observation that both StNTPs possess predicted C-terminal TM domains. However, GFP-Pi03192 is also ER localised despite the absence of a predicted TM domain. Western blots hybridised with an antibody specific to GFP demonstrated that the GFP-StNTP1, GFP-StNTP2 and GFP-Pi03192 fusion proteins were stable, showing bands of the predicted sizes, respectively 111 kDa, 106 kDa and 39 kDa (Figure 3C).

Bottom Line: This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192).Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity.Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

View Article: PubMed Central - PubMed

Affiliation: The Division of Plant Sciences, College of Life Science, University of Dundee at the James Hutton Institute, Invergowrie, Dundee, United Kingdom ; Dundee Effector Consortium, James Hutton Institute, Invergowrie, Dundee, United Kingdom.

ABSTRACT
The potato late blight pathogen Phytophthora infestans secretes an array of effector proteins thought to act in its hosts by disarming defences and promoting pathogen colonisation. However, little is known about the host targets of these effectors and how they are manipulated by the pathogen. This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192). The effector interacts with NAC Targeted by Phytophthora (NTP) 1 and NTP2 at the endoplasmic reticulum (ER) membrane, where these proteins are localised. Transcripts of NTP1 and NTP2 rapidly accumulate following treatment with culture filtrate (CF) from in vitro grown P. infestans, which acts as a mixture of Phytophthora PAMPs and elicitors, but significantly decrease during P. infestans infection, indicating that pathogen activity may prevent their up-regulation. Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity. Transient expression of Pi03192 in planta restores pathogenicity of the Pi03192-silenced line. Moreover, colonisation by the Pi03192-silenced line is significantly enhanced on N. benthamiana plants in which either NTP1 or NTP2 have been silenced. StNTP1 and StNTP2 proteins are released from the ER membrane following treatment with P. infestans CF and accumulate in the nucleus, after which they are rapidly turned over by the 26S proteasome. In contrast, treatment with the defined PAMP flg22 fails to up-regulate NTP1 and NTP2, or promote re-localisation of their protein products to the nucleus, indicating that these events follow perception of a component of CF that appears to be independent of the FLS2/flg22 pathway. Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

Show MeSH
Related in: MedlinePlus