Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

Abstract

Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

Keywords: CRN; Phytophthora capsici; cell death; effector; immunity; nucleus.

Figure 1

CRN induced cell death and nuclear localization is conferred by the C-terminus. (A) Localization of ectopically expressed CRN-GFP fusion products. The panels show the localization for free eGFP, CRN20_624 N-terminus, CRN20_624 C-terminus, and mature CRN20_624 protein, 2 days post infiltration at OD 0.1 Scale bar = 25 μm. (B) Immunoblot analysis of CRN20_624 upon over-expression in plant tissue. The blot was probed with anti-GFP antibody. 70 and 25 kDa markers are indicated on the right hand side. Lower panel shows coomassie brilliant blue staining loading control. (C) Cell death inducing activity of CRN-GFP fusion products 7 days after infiltration at an OD of 1.0.

Figure 2

Necrosis inducing CRNs show distinct cell death inducing dynamics. (A) Progression of cell death in N. benthamiana leaves infiltrated with CRNs. Cell death was scored every 24 h on a scale of 0–6. Example lesions for each score are shown on the left. The graph shows average values ± standard deviation for one representative experiment. (B) Ion leakage measurements confirming differences in cell death response. Each data point is an average of 6 measurements ± standard deviation. TDS ppm: total dissolved solids in parts per million. (C) Graphical representation of the experimental set-up (left) and a typical leaf 4 days post inoculation. (D) Western blots and loading control for CRNs and control samples showing protein levels up to 4 dpi. EV: GFP antibodies, CRNs: strepII antibodies.

Figure 3

Expression of CRN20_624, but not CRN79_188 and CRN83_152 has an additive effect on PAMP induced cell death. (A) qRT-PCR analyses on cDNA derived from leaf panels transiently expressing EGFP and treated with PB and CF. Each bar represents the fold change in gene expression upon CF treatment relative to PB ± standard deviation. Expression was examined for known PTI marker genes NbPti5, NbAcre31, and NbGras2 at 1 and 12 h post infiltration (hpi). (B) Graph showing average necrosis scores ± standard deviation for three independent experiments. (C) Graphical representation of the experimental set-up. CRN-GFP fusion constructs were infiltrated into N. benthamiana plants and after 48 h, leaves were infiltrated with either a PAMP cocktail (Phytophthora capsici culture filtrate) or a control solution of pea broth. Cell death was scored on a scale of 0–6, 48 h after CF treatment. (D) Examples of representative leaves for each treatment on day of scoring.

Figure 4

Necrosis inducing CRNs do not cause altered ETI responses within the plant. (A) Graphical representation of the experimental set-up. CRN-GFP fusion and EV constructs were co-infiltrated into N. benthamiana leaves with Avr3a^KI and R3a (ETI), and Avr3a^EM and R3a (ΔETI) to monitor ETI responses. After 48 h, leaves were infiltrated with dexamethasone and incubated for a further 24 h for induction of Avr3a expression. (B) Cell death in response to CRNs in ETI and non-ETI induced leaves scored on a scale of 0–6. Graph shows average necrosis scores ± standard deviation for one representative experiment. (C) Examples of representative leaves for each treatment on day of scoring.

Figure 5

CRN 83_152 causes re-localization of DNA within the nucleus. CRN-GFP fusion constructs were over expressed in N. benthamiana plants and imaged by confocal microscopy 48 h post-infiltration. (A) Leaves were co-infiltrated with RFP-fibrillarin and were DAPI stained by infiltrating with 4′,6-Diamidino-2-Phenylindole dilactate at a final concentration of 5 μg/ml. (B) N. benthamiana plants stably expressing histone RFP were infiltrated with CRN-GFP fusion constructs as described above. Scale bars = 5 μm.

Figure 6

3D-SIM imagining of CRN C-terminal constructs on OMX confirms distinct localization within the nucleus. (A) Projection view of 3D-SIM images on epidermal peels of N. benthamiana H2B-RFP transgenic lines infiltrated with CRN-GFP fusion constructs. Histone 2B distribution is shown in RFP channel whereas CRN protein distribution is in the GFP channel. Merged channel confirms impairment of chromatin distribution in CRN83_152 expressing cells. (B) Single plane views of 3D-SIM images on epidermal peels of N. tabacum infiltrated with CRN-GFP fusion constructs show distinct nuclear distribution patterns for CRN effector proteins. All images were taken 48 h after infiltration. Scale bars = 5 μm.