Phytophthora palmivora establishes tissue-specific intracellular infection structures in the earliest divergent land plant lineage

Abstract

The expansion of plants onto land was a formative event that brought forth profound changes to the earth's geochemistry and biota. Filamentous eukaryotic microbes developed the ability to colonize plant tissues early during the evolution of land plants, as demonstrated by intimate, symbiosis-like associations in >400 million-year-old fossils. However, the degree to which filamentous microbes establish pathogenic interactions with early divergent land plants is unclear. Here, we demonstrate that the broad host-range oomycete pathogen Phytophthora palmivora colonizes liverworts, the earliest divergent land plant lineage. We show that P. palmivora establishes a complex tissue-specific interaction with Marchantia polymorpha, where it completes a full infection cycle within air chambers of the dorsal photosynthetic layer. Remarkably, P. palmivora invaginates M. polymorpha cells with haustoria-like structures that accumulate host cellular trafficking machinery and the membrane syntaxin MpSYP13B, but not the related MpSYP13A. Our results indicate that the intracellular accommodation of filamentous microbes is an ancient plant trait that is successfully exploited by pathogens like P. palmivora.

Keywords: Phytophthora; bryophyte; haustoria; liverworts; oomycetes.

Fig. 1.

P. palmivora colonizes the photosynthetic layer of M. polymorpha. (A) Disease symptoms of 3-wk-old M. polymorpha TAK1 (male) thalli inoculated with P. palmivora ARI-td zoospores or water (Mock) over a 7-d time course. (B) Epifluorescence microscopy demonstrating the spread of P. palmivora growth across TAK1 thalli from 1 to 4 dpi. Epifluorescence (Epifluor.) from the pathogen is displayed alongside bright-field (BF) images. (Scale bars, 500 μm.) (C) Confocal fluorescence microscopy of sectioned TAK1 thalli infected with P. palmivora at 7 dpi. Z-stack projections of red fluorescence from the pathogen are displayed alone (Left, tdTomato) or merged with all channels (Right, bright-field and plastid autofluorescence in turquoise). Arrowheads indicate air pores. (Scale bars, 100 μm.) (D) Cryo-SEM image of TAK1 thalli colonized by P. palmivora at 7 dpi. Mechanically fractured air chamber demonstrating hyphal growth within the chamber (yellow arrows) and sporangia (Sp) at the air pore. (E) Cryo-SEM image showing intercellular (yellow arrows) and intracellular (red arrow) associations between P. palmivora hyphae and photosynthetic filaments within M. polymorpha air chambers at 7 dpi. (Scale bars, 20 μm.) All experiments were performed at least three times, with similar results.

Fig. 2.

P. palmivora completes a full infection cycle that includes the intracellular colonization of living Marchantia cells. (A) Confocal fluorescence microscopy demonstrating key morphological transitions in P. palmivora lifestyle during the colonization of TAK1 plants from 1 to 3 dpi. Z-stack projections of pathogen fluorescence merged with plastid autofluorescence (turquoise). Intracellular infection structures are denoted by an asterisk. Sporangia are indicated by dashed arrows. (Scale bars, 10 μm.) (B) Quantification of P. palmivora lifestyle marker genes during the colonization of TAK1 thalli from 1 to 4 dpi via qRT-PCR analysis. Pathogen biomass (PpEF1a) was quantified relative to M. polymorpha biomass markers (MpACT and MpEF1a). Haustoria (PpHmp1) and sporulation (PpCdc14) marker genes were quantified relative to pathogen biomass controls (PpEF1a and PpWS21). Different letters signify statistically significant differences in transcript abundance [ANOVA, Tukey’s honest significant difference (HSD), P < 0.05]. (C) Quantification of P. palmivora RXLR effector gene transcripts during the colonization of TAK1 thalli from 1 to 4 dpi via qRT-PCR analysis. RXLR effector (REX1, REX3, and REX4) gene expression was quantified relative to P. palmivora biomass (PpEF1a and PpWS21). Different letters signify statistically significant differences in transcript abundance (ANOVA, Tukey’s HSD, P < 0.05). (D) P. palmivora transcriptome. Hierarchical clustering of differentially expressed genes between in planta (3 and 4 dpi) and axenically grown mycelium transcriptomes (LFC of ≥2, P value of ≤10⁻³). rLog-transformed counts, median-centered by gene, are shown. (E) P. palmivora secretome. Summary of functional categories of 394 genes encoding putative secreted proteins up-regulated during P. palmivora infection of M. polymorpha. Experiments in A–C were performed at least three times, with similar results.

