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. 2015 Sep;8(9):1385-95.
doi: 10.1016/j.molp.2015.04.012. Epub 2015 Apr 30.

A Host KH RNA-Binding Protein Is a Susceptibility Factor Targeted by an RXLR Effector to Promote Late Blight Disease

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Free PMC article

A Host KH RNA-Binding Protein Is a Susceptibility Factor Targeted by an RXLR Effector to Promote Late Blight Disease

Xiaodan Wang et al. Mol Plant. .
Free PMC article

Abstract

Plant pathogens deliver effector proteins that alter host processes to create an environment conducive to colonization. Attention has focused on identifying the targets of effectors and how their manipulation facilitates disease. RXLR effector Pi04089 from the potato blight pathogen Phytophthora infestans accumulates in the host nucleus and enhances colonization when transiently expressed in planta. Its nuclear localization is required for enhanced P. infestans colonization. Pi04089 interacts in yeast and in planta with a putative potato K-homology (KH) RNA-binding protein, StKRBP1. Co-localization of Pi04089 and StKRBP1, and bimolecular fluorescence complementation between them, indicate they associate at nuclear speckles. StKRBP1 protein levels increased when it was co-expressed with Pi04089. Indeed, such accumulation of StKRBP1 was observed also on the first day of leaf colonization by the pathogen. Remarkably, overexpression of StKRBP1 significantly enhances P. infestans infection. Mutation of the nucleotide-binding motif GxxG to GDDG in all three KH domains of StKRBP1 abolishes its interaction with Pi04089, its localization to nuclear speckles, and its increased accumulation when co-expressed with the effector. Moreover, the mutant StKRBP1 protein no longer enhances leaf colonization by P. infestans, implying that nucleotide binding is likely required for this activity. We thus argue that StKRBP1 can be regarded as a susceptibility factor, as its activity is beneficial to the pathogen.

Keywords: effector-triggered susceptibility; late blight; oomycete; plant disease.

