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, 12 (9), 1703-18

MAP Kinase and Protein Kinase A-dependent Mobilization of Triacylglycerol and Glycogen During Appressorium Turgor Generation by Magnaporthe Grisea

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MAP Kinase and Protein Kinase A-dependent Mobilization of Triacylglycerol and Glycogen During Appressorium Turgor Generation by Magnaporthe Grisea

E Thines et al. Plant Cell.

Abstract

Magnaporthe grisea produces an infection structure called an appressorium, which is used to breach the plant cuticle by mechanical force. Appressoria generate hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control and biochemical mechanism for turgor generation, we assayed glycerol biosynthetic enzymes during appressorium development, and the movement of storage reserves was monitored in developmental mutants. Enzymatic activities for glycerol generation from carbohydrate sources were present in appressoria but did not increase during development. In contrast, triacylglycerol lipase activity increased during appressorium maturation. Rapid glycogen degradation occurred during conidial germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. Lipid droplets also moved to the incipient appressorium and coalesced into a central vacuole before degrading at the onset of turgor generation. Glycogen and lipid mobilization did not occur in a Deltapmk1 mutant, which lacked the mitogen-activated protein kinase (MAPK) required for appressorium differentiation, and was retarded markedly in a DeltacpkA mutant, which lacks the catalytic subunit of cAMP-dependent protein kinase A (PKA). Glycogen and lipid degradation were very rapid in a Deltamac1 sum1-99 mutant, which carries a mutation in the regulatory subunit of PKA, occurring before appressorium morphogenesis was complete. Mass transfer of storage carbohydrate and lipid reserves to the appressorium therefore occurs under control of the PMK1 MAPK pathway. Turgor generation then proceeds by compartmentalization and rapid degradation of lipid and glycogen reserves under control of the CPKA/SUM1-encoded PKA holoenzyme.

