The Metarhizium anisopliae Perilipin Homolog MPL1 Regulates Lipid Metabolism, Appressorial Turgor Pressure, and Virulence

Abstract

Cells store lipids in droplets. Studies addressing how mammals control lipid-based energy homeostasis have implicated proteins of the PAT domain family, such as perilipin that surrounds the lipid droplets. Perilipin knock-out mice are lean and resistant to obesity. Factors that mediate lipid storage in fungi are still unknown. Here we describe a gene (Mpl1) in the economically important insect fungal pathogen Metarhizium anisopliae that has structural similarities to mammalian perilipins. Consistent with a role in lipid storage, Mpl1 is predominantly 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. 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. Mpl1 therefore acts in a perilipin-like manner suggesting an evolutionary conserved function in lipid metabolism. However, reflecting general differences between animal and fungal lineages, these proteins have also been selected to cope with different tasks. Thus, turgor generation by ⌬Mpl1 appressoria is dramatically reduced indicating that lipid droplets are required for solute accumulation. This was linked with the reduced ability to breach insect cuticle so that Mpl1 is a pathogenicity determinant. Blast searches of fungal genomes revealed that perilipin homologs are found only in pezizomycotinal ascomycetes and occur as single copy genes. Expression of Mpl1 in yeast cells, a fungus that lacks a perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions. Eukaryotic cells contain droplets of triglycerides encased in phospholipid membranes. These lipid droplets (LDs) 3 were once considered to be just inert storage vessels for energy-rich fat, but recent studies have shown they have additional roles in maintaining membranes and moving components around cells. The lipid droplets have also been implicated in lipid diseases, inflammation, diabetes, cardiovascular disease, and liver disease (1). Consistent with fat droplets being metabolically active, the membranes encasing them have proteins with wide ranging biochemical activities. The best studied are mammalian proteins of the perilipin family (also called the PAT family) such as perilipin, adipocyte differentiation-related protein, and TIP47. These proteins have a characteristic series of hydrophobic sequences (the PAT domain) that facilitate their localization to the surface of lipid droplets. By coating droplets, perilipin forms a barrier that restricts the access of cytosolic lipases. During food deprivation, perilipin is phosphorylated by protein kinase A; the barrier function of perilipin is attenuated, and lipolysis increases (2). In comparison with normal mice, perilipin-deficient mice have less fat, more muscle, a higher metabolic rate, and are resistant to diet-induced and genetic obesity (3...

Linkage of autophagy to fungal development, lipid storage and virulence inMetarhizium robertsii

“…To some extent, it is similar to the reduced virulence of M. robertsii with deletion of the perilipin-like protein gene, MPL1, that leads to decreased turgor pressure within the appressoria. 27 However, in our injection assays that bypass insect cuticle Mratg8Δ showed a significantly reduced virulence while Mpl1Δ killed insects like the WT strain. 27 This could be due to the inability of Mratg8Δ to counteract insect defense responses (Fig.…”

“…27 However, in our injection assays that bypass insect cuticle Mratg8Δ showed a significantly reduced virulence while Mpl1Δ killed insects like the WT strain. 27 This could be due to the inability of Mratg8Δ to counteract insect defense responses (Fig. 9).…”

“…26 We previously identified a mammalian perilipin-like protein Mpl1 that regulates LD storage, appressorium turgor pressure and virulence in Metarhizium. 27 To investigate whether the impaired LD accumulation in Mratg8Δ is connected to MPL1 gene transcription and protein expression, we performed RT-PCR and western blot analyses. Interestingly, the transcription of MPL1 in Mratg8Δ was not significantly different when compared with the WT and Comp (Fig.…”

“…The wild-type strain and mutants of M. robertsii ARSEF 2575 (previously named as M. anisopliae) were routinely cultured on potato dextrose agar (PDA, Difco, 213200) at 25°C. For characterization of growth, fungi were additionally cultured on a minimum medium (MM) 27 and a complete medium (CM). 24 For liquid incubation, fungi were grown in Sabouraud dextrose broth (SDB, Difco, 238220) and MM-N (MM without the nitrogen source and agar).…”

“…The resultant vector pYES-MrATG8 was then transformed into the yeast KVY5 strain using a small-scale yeast transformation protocol. 27 The uracil prototrophic transformants were selected and verified by PCR. The complementation effect of Metarhizium MrATG8 gene was tested in a starvation challenge as described previously.…”