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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
Discussion
MPL1
- produced by M. anisopliae
- has structural elements in common mammalian perilipin
- include N-terminal B-stands & central 3 hydrophobic regions that target and anchor Per A to LDs (critical for TAG storage
- MPL1 has multiple phosphorylation sites
- operates in a peripilin-like manner by localizing to lipid droplets & modulating the rate of hydrolysis
periliphins
- first identified mammalian lipid-associated proteins
Phosphorylation of peripilin by CAMP-dependent protein kinase
- results in the conformational change that initiates lipolysis by hormone-sensitive lipase
the lean Per A knock-out mouse under diet control : germ tubes of deltaMpl1 are slimmer than those of the wild type Metarhizium
- means peripilin-like proteins in the most diverse eukaryotes share an ancesteral function
homologes of MPL1
- only present in pezizomycotinal ascomycetes
pezizomycotinal fungi
- include most important and well known animal and plant pathogens
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Yeast cells expressing Mpl1
- unable to make use of their stored lipids during starvation
- yeast has lost or never acquired
- phosphorylation mechanism for removing perilipin-like proteins to access LDs
- responsible for lipid metabolism in non-peripilin producing majority of fungi
differences between animal and fungal lineages
- 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
- decreased virulence against an insect host
appressoria of the rice blast fungus Magnaporthe grisea
- take up water to generate turgor for mechanical penetration of host surface
turgor
- generated by accumulation of solutes (lipid-breakdown product glycerol)
cell compartmentalization
- required in rice blast fungus M. grisea to generate
appressorial turgor
- single MPL1 homolog in M. grisea candidate regulation of these process
- general phenomenon among pathogenic ascomycetes as Cap20 mutants of the plant pathogen C. gloeosporioides
:red_flag: although
normal looking the mutant appressoria might not be fully functional
role for Mpl1
- virulence of insects
- important for understanding the molecular and
biochemical basis of pathogenicity
- should be relevant to innovating new measures of pest control
- up-regulated (>2-fold) in aging fungal sectors
- degenerated fungus demonstrates decreases level of cAMP
- variable rates of lipid metabolism
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Experimental Procedures
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(5) Lipid Quantification
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Spore suspensions were added to glass tubes, add H2SO4, boiled.
After cooled, 5 ml of phosphoric acid-vanillin reagent added and incubated at 37 °C for 15 min
samples were centrifuged, and the absorbance of supernatants was measured at 530 nm
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for yeast studies
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Differences in lipid content between treatments were compared using the Duncan’s analysis of variance analysis
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(7) Insect Bioassay
Virulence of the wild type and mutant Mpl1 was
assayed against newly emerged 5th instar larvae of M. sexta*
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Expression Profile and Localization of MPL1 - analyzed Mpl1
expression in M. anisopliae grown in different
media.
- RT-PCR: strong expression of Mpl1 (on nutrient rich medium- insect hemolymph or SDB)
- addition of fatty acids to mammalian cells stimulates lipid accumulation and increases intracellular levels of perilipin
- transcription of Mpl1 by M. anisopliae germlings was up-regulated within 30 min following the addition of oleic acid to minimal
medium.
- Conidia
contained many large LDs, strong
enrichment of Mpl1 transcripts.
- Transcription levels decreased
during germination.
- LD numbers dropped by 40%
between 6 and 12 h ( lipid stores are mobilized during
germ tube elongation)
- Mpl1 regulation parallels
lipid storage.
Intracellular targeting MPL1 in vivo; expressed MPL1 GFP fusion protein in M. anisopliae using the Aspergillus GpdA promoter.
- Perilipin A , TIP47 , & adipocyte differentiation-related protein , N-terminal fusion of MPL1 with GFP did not disrupt the ability of the protein to localize to lipid vesicles.
- Transformed yeast and Metarhizium cells treated with the neutral lipid stain NR colocalized with the GFP signal confirming that MPL1 is binding to LDs
- No additional diffuse cytoplasmic signal was seen with either GFP or NR
- The expression patterns & intracellular localization of MPL1 ; consistent with the proposal that the protein plays a regulatory role in global triacylglycerol (TAG) storage by acting at the level of LDs.
Introduction
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In this study- ascomycete Metarhizium anisopliae, a ubiquitous insect pathogen and biocontrol
agent
- Produces a single mammalian perilipin homolog we
designated as Mpl1 for Metarhizium perilipin-like protein