Figures

Figure 1.
Figure 1.
Cellular Distribution of Glycogen during Appressorium Morphogenesis by M. grisea. Conidia from the wild-type M. grisea strain Guy11 were allowed to germinate in water on hydrophobic plastic cover slips (see Methods) and form appressoria. Sample cover slips were removed every 2 hr and incubated in a glycogen staining solution containing 60 mg of KI and 10 mg of I2 per milliliter of distilled water (Weber et al., 1998). Yellowish-brown glycogen deposits became visible immediately in bright-field optics with a Nikon Optiphot-2 microscope. (A) to (H) Dense deposits of glycogen were visible in ungerminated conidia (0 hr) (A) but were greatly reduced in the germinating conidial cell and its germ tube after 2 hr (B) and in the nascent appressorium after 4 hr (C). After formation of the basal septum, iodine staining reappeared for a time after 6 (D) and 8 hr (E). Glycogen disappeared again during appressorium maturation after 12 (F), 24 (G), and 48 hr (H). Note that the process of appressorium formation in M. grisea occurs considerably faster on plastic cover slips than on onion epidermis (see Figure 4). Bar in (H) = 20 μm for (A) to (H).
Figure 2.
Figure 2.
Cellular Distribution of Glycogen in Developmental Mutants of M. grisea. Conidia from the regulatory mutants of M. grisea were allowed to germinate in water on hydrophobic plastic cover slips and form appressoria. Sample cover slips were removed every 2 hr and stained for the presence of glycogen (see Methods and legend to Figure 1). Conidia and developing appressoria are shown at 0 and 8 hr. (A) and (D) Δpmk1 mutant nn95. (B) and (E) ΔcpkA mutant DF51. (C) and (F) Δmac1 sum1-99 mutant DA-99. The mobilization of glycogen was severely impaired in the Δpmk1 mutant with substantial amounts of glycogen still present in conidia after 8 hr. In the ΔcpkA mutant, glycogen mobilization was retarded and glycogen was not accumulated in the developing appressorium after 8 hr. The Δmac1 sum1-99 mutant produced conidia with depleted glycogen contents, particularly when harvested from cultures >10 days old. Glycogen degradation in the appressorium occurred more rapidly than in Guy11 and was complete in 8 to 10 hr. Bar in (F) = 10 μm for (A) to (F).
Figure 3.
Figure 3.
Triacylglycerol Lipase Activity in Conidia and Developing Appressoria of M. grisea. Conidia were allowed to form appressoria on the hydrophobic surface of Petri dishes, and protein was extracted as described in Methods. Triacylglycerol lipase activity was assayed by measuring the liberation of oleic acid from the substrate triolein, which was determined by densitometric analysis of the liberated oleic acid resolved by thin-layer chromatography. Enzymatic activity is expressed as relative lipase activity compared with the activity expressed by 2.5 units of purified triacylglycerol lipase from Rhizopus arrhizus (Roche Molecular Biochemicals). Each data point is the mean activity determined from three independent replications of the experiment. The error bars show the standard deviation. Triacylglycerol lipase activity is shown from the wild-type M. grisea strain Guy11 (diamonds), from the ΔcpkA mutant DF51 (squares), and the Δmac1 sum1-99 mutant DA-99 (circles with dotted line).
Figure 4.
Figure 4.
Cellular Distribution of Lipid Droplets during Appressorium Morphogenesis by M. grisea. Conidia were allowed to germinate on onion epidermis in water drops and form appressoria. Sample preparations were removed at various intervals during a 96-hr period and stained for the presence of triacylglycerol by using Nile Red, as described in Methods. Photographs were taken at 0, 4, 12, 24, 48, and 96 hr after inoculation; for each time, a Hoffman modulation contrast image (left panel) and an epifluorescence image of Nile Red–stained material (right panel) are presented. Shown are the results for preparations of the wild-type strain Guy 11 and the buf1 mutant 6-R-10. Because melanin masked the epifluorescence somewhat, a nonmelanized mutant was examined for better quality resolution of lipid bodies. In both strains, numerous small lipid droplets were seen in ungerminated conidia (0 hr) and in nascent appressoria (4 hr), whereas later (12, 24, and 48 hr), fewer but larger lipid droplets were observed coalescing into vacuolar structures. Lipid bodies were degraded at the onset of turgor generation and cuticle penetration (24 to 96 hr after inoculation). Bar = 20 μm for all images.
Figure 5.
Figure 5.
Ultrastructure of an Appressorium of M. grisea. Appressoria from the wild-type strain Guy11 were fixed by freeze-substitution 24 hr after inoculation of conidia onto onion epidermis. (A) Median section of a whole appressorium formed on the surface of onion epidermis. The basal septum (arrow) is visible. (B) Ultrastructural detail of another appressorium. Several electron-light lipid droplets (L), dark crystalline glycogen granules (arrowheads), and vacuoles with heterogenous contents (V) are visible, in addition to a nucleus (N) and mitochondria (M). Bar in (A) = 2 μm; bar in (B) = 1 μm.
Figure 6.
Figure 6.
