The Ustilago hordei-Barley Interaction Is a Versatile System for Characterization of Fungal Effectors

Abstract

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

Keywords: CRISPR-Cas9; Ustilago hordei; effectors; haustoria; heterologous gene expression.

Figure 1

Generation of a solopathogenic Ustilago hordei strain. (A) Filamentation test on charcoal plate. U. hordei wild-type strains 4857-4 MAT-1, 4857-5 MAT-2, mating of 4857-4 MAT-1 × 4857-5 MAT-2, solopathogenic DS199, and DS200 strains. Pictures were taken after 3 days incubation at RT. (B) Appressoria formation ability of U. hordei strains on parafilm. Mating of U. hordei wild-type 4857-4 MAT-1 and 4857-5 MAT-2, solopathogenic DS199, solopathogenic DS200. Yellow arrowheads indicate appressoria. Pictures were taken after 24 h (hours) incubation (C) Disease development of different U. hordei strains on barley. Mating of U. hordei wild-type 4857-4 MAT-1 and 4857-5 MAT-2 at 3 dpi (days post inoculation), solopathogenic DS199 at 3 dpi, solopathogenic DS200 at 3 dpi. Following wheat germ agglutinin (WGA)-AF488/propidium iodide (PI) staining, fungal cell walls are shown in green and plant cell walls in red. (D) Quantification of appressoria formation for U. hordei wild-type 4857-4 MAT1, solopathogenic DS199, and DS200 on plant. (E) Quantification of penetration efficiency for U. hordei wild-type 4857-4 MAT1, solopathogenic DS199, and DS200 on plant.

Figure 2

(A–H) Transmission electron microscopy micrographs of wild-type Ustilago hordei-infected barley leaves. (A,B) Biotrophic interphase in the U. hordei–barley interaction. During host colonization, U. hordei invaginates the host cell membrane without breaching it. The host–pathogen interaction mainly takes place within this biotrophic interphase (BIP), which consists of the fungal cell wall (FCW), electron-dense extracellular matrix (edECM), and electron-translucent extracellular matrix (etECM). (C,D) Formation of vesicles at the hyphal tip of U. hordei. Fungal vesicles (Ve) with cores of different electron densities and plant multivesicular bodies (MVB) were detected at hyphal tips and in the plant cytoplasm close to fungal penetration sites, respectively. (E,F) Immunogold labeling of callose with a monoclonal antibody recognizing (1-3)-β-glucan epitopes. Callose accumulation was detected at the electron-translucent ECM (etECM) site (yellow arrowheads). (G,H) Cell-to-cell penetration of U. hordei. U. hordei primarily grows intracellularly at 8 dpi in barley leaves. When the fungal hyphae penetrate a new plant cell, the hypha gets thickened at the site of cell-to-cell passage, resembling appressorial structures (G). The edECM gets thicker at the site of hypha contact with the plant cell wall (yellow arrowheads) (H), while electron-dense material can also diffuse into adjacent parts of the plant cell wall (red arrowhead) (H). (I–M) Haustoria formation during host colonization. U. hordei grows intracellularly and forms haustorial structures in barley cells. (I,J) Wheat germ agglutinin (WGA)-AF488/propidium iodide (PI) staining was performed to visualize U. hordei at 8 days post inoculation (dpi) under confocal/fluorescent microscopy. (K–M) Transmission electron micrographs showing different planes of the section through haustoria. Haustorial structures were distinguished from hyphae by their bigger size and interconnected lobular shapes. Yellow arrowheads (L) point out the connections between haustorial lobes. U. hordei haustoria possess large vacuoles with a granular lumen containing vesicles of different sizes (M) (yellow arrowheads; magnification of inset in (L)). BIP: biotrophic interphase; FCW: fungal cell wall; FPM: fungal plasma membrane; H: hypha; Ha: haustorium; edECM: electron-dense extracellular matrix; etECM: electron-translucent extracellular matrix; LB: lipid bodies; MVB: multi-vesicular body; PCW: plant cell wall; PPM: plant plasma membrane; Ve: vesicles.

