Rice blast is the most serious disease of cultivated rice, the staple food for one-third of the world's population3,4. The fungusforms heavily melanin-pigmented appressoria that generate enormous turgor. The pressure is applied as a physical force to break the rice leaf cuticle5,6. Turgor can be as great as 8.0 MPa (modal value, 6.0 MPa)5, equivalent to 40 times the pressure in a car tyre and is, to our knowledge, the highest recorded in any living organism5.

We extracted the contents of appressoria and biochemically characterized them, searching for a metabolically compatible solute responsible for generating the hydrostatic pressure. We grew spores of M. grisea in water drops on hydrophobic plastic membranes and allowed them to form appressoria. These generated full turgor over a period of 24-48 h. Gas-liquid chromatography showed that the most abundant solute in appressoria is glycerol.

Glycerol is generated rapidly during germination and germ-tube elongation (Fig. 1a). Here it probably contributes to plasma membrane biosynthesis during initial fungal growth7. Glycerol levels decrease at the onset of appressorium formation but rise sharply during turgor generation. This coincides with the collapse of the conidium and germ tube, and concentration of the cytoplasm within the unicellular appressorium (Fig. 1b). Intracellular glycerol concentrationis considerably higher at this time (48 h), being contained in a small volume.

Figure 1: Intracellular glycerol increases during Mgrisea appressorium turgor generation.
figure 1

a, Change in glycerol contents of extracts made from germinating conidia and developing appressoria with time, determined enzymatically using a glycerol-specific assay (Boehringer). b, Low-temperature scanning electron microscope images of developing appressoria at corresponding times. Appressoria were allowed to form in water drops on polytetrafluoroethylene (PTFE-Teflon, DuPont) membranes. As the appressorium develops turgor the conidium and germ tube collapse6. Cytoplasm is present only in the unicellular appressorium. Scale bars, 20 μm.

We estimate the mean internal volume of an appressorium as 64 μm3 assuming that the cell is hemispherical with an internal radius of 3.1 μm (n=100, s.d.=0.5). The mean concentration of glycerol in an appressorium rose from 0.50 M at 24 h of development to 3.22 ± 0.40 M after 48 h. This is a conservative estimate because not all of the cell volume is available for glycerol accumulation. The osmotic potential generated by this concentration of glycerol would be −8.7 MPa at 20 °C (assuming that glycerol is an ideal solute).

Glycerol solutions deviate from ideal at high concentrations. The maximum concentration of glycerol for which osmotic potential can be assayed psychrometrically8 is 2.0 M, but by extrapolation we estimate that 3.22 M glycerol would generate an osmotic potential of −5.8 MPa. This represents a minimum osmotic potential because living appressoria contain other solutes. The appressorium forms in a drop of water, so the turgor produced is at least 5.8 MPa.

To validate these estimates we incubated appressoria in a series of glycerol solutions of varying molarity and measured the frequencyof cytorrhysis5 (cell collapse). The frequency was dependent on external glycerol concentration. A concentration of 1.75 M glycerol (−3.7 MPa) caused the collapse of 52% of appressoria (data not shown). This supports the link between turgor and glycerol accumulation but also implies that the melanized wall is largely impermeable to glycerol.

Appressoria of M. grisea are heavily melanized9,10 and it has been shown genetically11 that non-melanized appressoria fail to generate turgor and are non-pathogenic5. We found much lower levels of intracellular glycerol in appressoria from non-melanized strains carrying single gene mutations at ALB1 and RSY1 (Fig. 2), genes encoding enzymes required for dihydroxynaphthalene-melanin biosynthesis10. We found a similar reduction in glycerol accumulation after treatment of M. grisea with tricyclazole, a melanin biosynthesis inhibitor12,13 (Fig. 2a). Thus, melanin biosynthesis is required for efficient glycerol accumulation.

Figure 2: Non-melanized appressoria are permeable to glycerol.
figure 2

a, Intracellular glycerol levels in appressoria of alb1 and rsy1 mutants9; and of strain Guy-11 treated with tricyclazole, compared with wild-type untreated appressoria. We measured intracellular glycerol concentration after 24 h. b, We allowed appressoria to form in water for 24 h on PTFE membranes and then replaced the water with 4 M aqueous glycerol. We determined the rate of cytorrhysis and the proportion of appressoria that had recovered from cytorrhysis at each time point. Mean (± s.d.) values from 100 appressoria in two independent experiments are represented. Melanized and non-melanized appressoria recovered fully after 60 min incubation in water (not shown).

In cytorrhysis experiments we found that alb1 mutant appressoria collapsed in hyperosmotic solutions of glycerol but quickly recovered (in under 1 min) and instead became plasmolysed5. This indicates that the non-melanized wall is permeable to glycerol and after initially causing cytorrhysis, glycerol diffuses through the cell wall and induces plasmolysis of the appressorial protoplast. This is in marked contrast to wild-type melanized appressoria which showed only limited recovery from cytorrhysis even after 48 h incubation in hyperosmotic glycerol (Fig. 2b). Maintenance of the enormous glycerol concentrations within appressoria is likely to be a consequence of the reduced permeability of melanized cell walls to glycerol preventing rapid leakage of the solute.

Several important plant pathogens form appressoria1,2 and although secretion of enzymes may aid cuticular degradation2, mechanical infection of plant tissues is probably widespread. The infinite solubility and metabolic compatibility of glycerol therefore provides a simple and durable mechanism for plant infection which may be widely applied by pathogenic fungi.