The intracellular delivery of TAT-aequorin reveals calcium-mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita.
Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi.
Department of Biology, University of Padova, Via U. Bassi 58/B, 35131, