Fig 1.
Putative Fusarium graminearum GPCRs expression patterns during infection and contribution to virulence on wheat.
A) Classification of the ten putative GPCR classes in F. graminearum, according to structural similarities [27]. Schematics depict the putative protein structures and distinct characteristics of each class. B) Distribution of putative classical (black) and non-classical (blue) GPCR-encoding genes across the four F. graminearum chromosomes alongside a chromosomal recombination frequency heatmap: Red high level–blue low level recombination. Displayed using OmniMap [50]. C) Expansion of class X receptors in F. graminearum in comparison to other ascomycete fungi, including model non-pathogenic and pathogenic yeasts Saccharomyces cerevisiae and Candida albicans, plus saprophytic (Aspergillus nidulans, Neurospora crassa and Trichoderma reesei) and plant pathogenic (Magnaporthae oryzae) fungi from the subphylum Pezizomycotina [26,27]. D) Affymetrix expression heatmap for selected F. graminearum classical (classes I-V) and non-classical (class X with or without a CFEM domain) receptors in axenic culture or during wheat infection [4]. Axenic culture represented by complete media (CM), carbon (MM-C) and nitrogen (MM-N) starvation. Wheat infection represents the distinct phases of infection at 7 days post infection (dpi), namely symptomless rachis infection, intermediate rachis infection, symptomatic rachis and symptomatic spikelet infection. * denotes GPCR encoding genes with either the highest absolute level, or fold change, in expression during infection. E) Identification of F. graminearum GPCRs that contribute to virulence. Presented are mutants lacking either FGRRES_16221 or FGRRES_07792, compared to the parental PH-1 strain and the mock non-infected controls at 15 dpi. F) Disease progression throughout the 15 day time course. G) Boxplot showing the number of diseased spikelets at 15 days. H) Quantification of fungal burden, within wheat at 15 dpi, presented as the ratio of F. graminearum tri5 to wheat cdc48 DNA. I) DON mycotoxin contamination within wheat heads at 15 dpi. G-I) *** = p<0.001, ** = p <0.01.
Fig 2.
The discontinuous distribution of bona fide Fusarium graminearum non-classical G-protein coupled receptors.
A) Phylogenetics of putative F. graminearum GPCR encoding genes. The identification of FGRRES_16221 and FGRRES_07792 orthologues shows a discontinuous distribution throughout the fungal tree of life. Diagrammatic representation of the fungal tree of life (not indicative of evolutionary time) adapted from the JGI MycoCosm [52]. Pie charts show the number of species genomes within each taxonomic class with at least one FGRRES_16221 or FGRRES_07792 orthologue as predicted by PhytoPath [51]. The number of species genomes present in each group analysed is shown in brackets. * No Lecanoromycete genomes were represented in the PhytoPath dataset. Therefore, BlastP analyses using the mature FGRRES_07792 and FGRRES_16221 protein sequences were performed, at the expected (e-value) cut-off thresholds of 1x10-50 and 1x10-100, on the predicted proteomes of four Lecanoromycetes presented on the JGI Mycocosm portal [52]. B) A phylogenetic tree based on the alignment of the eight F. graminearum class X receptors, which possess the extracellular CFEM domain, with the original fungal class X receptor, PTH11 from Magnaporthae oryzae, labelled MoPTH11. C) Alignment of the CFEM domain from the eight F. graminearum class X GPCRs and MoPTH11, showing the conservation of the cysteine residues. * denotes Asp residue conserved in heme-binding CFEM proteins [34]. D) Left panel—Schematic depicts the yeast split ubiquitin approach for identification of receptor-G-protein interactions at the cell membrane. Right panel—Assay demonstrates class X non-classical (FGRRES_07792 and FGRRES_16221) receptors specifically interact with multiple Gα-proteins at the cell membrane. Yeast serial dilutions (1:1, 1:10, 1:100, 1:1000) were grown on non-selective SD media lacking tryptophan (T) and leucine (L), plus selective media also lacking histidine (H) and adenine (A). The Alg5 membrane protein is a non-GPCR interacting control.
Fig 3.
FGRRES_16221 receptor sub-domains are important to virulence.
A) The putative protein structure of GPCR FGRRES_16221, as predicted by the Phyre2 software [61], showing the presence of a cysteine-rich extracellular CFEM domain, plus the 3rd cytoplasmic loop (CL) and extended cytoplasmic tail (CT) potentially involved in interactions with downstream Gα-proteins. Schematic initially depicts the organisation of the native FGRRES_16221 protein and then shows the three FGRRES_16221 truncations generated. This includes ΔCFEM lacking the CFEM domain, ΔCT lacking the cytoplasmic tail and ΔTM3-7+CT lacking the majority of the protein (containing the 3rd cytoplasmic loop and cytoplasmic tail), leaving the extracellular CFEM domain tethered to the plasma membrane. In addition, the Δ16221_1 and Δ16221_3 mutants were both complemented with the native FGRRES_16221 gene. B) A boxplot showing the number of diseased spikelets at 15 days. The three types of FGRRES_16221 truncations all showed reduced disease symptoms on wheat at 15 days post infection (dpi). Truncations had a more severe impact on fungal virulence than the entire FGRRES_16221 gene deletion. Complementation of the Δ16221_1 and Δ16221_3 mutants (labelled—COM) restored fungal virulence to level equivalent to the parental PH-1 strain. *** = p<0.001, ** = p<0.01. C) The appearance of wheat disease symptoms at 15 dpi. Presented are two independent F. graminearum mutants of the deletion, truncation and complementation of the FGRRES_16221 receptor, compared to the parental PH-1 strain and the mock non-infected controls.
