An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus - PubMed (original) (raw)

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

Hazel McLellan et al. PLoS Pathog. 2013.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Transient over-expression of Pi03192 in planta increases P. infestans virulence.

Agrobacterium was used to transiently over-express Pi03192 on one half of a leaf and empty vector on the other. Leaves were subsequently infected with P. infestans. (A) Shows a typical leaf with larger lesions on the half of the leaf expressing Pi03192. (B) The leaf in (A) stained with trypan blue to show increased P. infestans mycelial growth on the half of the leaf expressing Pi03192. (C) Graph quantifying lesion area in the presence or absence of Pi03192 expression. Error bars show standard error and asterisks indicate a highly significant difference (p<0.001, t-test) (from 3 biological replicates each comprising 17 inoculations per treatment).

Figure 2

Figure 2. Pi03192 interacts with two putative NAC transcription factors from potato.

(A) StNTP1 and StNTP2 grow on Δ histidine (-HIS) media and show β-galactosidase (B-Gal) activity when co-transformed with Pi03192 but not with control RXLR effector PiAvr2. (B) Schematic representations of StNTP1 and StNTP2 showing NAM DNA-binding domains (NAC domain) and TM domains and the number, length in amino acids (aa) and position of the interacting fragments identified in Y2H.

Figure 3

Figure 3. 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.

Figure 4

Figure 4. Interactions between Pi03192 and StNTP1 or StNTP2 occur at the ER membrane.

(A) BiFC images showing that YFP fluorescence is reconstituted at the ER when YC-StNTP1 and YC-StNTP2 are co-expressed with YN-Pi03192, but not with YN-Avr2. The arrowheads indicated the ER membrane surrounding the nucleus. Scale bars indicate 35 µm. (B) Graph shows relative fluorescence quantified using a fluorimeter. Error bars show standard error and asterisks indicate a very highly significant difference (p<0.001, _t_-test) in fluorescence of YC-StNTP1 or YC-StNTP2 co-expressed with YN-Pi03192, compared to co-expression with YN-Avr2. (C) Immunoblots showing stability of full length YN-Pi03192 and YN-Avr2 (myc antibody) fusions and YC-StNTP1 and YC-StNTP2 (HA antibody) fusions.

Figure 5

Figure 5. VIGS of NbNTP1 and NbNTP2 increases susceptibility to P. infestans.

(A) Typical confocal images of P. infestans 88069-tdT growth (observed as Td-tomato fluorescence) at 3 dpi in unsilenced (GFP) or silenced (NTP1 II or NTP2 I as examples) for comparison with categories of infection in Figure S7). (B) Graph shows the percentage of P. infestans 88069-tdT infection sites on plants that are unsilenced (GFP) or expressing each VIGS construct (as indicated) which belong to each infection category (surface growth, initial colonisation, extensive mycelial growth) as visualised by confocal microscopy at 3 dpi. The results are combined data for 2 biological reps (n = 8 per construct per rep). (C) Graph shows the percentage increase in the number of inoculation points that are sporulating at 7 dpi, with the levels of sporulation in the GFP control set to zero. Error bars are standard error and the graph represents the combined data from 10 biological reps (n = 32 per construct per rep). Significant differences to the GFP control by _t_-test are indicated by asterisks and p values. (D) Photographs of leaves from the indicated VIGS plants infected with WT P. infestans at 10 dpi. (E). Sporangia per ml recovered at 10 dpi from each VIGS line. Error bars are standard error and significant difference to the GFP control by _t_-test are indicated by asterisks and p values.

Figure 6

Figure 6. Silencing of Pi03192 compromises pathogenicity which can be restored in TRV:NTP plants.

(A) Graph shows expression levels of Pi03192 are severely down regulated in 03192_IR compared to WT (strain 88069) while three other known P. infestans avirulences are unaffected. (B) Graph shows 03192_IR is significantly less able to infect both potato and N. benthamiana compared to WT (two tailed _t_-test p<0.005,n = 8). Images show symptoms of WT and 03192_IR infection on (C) potato and (D) N. benthamiana. (E) Graph shows that in planta expression of Pi03192 is able to restore virulence of 03192_IR to WT levels (strain 88069 inoculated with transient expression of empty vector control; vec) (no significant difference with one way anova p = 0.83, n = 55). In contrast, colonisation by 03192_IR with expression of the empty vector control (vec) is significantly reduced (one way anova p<0.05, n = 73). (F) Graph shows significant increases in lesion diameter for 03192_IR on each TRV:NTP silenced line compared to the TRV:GFP control (two tailed t-test p<0.05, n = 30). (G) Images show 03192_IR symptoms on TRV:NTP silenced plants and the TRV:GFP control. Error bars are standard error.

Figure 7

Figure 7. Differential transcript accumulation of NbNTP genes.

Transcript abundance of (A) Pi03192 (relative to expression in sporangia, which is given a value of 1) and (B) NbNTP1 and NbNTP2 (relative to uninoculated plant, which is given a value of 1) at 16, 24 and 48 hours post-inoculation of N. benthamiana plants infected with wildtype P. infestans. Error bars are standard error. Expression of (C) NbNTP1 and (D) NbNTP2 at 3, 16, 24 and 48 hours post-inoculation with either culture media or P. infestans culture filtrate (CF), relative to untreated plant (0). Error bars are standard error.

Figure 8

Figure 8. Pi03192 prevents CF-mediated accumulation of StNTP1 and StNTP2 in the nucleus.

(A) Confocal images of localisation of either GFP-StNTP1 or GFP-StNTP2 co-expressing either pFlub (vec) or pFlub-03192 and treated with Media or culture filtrate, plus and minus MG132. Scale bar is 10 µm. (B) Graph shows the percentage of nuclei containing either GFP-StNTP1 or GFP-StNTP2 fluorescence with each treatment. Error bars are standard error and one-way ANOVA analysis shows that treatment with CF and MG132, when co-expressed with empty vector (vec), is statistically different from the other treatments, which are not different from each other. Significance is indicated by p-value and asterisks. (C) Immunoblots show the stability of GFP-StNTP1 and GFP-StNTP2 with different treatments as indicated, probed with a specific GFP antibody. PS is Ponceau stain.

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