Infection of Arabidopsis thaliana by Phytophthora parasitica and identification of variation in host specificity

Abstract

Oomycete pathogens cause severe damage to a wide range of agriculturally important crops and natural ecosystems. They represent a unique group of plant pathogens that are evolutionarily distant from true fungi. In this study, we established a new plant-oomycete pathosystem in which the broad host range pathogen Phytophthora parasitica was demonstrated to be capable of interacting compatibly with the model plant Arabidopsis thaliana. Water-soaked lesions developed on leaves within 3 days and numerous sporangia formed within 5 days post-inoculation of P. parasitica zoospores. Cytological characterization showed that P. parasitica developed appressoria-like swellings and penetrated epidermal cells directly and preferably at the junction between anticlinal host cell walls. Multiple haustoria-like structures formed in both epidermal cells and mesophyll cells 1 day post-inoculation of zoospores. Pathogenicity assays of 25 A. thaliana ecotypes with six P. parasitica strains indicated the presence of a natural variation in host specificity between A. thaliana and P. parasitica. Most ecotypes were highly susceptible to P. parasitica strains Pp014, Pp016 and Pp025, but resistant to strains Pp008 and Pp009, with the frequent appearance of cell wall deposition and active defence response-based cell necrosis. Gene expression and comparative transcriptomic analysis further confirmed the compatible interaction by the identification of up-regulated genes in A. thaliana which were characteristic of biotic stress. The established A. thaliana-P. parasitica pathosystem expands the model systems investigating oomycete-plant interactions, and will facilitate a full understanding of Phytophthora biology and pathology.

Figure 1

Infection of Arabidopsis thaliana leaves by Phytophthora parasitica. Four‐week‐old leaves of A. thaliana were drop inoculated with zoospores of P. parasitica strain Pp016. Inoculated leaves were collected at different time points post‐inoculation and stained with trypan blue before being subjected to microscopic characterization. (A) Water‐soaked lesions developed 3 days post‐inoculation (dpi) of strain Pp016 zoospores. (B) Germ tube (GT) differentiated from cyst (C), appressorium (A) developed and penetration peg (PP) formed to invade the plant at the anticlinal walls (AW) of adjacent plant epidermal cells (bar, 5 µm). (C) Appressorium‐like structures (A) mostly differentiated at the anticlinal walls (AW) of epidermal cells (bar, 10 µm). (D) Haustoria‐like structures (Ha) formed by intercellular hyphae (Hy) in epidermal cells 6 h post‐inoculation (bar, 50 µm). (E) Haustoria (H) developed on invasive hyphae in mesophyll cells (MC) (bar, 20 µm). (F) Infection hyphae (Hy) with multiple branches, as well as abundant haustoria (Ha), were evident in both epidermal cells and mesophyll cells (bar, 20 µm). (G) Sporangia (S) were abundant on the leaf surface within 4 dpi (bar, 20 µm).

Figure 2

Root infection and colonization of Arabidopsis thaliana by Phytophthora parasitica. Live root tissues were inoculated by dipping into a zoospore suspension (10⁵ spores/mL) for 5 s, followed by removal of excess solution on sterile Whatman paper and transfer to Petri dishes containing half‐strength Murashige and Skoog (MS) medium without sugar. (A) Wilt and collapse of whole plant seedlings 4 days post‐inoculation of strain Pp016 zoospores (A2); (A1) is the water control plate. (B) Haustoria‐like structures (Ha) developed by intercellular hyphae (Hy) in the cortex 24 h post‐inoculation of zoospores (bar, 50 µm). (C) Heavy colonization of root tissues as shown by the dramatic increase in pathogen biomass by 48 h post‐inoculation of zoospores; Hy, hyphae; S, sporangia.

Figure 3

Microscopic characterization of the incompatible interaction between Arabidopsis thaliana Col‐0 and Phytophthora parasitica Pp009. (A) Cell wall deposition (Cd) at the attempted infection site of epidermal cells by strain Pp009, as revealed by aniline blue staining and examination under UV light, in which the green fluorescence indicates the accumulation of callose (A2); (A1) is the same image under visible light to show the infection site; A, appressorium; C, cyst. (B) Rapid hypersensitive response (HR) of a Col‐0 epidermal cell in response to penetration by strain Pp009; C, cyst. (C) Penetration of mesophyll cells by progressive hyphae (Hy) is stopped by the cell undergoing HR. The leaf was stained with trypan blue and deeply blue‐stained tissues indicate cell necrosis (Cn). (D) Germinated cyst (C) with development of secondary appressoria (SA); PA indicates primary appressorium.

Figure 4

Host specificity in Arabidopsis thaliana Sap‐0 to infection by Phytophthora parasitica. Two‐week‐old sterile Sap‐0 seedlings were inoculated with P. parasitica mycelial cultures and the results were scored 7 days post‐inoculation. (A) Water agar plug control. (B) Constricted seedling hypocotyls inoculated with strain Pp016. (C) No visible symptoms appeared in hypocotyls in seedlings inoculated with Pp009.

Figure 5

A comparison of Functional Catalogue annotation of six cDNA libraries representing different developmental stages and biotic and abiotic stresses. Five non‐normalized, non‐subtracted Arabidopsis thaliana cDNA libraries were downloaded from DFCI Arabidopsis Gene Index Build 14.0 (AtGI) and compared with expressed sequence tags (ESTs) derived from Phytophthora parasitica Pp016‐infected A. thaliana Col‐0 leaf tissues. The libraries were derived from A. thaliana adult vegetative tissues of Col‐0 (Castelli et al., 2004) (AtGI cat #F0G, V), A. thaliana leaf at senescence stage (Guo et al., 2004) (AtGI cat #DGL, L), Erysiphe cichoracearum‐infected A. thaliana leaf tissues (AtGI cat #BKG, E), hormone‐treated A. thaliana Col‐0 callus (Castelli et al., 2004) (AtGI cat #F0H, H) and A. thaliana Col‐0 flowers and buds (Castelli et al., 2004) (AtGI cat #F0J, F).

Figure 6

Differential gene expression profiles in the six Arabidopsis thaliana cDNA libraries. The cDNA libraries represent different plant developmental stages and biotic and abiotic stresses. Genes with a log‐likelihood ratio R of over 12 were included in the figure. R, which measures the extent to which the differences in gene expression correspond to specific or random, was derived from the entropy of partitioning of genes among cDNA libraries. Colours from dark to bright represent low to high levels of gene expression in the corresponding libraries, respectively.