Figure 3

(A,B) Heterologous expression of GusA-mCherry in Ustilago hordei. (A) GusA-mCherry was heterologously expressed in solopathogenic strain DS200 under control of the UHOR_02700 promotor with or without signal peptide (SP) for extracellular secretion. The ± SP-GusA-mCherry DS200 strains were inoculated on barley seedlings, then at 4 days post inoculation (dpi) confocal microscopy was performed to monitor expression and localization of recombinant proteins. While +SP-GusA-mCherry is secreted around the tip of the invasive hyphae, -sp-GusA-mCherry localizes in the fungal cytoplasm. The white graphs indicate the mCherry signal intensity along the diameter of the hyphae (illustrated by white lines in the image). (B) Western blot analysis was performed with apoplastic fluid isolated from barley leaves infected with ± SP-GusA-mCherry DS200 strains. While a band corresponding to secreted +SP-GusA-mCherry (at ~100 kDa) and free mCherry (at 27 kDa) in isolated apoplastic fluid was detected, no band corresponding to cytoplasmic -sp-GusA-mCherry could be detected. Anti-RFP antibody was used for Western blot analysis. (C,D) Establishment of CRISPR/Cas9 gene editing system for Ustilago hordei. (C) Codon-optimized Cas9 was cloned into the p123 plasmid under the control of the Hsp70 promoter. The U. hordei pU6 promotor was used to express sgRNA for the targeted gene. Carboxin resistance was used as selection marker. (D) U. hordei Fly1 gene, a fungalysin metalloprotease involved in fungal cell separation, was edited via the CRISPR/Cas9 system for knock-out. While DS200 sporidia showed normal growth, DS200Δuhfly1 cells were impaired in cell separation.

Figure 4

Heterologous expression of fungal effectors in Ustilago hordei. (A) Heterologous expression and secretion of CfAvr4 in U. hordei DS200 strain in vitro. U. hordei strain DS200 expressing CfAvr4 of Cladosporium fulvum with UHOR_02700 signal peptide and under the control of pActin promoter (for constitutive expression), as well as FvRibo1 of Fusarium verticillioides with UHOR_02700 signal peptide and under the control of pUHOR_02700 promoter (for expression in planta only) were grown in YEPS_light liquid medium till OD:1.0. The U. hordei cell suspensions were centrifuged and the culture filtrates (CF) of each sample were infiltrated into tobacco leaves expressing Cf4-resistant protein, which can recognize CfAvr4 and induce cell death by means of hypersensitive response (HR). The culture filtrates from U. hordei DS200 and DS200-FvRibo1 strains were used as negative controls. Pictures were taken at 5 days post infiltration (dpi). Autofluorescence of infected leaves was imaged to more easily see sites of cell death by using Gel-Doc (Bio-Rad). (B) Biomass quantification of DS200-FvRibo1 in barley leaves. The virulence of the U. hordei DS200 and two independent DS200-FvRibo1 strains was assessed by fungal biomass quantification from DNA isolated from infected barley leaves at 6 days post inoculation (dpi). The Ppi1 gene of U. hordei was used as a standard for qPCR. The fungal biomass was deduced from a standard curve. A student t-test was performed to determine significant differences, which are indicated as asterisks (***, p < 0.001). Error bars represent the standard deviation of three biological repeats. (C) Heterologous expression and secretion of FvRibo1 in U. hordei strain DS200 in planta. Ustilago hordei strain DS200 and DS200 expressing FvRibo1 (encoding a secreted ribotoxin) of Fusarium verticillioides with UHOR_02700 signal peptide and under the control of the UHOR_02700 promoter (for only in planta expression) were inoculated on susceptible 12-day-old barley seedlings. Macroscopic pictures were taken at 6 dpi. Autofluorescence pictures were taken to see better cell death by using Gel-Doc (Bio-Rad). (D) Wheat germ agglutinin (WGA)-AF488/propidium iodide (PI) staining was performed to visualize the colonization of DS200-FvRibo1 in barley leaves compared to DS200. While green signal indicates fungal colonization, the red signal represents the plant cell walls.