Figure 1.
Replacement of F. graminearum gpi7 with a hygromycin phosphotransferase (hph) marker.
A. Gene replacement of gpi7 using a split-marker approach. White bars represent genomic regions upstream and downstream of the gpi7 coding sequence that were amplified and fused to segments of the hph cassette. Black bars represent genomic regions outside of the replacement construct. The deletion constructs were amplified with primers gpi7KO_P1-gpi7KO_P4 and H1-H4. Confirmation of gene replacement was tested with primers gpi7KO_chk1-gpi7KO_chk6. B. Results of diagnostic PCRs performed with primers gpi7_chk1-gpi7_chk6 (PCR reactions 1-3; see panel A). ‘gpi7-11’ and ‘gpi7-92’ are two independent Δgpi7 mutants. ‘gpi7-11+gpi7’ is strain gpi7-11 complemented with a complete gpi7 coding sequence. ‘wt’ refers to strain PH-1. Numbers on the left represent the migration of standard DNA markers. C. Colonial phenotypes of Δgpi7 mutants after 3 days of growth on V8 medium at room temperature.
Figure 2.
Macroconidia abnormalities of Δgpi7 mutants.
A. Reduced length of macroconidia produced by Δgpi7 mutants gpi7-11 and gpi7-92. All micrographs in panel A are the same scale. B. Cell separation defect of Δgpi7 macroconidia. Some macroconidia appeared to be two macroconidia ‘fused’ together (black arrows). Roughly 10% (see Table 2) of the macroconidia from both gpi7-11 and gpi7-92 displayed macroconidia of this phenotype. Note that the macroconidia scored ‘aberrant’ for wt:hyg+ strain P2 were restricted to small protrusions at the macroconidial tip, whereas the phenotypes seen in the Δgpi7 mutants were more drastic. All micrographs in panel B are at the same scale. Scale bars = 10 μm.
Figure 3.
Conidiophores of Δgpi7 mutants.
Note the long chains produced by Δgpi7 strains gpi7-11 and gpi7-92 (black arrows), compared to the short chains produced by the complemented strain (gpi7-11 + gpi7) and wildtype strain (wt). Also, conidiophores of the Δgpi7 mutants displayed septation within the conidiophores (white arrows), while such septation sites are not typical within the conidiophores of wildtype strains. All panels in same scale. Scale bar = 10 μm.
Figure 4.
Cell wall defects of Δgpi7 mutants.
A. Increased sensitivity of Δgpi7 mutants gpi7-11 and gpi7-92 to the cell wall disturbing agent calcofluor white compared to the complemented strain (gpi7-11 + gpi7) and hygromycin-resistant PH-1 strain (wt:hyg+). 7 μl of macroconidial suspensions of different concentration were serially spotted onto media. B. Transmission electron micrographs of cell walls in wildtype and Δgpi7 mutant macroconidia and hyphae. Black arrows point to the outer protein layer of the cell wall. There were no gross morphological changes observed in the cell wall of the Δgpi7 mutant. Scale bar = 500 nm.
Figure 5.
Virulence of Δgpi7 mutants gpi7-11 and gpi-92.
A. Inoculated wheat heads two weeks after inoculation. Tween=mock inoculated negative control. Regarding Δgpi7 mutants, “-“ refers to heads exhibiting some black scabbing on the inoculated spikelet, but little to no chlorosis, while “+” refers to heads that displayed chlorosis at the inoculated spikelet. White arrows indicate inoculated spikelets. B. Mean percentage of symptomatic spikelets per inoculated head. Across two experiments, n = 13, 19, 8, 8, 19, 19 for gpi7-11-, gpi7-92-, gpi7-11+gpi7-, wt:hyg+ -, wt-, and tween-inoculated heads, respectively. Error bars = +/- standard deviation. C. Spread of symptoms through the rachis of wheat heads. Florets were removed from the inoculated side of the wheat head. Note that symptoms in those heads scored “-“ did not advance into the rachis and that in those scored “+”, the advancement was less than that of control strains. White arrows indicate nodes where inoculated florets were located.
Figure 6.
Hyphal invasion of rachis tissue.
Rachis tissue adjacent to the inoculated spikelet was stained with lactophenol trypan blue and observed with bright-field microscopy. Both pith cells and parenchyma cell adjacent to vascular tissue were observed for the presence of hypha (note: vascular tissue stained with lactophenol blue, even in the mock-inoculated control, which made the observation of hyphae within vascular tissue difficult). ‘Tween’ represents mock-inoculated wheat heads; ‘gpi7-11’ and ‘gpi7-92’ were inoculated with these two Δgpi7 mutants; ‘gpi7-11 + gpi711’ were inoculated with the a complemented gpi7-11 strain; ‘wt’ were inoculated with strain PH-1.
Figure 7.
Saprophytic growth of Δgpi7 mutants and development of infection-related hyphae.
A. Previously-frozen wheat heads that were inoculated at a single spikelet and observed 5 days post inoculation. Note the white aerial mycelia profilerating on wheat heads. B. Hyphal development within the rachis tissue of wheat heads depicted in panel A. Micrographs in panel B show the parenchyma cell adjacent to vascular tissue, which were not readily invaded by Δgpi7 mutants in living wheat plants. C. Development of infection-related hyphae on detached wheat glumes. SCH=subcuticular hyphae. BIH=bulbous infection hyphae. Scale bar = 10 μm in all micrographs.
Figure 8.
Hypersensitivity of Δgpi7 mutants to fungal cell wall degrading enzymes.
A. Macroconidia were germinated in liquid media containing various concentrations of Glucanex. Images were captured 10 hour post inoculation. All micrographs in panel A are at the same scale. B. The hyphal phenotypes observed in Δgpi7 mutants at a Glucanex concentration of 1 mg/ml. All micrographs in panel B are at the same scale. Scale bars for panels A and B = 10 μm.
Figure 9.
Intragenic variability in genes FGSG_01588 and FGSG_08844 among various F. graminearum strains collected in Nebraska (Fg1-Fg48, NE1-NE2, IN1).
M=Invitrogen 1kb plus mass ladder. PH-1 = standard laboratory strain (genome sequenced). Those marked with an asterisk were cloned and sequenced (see Fig. 9).
Figure 10.
Differences in gene length of FGSG_01588 and FGSG_08844 are due to gaps within the variability region of the genes.
Importantly, the gaps in DNA sequence occur in multiples of three, so that the rest of the coding region remains in-frame.