Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch)

Abstract

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant-Microbe Interact. 18, 110-115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.

Figure 1. Southern analysis using a probe homologous to Mdh1 (A) and Mpd1 (B)

Digested genomic DNA of representative strains is shown: lanes 1 and 8, SN15; lane 2, mdh1-71; lane 3, mdh1-73; lane 4, mdh1-79; lanes 5 and 11, mpd1mdh1-102; lanes 6 and 12, mpd1mdh1-107; lane 7, mdh1-63; lane 9, mpd1-1; lane 13, mpd1mdh1-101.

Figure 2. Growth assays for strains SN15, mpd1-1, mdh1-71, mpd1mdh1-102 of S. nodorum on 10 mM glucose (grey bars) and 10 mM mannitol (black bars)

S. nodorum SN15 also grown in the absence of a carbon source is shown as a control (‘No C’). The growth data shown is the average of eight attenuances (OD₆₀₀=D₆₀₀) collected for each strain growing on each carbon source. The experiment was performed in triplicate with the S.E.M. bars shown.

Figure 3. Sporulation assays on complex CzV8CS medium (light grey) and defined MM (dark grey)

Strains are as follows: SN15, S. nodorum SN15; md71, mdh1-71; md73, mdh1-73; Md63, mdh1-63; mp1, mpd1-1; m102, mpd1mdh1-102; m107, mpd1mdh1-107; M101, mpd1mdh1-101. The assays were performed in triplicate with S.E.M. bars shown.

Figure 4. Sporulation assays using inoculum sourced from progressive subculturing on MM

MM1 refers to spore counts on MM plates inoculated with spores harvested from complex CzV8CS plates, MM2 represents MM plates not containing mannitol that were inoculated with spores harvested from progressive subculturing on MM plates, and MM3 is identical with MM2 with the exception that the MM plates contained 3 mM mannitol. The white bars represent S. nodorum SN15, light-grey bars represent mdh1-71, dark-grey bars represent mpd1-1, and the black bars represent mpd1mdh1-102. The grey shaded area to the right of the graph represents the mannitol abundance of the spores harvested from the plates for each of the strains under the different conditions: +++, high abundance; ++, medium abundance; +, low abundance; –, trace amounts detected.

Figure 5. Infected wheat leaves at 8 dpi in a detached-leaf assay

(A) S. nodorum SN15; (B) S. nodorum mdh1-71; (C) S. nodorum mpd1-1; (D) S. nodorum mpd1mpd1-102.

Figure 6. Detached wheat leaves infected with S. nodorum SN15 in the absence (A) or presence (B) of 3 mM mannitol, and S. nodorum mpd1mdh1-102 in the absence (C) or presence (D) of 3 mM mannitol

Photographs were taken when the plants were at 19 dpi.

Figure 7. Normalized gene expression for each of Mpd1, Mdh1 and Gpd1 at 3 (mid-grey bars) and 8 (dark-grey bars) dpi on detached wheat leaves

The scale shown represents the calculated number of copies of each gene divided by the number of determined actin transcripts for that particular sample. The experiment was performed in triplicate on biologically independent samples and average values with S.D. values are shown.