Ancient class of translocated oomycete effectors targets the host nucleus

Abstract

Pathogens use specialized secretion systems and targeting signals to translocate effector proteins inside host cells, a process that is essential for promoting disease and parasitism. However, the amino acid sequences that determine host delivery of eukaryotic pathogen effectors remain mostly unknown. The Crinkler (CRN) proteins of oomycete plant pathogens, such as the Irish potato famine organism Phytophthora infestans, are modular proteins with predicted secretion signals and conserved N-terminal sequence motifs. Here, we provide direct evidence that CRN N termini mediate protein transport into plant cells. CRN host translocation requires a conserved motif that is present in all examined plant pathogenic oomycetes, including the phylogenetically divergent species Aphanomyces euteiches that does not form haustoria, specialized infection structures that have been implicated previously in delivery of effectors. Several distinct CRN C termini localized to plant nuclei and, in the case of CRN8, required nuclear accumulation to induce plant cell death. These results reveal a large family of ubiquitous oomycete effector proteins that target the host nucleus. Oomycetes appear to have acquired the ability to translocate effector proteins inside plant cells relatively early in their evolution and before the emergence of haustoria. Finally, this work further implicates the host nucleus as an important cellular compartment where the fate of plant-microbe interactions is determined.

Fig. 1.

Expression of AVR3a in P. capsici results in avirulence on R3a N. benthamiana leaves. (A) Schematic of constructs used in translocation assays. Full-length Avr3a with a functional RXLR-dEER motif as well as its mutated full-length counterpart cloned in pTOR as described in Whisson et al. (14) were used to transform P. capsici. (B) Zoospore suspensions (50,000–100,000 spores/mL) of a P. capsici strain carrying the empty pTOR vector and of strains expressing either wild-type or mutated Avr3a were inoculated onto N. benthamiana wild-type and R3a leaves. Inoculation sites were scored for lesion formation 4 d after inoculation (dpi), and infection frequencies were calculated. Data represent three independent experiments. (C) Images showing representative wild-type and transgenic R3a leaves 4 dpi with strains analyzed in B. Avr3a expression was confirmed by RT-PCR analyses (Fig. S2). Lesions are marked by circles.

Fig. 2.

The CRN protein family forms a class of modular proteins that are translocated inside host cells. (A) Schematic representation of CRN2, CRN8, and CRN16 N-terminal domains identified in P. infestans and used for translocation assays presented. (B) Virulence assessment of transgenic P. capsici strains on wild-type and transgenic R3a N. benthamiana leaves. Infection rates of transgenic strains carrying empty vector (pTOR), CRN2:AVR3a, CRN16:AVR3a, and their mutated counterparts (CRN2mut:AVR3a, CRN16mut:AVR3a) were determined on wild-type and R3a leaves 4 dpi. Values represent the mean percentage of successful infections over three independent experiments. (C) Representative pictures showing wild-type and transgenic R3a leaves inoculated with strains carrying (Left to Right) the empty vector pTOR, CRN2:AVR3a, and CRN16:AVR3a as well as their mutated counterparts CRN2mut:AVR3a and CRN16mut:AVR3a. Pictures were taken 4 dpi. Crn-Avr3a expression was confirmed by RT-PCR analyses (Fig. S2). Lesions are marked by circles.

Fig. 3.

CRN N-terminal domains are widespread within the oomycete lineage and function as translocation domains. Previously, examinations of A. euteiches sequence collections identified the CRN5 homolog AeCRN5 (26). (A) The CRN protein family arose before acquisition of haustorium formation. Genome-wide surveys for effector genes (described in SI Text) were superimposed on a phylogenetic tree describing oomycete evolutionary relationships (adapted from ref. 24). (B) Comparisons between the N terminus of AeCRN5 and known P. infestans CRNs highlight similarity in domain architecture. An LXLFLAK-like motif (LXLYLALK) is present 47 residues from the N terminus, and the conserved HVLVXXP motif is present. (C) The AeCRN5 N terminus fused to C-terminal AVR3a conditions avirulence on R3a but not on wild-type N. benthamiana leaves. Quantification of infection rates across three independent experiments (4 dpi). (D) The wild-type and transgenic R3a leaves inoculated with strains analyzed in C. Pictures were taken 4 dpi with zoospore suspensions. Transgene expression was confirmed by RT-PCR for each strain tested (Fig. S2). Lesions are marked by circles.

Fig. 4.

CRN effector domains traffic to and function in the host nucleus. (A) Confocal imaging of GFP, GFP:CRN15, GFP:CRN16, GFP:CRN2, GFP:CRN8, and GFP:AeCRN5 in N. benthamiana epidermal cells 24 h after infiltration. (Scale bars: 10 μm.) (B) Immunoblot analyses of GFP fusion protein accumulation in planta 24 h after infiltration. Blots were probed with α-GFP antibody. Sizes in kDa are indicated on the left. (C) Nuclear localization is required for effector-induced cell death. N. benthamiana leaves infiltrated with Agrobacterium strains carrying PVX CRN8, PVX NES:CRN8, or PVX mNES:CRN8 and an empty vector (ev) control. A representative picture was taken 5 d after infiltration. (D) Quantification and direct comparisons of cell death (CD) induced by nuclear-localized and nuclear-excluded CRN8. For each experiment, 14 leaves were infiltrated with strains as shown in C. Leaf panels were scored 3 d after infiltration, and the number of infiltration sites showing cell death was counted.