Figures

Figure 1
Figure 1
Pi04089 Contributes to P. infestans Virulence. (A)Pi04089 expression is up-regulated at 24 and 48 h post infection (hpi) of potato plants with P. infestans. (B) Increase in the area colonized by P. infestans following Agrobacterium-mediated expression of GFP-04089 in one half of a leaf compared with the expression of a GFP control. (C) Graph shows a significant increase (p < 0.001, t test, as indicated by asterisks) in mean diameter of P. infestans lesions following Agrobacterium-mediated expression of GFP-04089 compared with the expression of a GFP control. Error bars are SE, and the graph represents the combined data from three biological reps (n = 84 per construct). (D) Confocal projection of N. benthamiana leaf epidermal cells transiently expressing GFP-04089, showing that the fusion protein accumulates in the cytoplasm and nucleus. The inset is a magnified single optical section through the cell nucleus, showing that the effector fusion forms a distinct ring around the nucleolus. Scale bar represents 50 μm.
Figure 2
Figure 2
Nuclear Localization of Pi04089 Is Necessary to Promote P. infestans Colonization. (A) Images are confocal projections of N. benthamiana leaf epidermal cells transiently expressing the GFP-Pi04089 and the modified NESGFP-Pi04089, showing the reduction in nuclear fluorescence resulting from the addition of the nuclear export signal. Insets are magnified single optical sections of the cell nuclei, revealing that fluorescence associated with the nucleus in the stacked projection is primarily in cytoplasm surrounding the nucleus. Scale bar represents 20 μm. (B) Graph shows a significant decrease (p < 0.001 by analysis of variance [ANOVA, as indicated by lowercase letters]) in mean diameter of P. infestans lesions following Agrobacterium-mediated expression of NESGFP-Pi04089 compared with the expression of GFP-Pi04089. Error bars are SE, and the graph represents the combined data from three biological reps (n = 142 per construct).
Figure 3
Figure 3
Pi04089 Interacts with a Potato Predicted KH RNA-Binding Protein in Y2H and In Planta. (A) Yeasts co-expressing the StKRBP1 with Pi04089 grow on -histidine (-HIS) medium and have β-galactosidase (B-gal) activity, whereas yeasts co-expressing SFI3 or empty vector (Empty) do not. (B) Co-immunoprecipitation from leaf extracts using GFP-trap (GFP IP) confirmed that StKRBP1 specifically interacted with Pi04089 and not with SFI3. Expression of constructs is indicated by plus signs. Effector protein fusion bands are indicated by asterisks. Protein size markers are indicated in kDa, and protein loading is indicated by Ponceau stain.
Figure 4
Figure 4
StKRBP1 Specifically Relocalizes Pi04089 to Nuclear Speckles. (A) Single optical sections of N. benthamiana leaf epidermal cell nuclei transiently co-expressing the GFP-Pi04089 or GFP-SFI3 with RFP-StKRBP1, showing that GFP-Pi04089 is relocated to nuclear speckles, and the nucleolar ring is no longer observed while the localization of GFP-SFI3 remains unaffected. Scale bar represents 10 μm. (B) Low-magnification images collected with identical imaging parameters and a 10× lens of N. benthamiana leaves, showing that bimolecular fluorescence between YN-Pi04089 and YC-StKRBP1 is more frequently observed than with the YN-SFI3 control and is located in the nuclei of expressing cells. Insets are single optical sections of nuclei, showing that the fluorescence between YN-Pi04089 and YC-StKRBP1 is located in the nuclear speckles. Inset for the YN-SFI3 plus YC-StKRBP1 control combination indicates the level of background non-specific fluorescence observed in a small number of cells. (C) Graph shows the average number of nuclei observable per field of view using the 10× lens and identical settings for each of the combinations. Significantly more nuclei are observed for YN-Pi04089 and YC-StKRBP1 compared with YN-SFI3 and YC-StKRBP1 (p < 0.001, t test, as indicated by lowercase letters). Error bars are SE, and the graph represents the data from one biological rep (n = 11 fields of view per construct). (D) Immunoblots indicate that each of the constructs used for bimolecular fluorescence experiments are stable and of the expected size.
Figure 5
Figure 5
Overexpression of StKRBP1 Promotes P. infestans Colonization. (A) Graph shows a significant increase (p < 0.001, t test, as indicated by asterisks) in mean diameter of P. infestans lesions following Agrobacterium-mediated expression of myc-StKRBP1 compared with the expression of the empty vector. Error bars are SE, and the graph represents the combined data from three biological reps (n = 194 per construct). (B) An example leaf showing a larger water-soaked lesion on the side infiltrated with myc-StKRBP1.
Figure 6
Figure 6
StKRBP1 Is Stabilized by Pi04089 and P. infestans Infection. (A) Immunoblot showing an increased signal from myc-StKRBP1 in the presence of GFP-Pi04089 and not with GFP-SFI3. (B) Immunoblots from two biological replicates showing that the myc-StKRBP1 is more stable following infection with P. infestans compared with a water-inoculated control. 24, 48, and 72 indicate hours post treatment with water or P. infestans, Un, a non-agro-infiltrated control.
Figure 7
Figure 7
A KRBP1 Mutant No Longer Interacts with Pi04089 and Fails to Promote P. infestans Colonization. (A) Yeasts co-expressing the StKRBP1 with Pi04089 grow on -histidine (-HIS) medium and have β-galactosidase (B-gal) activity, whereas yeasts co-expressing StKRBP1 mutant (StKRBP1mut) do not. The control combination of StKRBP1mut with the empty prey vector indicates that it does not autoactivate. Controls show no (-), weak (+), and strong (++) interactions. (B) Co-immunoprecipitation confirmed that Pi04089 specifically interacted with StKRBP1 and not with the mutated protein. (C) Single optical sections of nuclei of cells co-expressing GFP-Pi04089 with either the wild-type StKRBP1 or the mutated form, showing that GFP-Pi04089 is only removed from the nucleolar periphery and relocated to speckles by the wild-type, and also that the mutated StKRBP1 does not locate to nuclear speckles. Scale bar represents 10 μm. (D) Graph shows that the mean diameter of P. infestans lesions following Agrobacterium-mediated expression of myc-StKRBP1 mutant was significantly decreased compared with the expression of the wild-type myc-StKRBP1 and did not significantly differ from the empty vector (myc-EV) control (p < 0.001, ANOVA, as indicated by lowercase letters). Error bars are SE, and the graph represents the combined data from three biological reps (n = 72 per construct). (E) Example infection sites on N. benthamiana leaves.

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