Cellular Distribution of Lipid Droplets during Infection-Related Development by ΔcpkA and Δpmk1 Mutants of M. grisea. Conidia were allowed to germinate on onion epidermis in water drops and were removed at intervals during a 96-hr period and stained with Nile Red for the presence of triacylglycerol (see Methods). Photographs were taken 0, 4, 12, 24, 48, and 96 hr after inoculation; for each time, a Hoffman modulation contrast image (left panel) and an epifluorescence image of Nile Red–stained material (right panel) are presented. In the ΔcpkA mutant DF51, lipid mobilization to the developing appressorium occurred as in wild-type strains of M. grisea except that appressorium formation was delayed until 12 hr after inoculation. The appressoria of ΔcpkA were small and sometimes misshapen. Lipid droplets enlarged during appressorium development (at 48 and 96 hr), but no degradation was observed during this experiment. In the Δpmk1 mutant nn95, no appressoria were formed; instead, lipid droplets were found evenly distributed into the developing germ tubes, which ultimately developed vacuolates (at 48 and 96 hr) and stopped growing. Bar = 20 μm for all images.
Figure 7.
Figure 7.
Quantitative Analysis of Lipid Distribution during Infection-Related Development by M. grisea. Conidia were allowed to germinate in water drops on the surface of onion epidermis and to undergo infection-related development. Samples were removed at intervals over a 12-hr period and stained for the presence of triacylglycerol by using Nile Red. The percentage of fungal structures that contained lipid bodies at a given time was recorded from a sample of 300 germinated conidia. The bar charts show the mean and standard deviation from three independent replications of the experiment. Solid black bars represent conidia, open bars represent germ tubes, and gray bars represent appressoria. (A) Wild-type M. grisea strain Guy11. (B) Δmac1 sum1-99 mutant DA-99. (C) ΔcpkA mutant DF51. (D) Δpmk1 mutant nn95.
Figure 8.
Figure 8.
Cellular Distribution of Lipid Droplets during Infection-Related Development by the Δmac1 sum1-99 Mutant DA-99. Conidia were allowed to germinate in water drops on the surface of plastic cover slips and undergo infection-related development. Samples were removed at intervals over a 12-hr period and stained for the presence of triacylglycerol with Nile Red; for each interval, a Hoffman modulation contrast image (left panel) and an epifluorescence image of Nile Red–stained material are presented. (A) and (D) At 4 hr after inoculation of conidia, lipid droplets have already migrated to the incipient appressorium and begun to coalesce. (B) and (E) At 8 hr after inoculation of conidia, lipid droplets are beginning to be degraded, before melanization of the appressorium. (C) and (F) At 12 hr after inoculation of conidia, lipid droplets have been almost completely degraded. Bar in (C) = 10 μm for (A) to (F).
Figure 9.
Figure 9.
Pathogenicity of the Δmac1 sum1-99 Mutant DA-99 Is Related to the Age and Storage Reserve Status of Conidia. Conidia from the Δmac1 sum1-99 mutant DA-99 were harvested from plate cultures that had been incubated for 6, 7, 9, 11, 13, and 15 days. Conidial suspensions were diluted to a uniform concentration of 104 mL–1 and sprayed onto 14-day-old rice seedlings of the blast-susceptible cultivar CO-39. The disease was allowed to progress for 96 hr, when disease lesions had not yet coalesced, and representative leaves were removed and photographed. An identical conidial suspension from the wild-type strain Guy11 was used in the positive control experiment. The severity of disease lesions decreased with increasing conidial age in the Δmac1 sum1-99 mutant.
Figure 10.
Figure 10.
Outline Model for the Genetic Control of Appressorium Turgor Generation by the Rice Blast Fungus M. grisea. In this model, the mobilization of glycogen and triacylglycerol reserves to the developing appressorium occurs under control of the PMK1 MAPK pathway. Glycerol generation is achieved by degradation of triacylglycerol by triacylglycerol lipase, which is regulated by CPKA/SUM1-encoded PKA. Fatty acids are processed by β-oxidation and may be used to produce glycerol by way of the glyoxylate cycle and gluconeogenesis. Glycogen degradation, which is similarly regulated within the appressorium at the onset of turgor generation, is used to fuel glycolysis during glycerol accumulation and may contribute to glycerol production by the metabolism of dihydroxyacetone 3-phosphate (DHAP), dihydroxyacetone (DHA), or glyceraldehyde (GAD). Glycerol accumulation results in hydrostatic turgor and is translated into the force required for cuticle penetration by reorientation of the cytoskeleton, localized dissolution of the cell wall, and formation of penetration pegs. These processes are likely to be regulated by the action of PKA and stimulation of a signal transduction pathway involving the MPS1 MAPK (Xu et al., 1998). In the model shown, genetic control points are shown in blue and putative enzymatic activities are given in green. GPH, glycogen phosphorylase (EC 2.4.1.1); TGL, triacylglycerol lipase (EC 3.1.1.3); GPD, NADH-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8); GD, NADPH-dependent glycerol dehydrogenase (EC 1.1.1.77). Multiple arrows indicate several intermediate steps in pathway not shown.

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