Fig. 3.

Host cellular responses to invading oomycete structures. (A) Detection of callose deposition at P. palmivora ARI-td intracellular infection structures at 3 dpi in M. polymorpha TAK1. Arrows indicate callose deposition at the peripheral neck region of the invading intracellular infection structure. (Scale bars, 5 μm.) (B) MpRab7 colocalization with invading P. palmivora ARI-td structures in 35S:mCitrine-MpRab7 plants at 3 dpi and (C) MpRab11A colocalization with invading P. palmivora ARI-td structures in 35S:mCitrine-MpRab11A plants at 3 dpi. Infection structures are indicated with asterisks, while intracellular hyphae are denoted by arrows. Z-stack projections are displayed. (Scale bars, 5 μm.) Experiments were performed three times, with similar results.

Fig. 4.

A colonization-induced host syntaxin accumulates at intracellular infection structures. (A) qRT-PCR analysis of MpSYP13A and MpSYP13B transcripts in mock-treated or P. palmivora-colonized (ARI-td) TAK1 plants from 1 to 4 dpi. Expression values are shown relative to internal MpACT and MpEF1a controls. Different letters signify statistically significant differences in transcript abundance (ANOVA, Tukey’s HSD, P < 0.05). (B) Confocal fluorescence microscopy demonstrating mCitrine-MpSYP13A/B localization in cells containing P. palmivora (ARI-td) intracellular infection structures at 3 dpi. Asterisks denote intracellular infection structures. (Scale bars, 10 μm.) (C) Patterns of mCitrine-MpSYP13B localization in P. palmivora-colonized (ARI-td) plants, including close-up images of the structure displayed in B. (Scale bars, 5 μm.) Experiments were performed three times, with similar results.

Fig. 5.

P. palmivora requires air chambers for the successful colonization of M. polymorpha thalli. (A) Disease symptoms of 3-wk-old M. polymorpha TAK1 (wild-type) and nop1 mutant plants inoculated with P. palmivora ARI-td zoospores at 1, 7, and 14 dpi. (B) Quantification of P. palmivora biomass (PpEF1a) and sporulation (PpCdc14) marker genes during the colonization of wild-type TAK1 and nop1 plants at 1, 3, and 5 dpi. PpEF1a expression was quantified relative to M. polymorpha biomass markers (MpACT and MpEF1a). PpCdc14 was quantified relative to pathogen biomass (PpEF1a). Different letters signify statistically significant differences in transcript abundances (ANOVA, Tukey’s HSD, P < 0.05). (C) Confocal fluorescence microscopy of sectioned TAK1 and nop1 thalli infected with P. palmivora ARI-td at 7 dpi. Micrographs display merged z-stack projections of red pathogen fluorescence and bright-field images. (Scale bars, 100 μm.) (D) Cryo-SEM images of TAK1 plants demonstrate ARI-td hyphae traveling between air pores (Left, arrows) at 4 dpi and hyphal growth (Right, arrow) within air chambers at 8 dpi. (Scale bars, 20 μm.) (E) Cryo-SEM images of nop1 plants demonstrate a network of ARI-td surface hyphae (Left) at 4 dpi, and collapsed epidermal cells (Right, asterisks) that are sometimes observed at 8 dpi. (Scale bars, 20 μm.) (F) Confocal fluorescence microscopy demonstrating invasive ARI-td hyphal growth (arrows) in nop1 epidermal cells at 3 dpi. Micrographs display z-stack projections of red pathogen fluorescence (tdTomato), plastid autofluorescence, bright-field images, and plastid and tdTomato fluorescence merged together. (Scale bars, 10 μm.) Experiments were performed three times (A–C and F) or twice (D and E), with similar results.