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The Metarhizium anisopliae Perilipin Homolog MPL1 Regulates Lipid…
The Metarhizium anisopliae Perilipin Homolog MPL1 Regulates Lipid Metabolism, Appressorial Turgor Pressure, and Virulence
ABSTRACT
Gene MPL1
Economically important insect
fungal pathogen Metarhizium anisopliae that has structural similarities to mammalian perilipins.
Expressed when M. anisopliae is engaged in accumulating lipids and ectopically expressed green fluorescent protein-tagged MPL1 (Metarhizium perilipin-like protein) localized to lipid droplets.
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Expression of Mpl1 in yeast cells, a fungus that lacks a perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions.
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Experimental Procedures
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4. Lipid Quantification
- Total lipid quantification was conducted using a phosphoric acid-vanillin method
- Conidia of both the wild type and mutant were harvested from newly mycosed Manduca larvae, potato dextrose agar (PDA), or PDA plus 600 uM oleic acid.
- Spore suspensions (0.5 ml containing ~1.5 x 10^8 conidia/ml) were added to glass tubes. To each tube, 2 ml of 18 M H2SO4 was added, and the tubes were boiled in a water bath for 10 min.
- After cooling (5 min at room temperature), 5 ml of phosphoric acid-vanillin reagent (0.12 g of vanillin, 20 ml of water and the volume adjusted to 100 ml with 85% H3PO4) was added, and the tubes were incubated at 37 °C for 15 min
For yeast studies
- a single colony of pYes2Per transformed yeast cells was incubated overnight in YPD (1% yeast extract, 2% peptone, and 2% dextrose), YPD plus 600 M OA, 2% raffinose
SC-U, or 2% raffinose SC-U plus OA
- Cells harvested from the different media were washed three times, adjusted to A600 = 1.0 with sterile water, and incubated for up to 20 h to induce starvation
- The cell concentration was adjusted to A600 = 2.0 and
0.5 ml used for lipid assay
- Differences in lipid content between
treatments were compared using the Duncan’s analysis of variance analysis (SPSS, 11.0.0)
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6. Insect Bioassay
- Virulence of the wild type and Mpl1 was
assayed against newly emerged 5th instar larvae of M. sexta
- Conidia were applied either topically by immersion of larvae in an aqueous suspension containing 2 x 10^7 conidia/ml for 20 s or by injecting the second proleg with 10 ul of an aqueous
suspension containing 5 x 10^6 spores per ml
- Each treatment
had three replicates with 10 insects each, and the experiments were repeated twice. Mortality was recorded every 12 h
INTRODUCTION
Lipid droplets (LDs)
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Implicated in lipid diseases, inflammation, diabetes, cardiovascular disease, and liver disease
Ascomycete Metarhizium anisopliae, a ubiquitous insect pathogen and biocontrol agen, produces a single mammalian perilipin homolog designated as Mpl1 for Metarhizium perilipin-like protein
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Discussion
- The fungus M.anisopliae produces a protein MPL1 that has structural elements in common with mammalian perilipin
Includes - N-terminal, Beta strands and central three hydrophobic regions that target and anchor Per A to LDs and critical for TAG storage
MPL1 operates in a perilipin-like manner by localizing to lipid droplets and modulating the rate of hydrolysis
This experiment demonstrates that perilipin-like proteins in the most diverse eukaryotes share an ancestral function
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Perilipin-like proteins have also been selected to cope with different tasks as the absence of MPL1 produced unique phenotypes, including reduced appressorial turgor pressure and decreased virulence against an insect host.
The data suggest that the single MPL1 homolog in M.grisea could be candidate for regulating turgor generation through accumulation of lipid-breakdown product glycerol and through cell compartmentalization
The functional demonstration of a role for Mpl 1 in virulence to insects is important for understanding the molecular and biochemical basis of pathogenicity and also for pest control
Mpl 1 is also up-regulated in aging fungal sectors and the degenerated fungus demonstrates decreased levels of cAMP and variable rates of lipid metabolism