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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
Experimental procedure
Gene Cloning and Deletion:
- has a high similarity with the virulence
factor CAP20 of plant fungal pathogen Colletotrichum gloeosporioides
- Gene deletion was conducted using the plasmid pGPS3Bar on the full cDNA
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GFP Fusion Constructs and Expression:
- BamHI sites (at both ends) and SmaI site (at 5' end) are introduced
- BamHI was digested to generate pYes2Per by inserting pYes2 (Invitrogen)
- GFP gene was intergated into the SmaI site of plasmid pYes2Per
RT-PCR Analysis of Gene Expression: To monitor gene
expression of Mpl1
- mycelia were collected for RNA extraction, cDNA conversion, and RT-PCR analysis
Fluorescence Microscopy: to detect neutral lipids
- Cells were then stained with Nile Red or Bodipy
Lipid Quantification: conducted using a phosphoric acid-vanillin method (phosphoric acid-vanillin reagent)
- conidia (wild-type & mutant) harvested from newly
mycosed Manduca larvae
Appressorial Turgor Assay: To examine the potential
involvement of MPL1 in turgor generation
- Individual wings were dipped in PEG solutions; the percentage of collapsed appressoria
was determined from 300 cells per PEG solution
Insect Bioassay: to test virulence
- conidia was applied on 5th instar larvae of M. sexta (newly emerged ); mortality was recorded
Results
A fungal Perilipin
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Expressed an MPL1-GFP using Aspergillus** GpdA promoter to:
- visualize the intracellular targeting of MPL1 in vivo
Determined whether LD localization is a general quality of MPL1 by expressing it with Gal1- inducible promoter in the yeast, S.cerevisiae - lack endogenous perilipin-like protein
PAT protein
- perilipin A
- N -terminal fusion of MPL1 with AFP x disrupt the ability of the protein to localize to lipid vesicles
Transformed yeast & Methrahizium cells treated with neutral lipid stain NR colocolized with the GFP signal confirming that MPL1 is binding to LDs
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Introduction
Discussion
:star: Phosphorylation of perilipin by cAMP dependent protein kinase results in the conformational change that initiates lipolysis by hormone-sensitive lipase.
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:star: MPL1 operates in a perilipin-like manner by localizing to lipid droplets and modulating the rate of hydrolysis.
:star: The functional demonstration of a role for Mpl1 in virulence to insects is important for understanding the molecular and biochemical basis of pathogenicity and should be relevant to innovating new measures of pest control.
:star: The appressoria of the rice blast fungus Magnaporthe grisea also take up water to generate turgor for mechanical penetration of the host surface.
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:star: A gene (Mpl1) in the economically important insect fungal pathogen Metarhizium anisopliae that has structural similarities to mammalian perilipins.
:star: Mpl1 is predominantly expressed when M.anisopliaeis engaged in accumulating lipids and ectopically expressed green fluorescent protein-tagged MPL1(Metarhiziumperilipin-likeprotein) localized to lipid droplets.
:star: Mutant M. anisopliae lacking MPL1 have thinner hyphae, fewer lipid droplets, particularly in appressoria (specialized infection structures at the end of germ tubes),and a decrease in total lipids.
:star: Mpl1 therefore acts in a perilipin-like manner suggesting an evolutionary conserved function in lipid metabolism.
:star: Blast searches of fungal genomes revealed that perilipin homologs are found only in pezizomycotinal ascomycetes and occur as single copy genes.
:star: Expressionof Mpl1 in yeast cells, a fungus that lacks a perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions.
