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. 2017 Apr 4;18(1):273.
doi: 10.1186/s12864-017-3619-4.

Cross-talk of the Biotrophic Pathogen Claviceps Purpurea and Its Host Secale Cereale

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Free PMC article

Cross-talk of the Biotrophic Pathogen Claviceps Purpurea and Its Host Secale Cereale

Birgitt Oeser et al. BMC Genomics. .
Free PMC article

Abstract

Background: The economically important Ergot fungus Claviceps purpurea is an interesting biotrophic model system because of its strict organ specificity (grass ovaries) and the lack of any detectable plant defense reactions. Though several virulence factors were identified, the exact infection mechanisms are unknown, e.g. how the fungus masks its attack and if the host detects the infection at all.

Results: We present a first dual transcriptome analysis using an RNA-Seq approach. We studied both, fungal and plant gene expression in young ovaries infected by the wild-type and two virulence-attenuated mutants. We can show that the plant recognizes the fungus, since defense related genes are upregulated, especially several phytohormone genes. We present a survey of in planta expressed fungal genes, among them several confirmed virulence genes. Interestingly, the set of most highly expressed genes includes a high proportion of genes encoding putative effectors, small secreted proteins which might be involved in masking the fungal attack or interfering with host defense reactions. As known from several other phytopathogens, the C. purpurea genome contains more than 400 of such genes, many of them clustered and probably highly redundant. Since the lack of effective defense reactions in spite of recognition of the fungus could very well be achieved by effectors, we started a functional analysis of some of the most highly expressed candidates. However, the redundancy of the system made the identification of a drastic effect of a single gene most unlikely. We can show that at least one candidate accumulates in the plant apoplast. Deletion of some candidates led to a reduced virulence of C. purpurea on rye, indicating a role of the respective proteins during the infection process.

Conclusions: We show for the first time that- despite the absence of effective plant defense reactions- the biotrophic pathogen C. purpurea is detected by its host. This points to a role of effectors in modulation of the effective plant response. Indeed, several putative effector genes are among the highest expressed genes in planta.

Keywords: Biotrophic pathogen; Claviceps purpurea; Effectors; Host-pathogen interaction; Transcriptome.

Figures

Fig. 1
Fig. 1
Infection stages of C. purpurea strains at the time point of sample collection. a The wild-type Cp20.1 (black line) enters the ovary at the stigmatic hairs (s), passes the transmitting tissue (tm), grows around the ovule (o) and almost reaches the base of the ovary. b The virulence-attenuated mutant Δcptf1 (grey continuous and dashed line) also penetrates the stigmatic hairs but reaches the base only sparsely. Δcpcdc42 (grey continuous line) penetrates the stigmatic hairs but does not enter the ovarian cap
Fig. 2
Fig. 2
Principal component analysis of de novo assembled transcripts: a plant sequences and b fungal sequences. Compared were the FPKM values of the reads matching the transcripts of all four samples (mock, Cp20.1 infected, ∆cptf1 infected, ∆cpcdc42 infected) plus a reference data set for plant and fungal sequences (ref; all data points set to zero)
Fig. 3
Fig. 3
Venn diagram of differentially expressed plant genes. Overlapping areas show the numbers of genes found to be congruently regulated comparing mock-Cp20.1 (green,45 up, 10 down), mock-Δcptf1 (blue, 19 up, 20 down), and mock-Δcpcdc42 (yellow, 23 up, 7 down), resp
Fig. 4
Fig. 4
C. purpurea CAZymes in comparison to CAZymes of other plant pathogenic fungi. C. purpurea data points (blue dots) were integrated in a plot showing the number of CAZymes (GH = glycosyl hydrolases, GT = glycosyl transferases, PL = polysaccaride lyases, CBMs = carbohydrate binding molecules) for biotrophic (red), hemibiotrophic (black) and necrotrophic (green) pathogens (modified after [28])
Fig. 5
Fig. 5
Validation of RNA-Seq data by qRT-PCR: Relative mRNA levels are comparable to RNA-Seq data in most cases. Comparison of FPKM values (left figure, RNA-Seq data) and relative expression (normalized to reference genes, right figure, qRT-PCR results) of various genes in the 2nd biological replicate. Shown are representative genes that were identified as induced during the infection or down-regulated in Δcptf1. cp1296: invertase; cp3570: xylanase; cp1105, cp1295, cp3095: effector candidate; cp2272: sge1-homologue; cp5492, cp5493, cp7156, cp8623: effector candidate; Sc1Loc01149658.2, Sc2Loc00096015.6: auxin-responsive genes; Sc5Loc00240479.1: chitinase; Sc2Loc02172093.1: flower development Sc3Loc01905034.2: plant defense; Sc4Loc00580338.2: xylanase inhibitor
Fig. 6
Fig. 6
Expression of effector candidates. Expression levels of cp1105 (a + g), cp3095 (b), cp3096 (c), cp5492 (d), cp5493 (e) and cp8623 (f) in the wild type Cp20.1 during infection 3, 5 and 10 dpi as well as in axenic culture was determined by qRT-PCR and normalized to housekeeping genes. Expression of cp1105 (a), cp3095 (b), cp3096 (c), cp5492 (d), cp5493 (e) was strongly induced 3 and 5dpi and decreased 10 dpi as well as in axenic cultures. No clear expression pattern was detectable for cp8623 (f). Here, comparable expression rates were observed 5dpi in planta and in axenic culture. Expression of cp1105 was also determined in strains harboring a cp1105:mCherry construct from axenic culture (g). The experiments were done in two independent biological replicates, exemplary results from the second replicate are shown. Error bars indicate standard deviation from technical replicates
Fig. 7
Fig. 7
Microscopical analyses of Δcp1105 (a + b) and localization of Cp1105 reporter fusion protein (c + d) in axenic culture. Strains were cultivated on medium covered objective slides and incubated for 3 days before they were microscopically analyzed. a The hyphal morphology of Δcp1105 shows mainly undulated hyphae. b Calcofluor White Staining of septa. The mutants show a uniform septation pattern which is similar to the wild-type. c + d Fluorescence can be observed in the medium surrounding the hyphae. No significant fluorescence was observed within medium inoculated with the wild-type Cp20.1. exp: exposure time (Bars = 20 μm)
Fig. 8
Fig. 8
Pathogenicity assays using the C. purpurea wild-type Cp20.1 strain and the Δcp1105 and Δcp8623, ΔΔcp3095/cp3096 and ΔΔcp5432/93 deletion mutants. a Rye ears were infected with conidia suspensions of the different strains, each deletion strain was simultaneously infected and compared to the wild-type. Honeydew formation and sclerotia formation were monitored for 3 weeks. b In vitro cultivated rye florets infected with the different strains. After 6 days, cross-sections of the ovaries were stained with aniline-blue (which emits green fluorescence), allowing the detection of fungal hyphae (indicated by arrows) within the plant tissue by fluorescence microscopy. The infection routes of the deletion strains are similar to those of the wild-type (bars: 1 mm)

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