Fig 4.
The Fusarium graminearum FGRRES_07792 and FGRRES_16221 mutants show reduced floral colonisation, but can penetrate the surface.
Presented are scanning electron micrographs of the inner surface of the palea for the non-infected (mock) wheat tissue, the parental PH-1 strain, plus the Δ16221_3 and Δ07792_3 mutants 2 days post infection. Inserts in each panel are higher magnification images of infection structures indicated. White arrow = infection structures. Black arrow = runner hyphae. Bar = 100 μm.
Fig 5.
The Fusarium graminearum mutants lacking FGRRES_07792 or FGRRES_16221 show a delay in disease progression.
Images depict the macro-dissection of individual spikelets (SP) and the associated rachis internode (RI) below the point of inoculation at 6, 9 and 12 days post infection (dpi), for the non-infected (mock) wheat tissue, the parental PH-1 strain, plus the mutant Δ07792_3 and Δ16221_3 strains. * denotes the increased browning of the rachis ahead of the last symptomatic spikelet in the absence of FGRRES_16221.
Fig 6.
The Fusarium graminearum FGRRES_16221 mutant shows defects in establishing symptomless rachis node infection, resulting in apoplastic occlusions.
A) Images depict non-infected wheat describing the anatomy of the wheat spikelet (SP), rachis node (RN) and internode (RI) tissues. B) Light micrographs of transverse sections through the rachis node of the mock and PH-1 infected tissues at 9 days post infection. The healthy mock tissue shows the branching of the vasculature within the rachis node. The fully symptomatic PH-1 infected tissue shows the destruction of the wheat vasculature, the absence of live plant cells and the presence of both inter- and intra-cellular fungal hyphae. Note that PH-1 infection has already advanced multiple rachis nodes ahead. C) Light micrographs of transverse sections of the Δ16221_3 infected 3rd rachis node revealing the appearance of infection at the advancing hyphal front, plus ahead (+100 μm) and behind (-100 μm) the hyphal front. Behind the hyphal front, Δ16221_3 accumulates within intercellular spaces between a mixture of live and dead plant cells. At the hyphal front, a limited amount of intercellular hyphae are surrounded by active live plant cells. In advance of the hyphal front, the plant cells are responding to infection resulting in cell fortifications and the occlusion of the intercellular spaces. Arrows: grey = fungal hyphae, yellow = wheat cells responding to infection. Bar = 50 μm. P = parenchyma. V = vasculature.
Fig 7.
The Fusarium graminearum mutant lacking FGRRES_16221 shows delayed rachis internode colonisation and vascular occlusions.
The delay in disease progression by the F. graminearum FGRRES_16221 mutant, in comparison to the parental PH-1 strain at 9 days post infection. Light micrographs of longitudinal sections of the 1st, 3rd and 5th rachis internode below the point of infection. In the 1st rachis internode, Δ16221_3 has established both inter- and intra-cellular infection, resulting in plant cell death and the destruction of the vasculature. In the 3rd rachis internode, Δ16221_3 has accumulated within the xylem vessel. This is the only infection beyond the apoplastic occlusion in the 3rd rachis node. In the 5th rachis internode, occlusions in the xylem vessels are observed ahead of infection. In contrast, the fully symptomatic PH-1 infected tissue shows the destruction of the wheat vasculature, the absence of live plant cells, and the presence of both inter- and intra-cellular fungal hyphae throughout the 1st, 3rd and 5th rachis internodes. Note rachis infection by the PH-1 strain has already progressed multiple rachis internodes ahead of infection by Δ16221_3. Arrows: grey = fungal hyphae, yellow = occlusions in the xylem vessel. Bar = 100 μm.
Fig 8.
RNA-sequencing reveals the role of FGRRES_16221 in coordinating fungal metabolism and virulence factors to promote infection without activating inhibitory wheat defences.
Wheat tissues sampled for RNA-sequencing to evaluate transcriptional differences in the pathogen and host during the infection establishment (spikelet, 3 day post infection; dpi) and the progression of infection (spikelet and sequential rachis internode pairs at 7 dpi). A) The ratio of wheat-to-fungal transcripts reflected differences in fungal burden, confirming the reduced progression of infection by the FGRRES_16221 mutant (Δ16221_3). B) The number of differentially expressed F. graminearum and wheat genes (DEGs; FDR <0.05, ±1log2 fold change in expression) identified in pairwise analyses between the parental PH-1 and Δ16221_3 during axenic culture and wheat infection. Overrepresented gene ontologies (GO) were identified within the DEGs and specific gene categories associated with pathogenesis (S1–S3 Files). Accumulative FPKM expression values for selected gene categories from F. graminearum (C) and wheat (D). Note absolute FPKM values presented for single FGL1 gene. Legend: YPD = axenic culture in YPD, SP_3d = spikelet 3 dpi, SP_7d = spikelet 7 dpi, R1-8_7d = pooled pairs of rachis internodes below inoculated spikelet at 7 dpi. E) Schematic representation of wheat infection by the parental PH-1 strain and the dysregulation of virulence mechanisms in the absence of FGRRES_16221. Green = live wheat cells. Yellow = Dead wheat cells. Brown = Wheat defence response including cell reinforcements, plus apoplastic and vascular occlusions. Purple = Invading F. graminearum hyphae.