Panellus stipticus is one of several dozen species of fungi that are bioluminescent. Strains from eastern North America are typically bioluminescent, but those from the Pacific regions of North America and from other continents are not. The luminescence is localized to the edges of the gills and the junction of the gills with the stem and cap. Bioluminescence is also observable with mycelia grown in laboratory culture, and the growth conditions for optimal light production have been studied in detail. Several chemicals have been isolated and characterized that are believed to be responsible for light production.Genetic analysis has shown that luminescence is controlled by a single dominant allele. The luminescent glow of this and other fungi inspired the term foxfire, coined by early settlers in eastern and southern North America. Modern research has probed the potential of P. stipticus as a tool in bioremediation, due to its ability to detoxify various environmental pollutants.
pictured above is a tube containing mitochondria isolated from the filamentous fungus and notorious plant pathogen, Fusarium oxysporum.
what is being viewed is a tube with three different layers of different percentage sucrose (53%, 44% and 20%) and two layers of isolated mitochondria; the upper (brownish yellow) and the lower (white/tan). differential sucrose gradient long-phase centrifugation is utilized to separate the fungal mitochondria from the rest of the fungal mass. the complete partially degraded fungal mass gets resuspended in 53% sucrose, and the 44% percent and 20% sucrose gets layered very slowly and carefully as to keep them separate entities. then, the tubes are spun at a 27k RPM for 2 hours and 10 minutes; the photo above is the product of that high speed spin using a swinging bucket steel rotor. the mitochondria is slowly and carefully pipetted out into specified smaller tubes and stored away in a -80C freezer. they can be used for mtDNA isolation, RNP purification and Isolation, among many other things all of which involve lysing the mitochondria.
upon inspection of the liquid fungal growth media for inventory purposes, this is what I found. so we make these giant liquid media to grow our fungi in, and we attempt to seal them firmly and sterilize them via autoclave, but some still get contaminated with stray fungal spores. after not using them or checking them in two weeks, this is what I find. a diverse array of fungi, none of which are the ones we use as models for our research in the lab. some are floaters others are not, some are fairly hydrophobic others are not, and the diversity is amazing!
Mitochondrial Isolation and Purification
one of the main techniques/procedures we do in my lab is isolate and purify mitochondria out of specific strains of fungi.
why? well, these different species, and strains of different species of fungi contain selfish genetic elements, or simply autonomous plasmids. these plasmids are found as separate entities existing and proliferating in the mitochondria of certain strains of fungi.
for example, let’s take the fungus I deal with, Fusarium oxysporum. this species has many strains, but I only deal with three, which are given numbers as well as names. we call them 699 (pFOXC2), 725 (pFOXC3) and 777 (pFOXC1) and each strain contains a different member of the pFOXC family of linear retroplasmids. retroplasmids are called that because they code for a reverse transcriptase, or an RNA dependent DNA polymerase.
let’s get back to the main point of this post before I get sidetracked into talking about my research in specific terms, not like haven’t lost most of you by now, but hey I’m trying here.
so, the conidia (spores if you will) are used to inoculate specific growth media used to grow the fungus, shaking over a period of three to four days. then, the fungus is harvested into a thick pad, which is cut up into little pieces and made into a slushy form via small glass beads. through many steps of resuspension and centrifugation, you get to the final step of differential sucrose gradient mitochondrial isolation via a high powered ultracentrifuge. after that, you carefully pipet your mitochondria out from between the different layers of sucrose (as seen above in the top left picture) and purify your mitochondria out of the sucrose with a special solution. in the end, you get a small pellet which fills up about .5 mls in a 1.5 ml eppendorf tube (top right picture). I start with 5 large flasks for 2 liters of media/fungus (10 liters total) and end up with 8 tubes of mitochondria. it takes a complete day to do.
well, that was pretty much my day today.
someone asked me how we isolate mitochondria in our lab from fungi…here’s the relevant post.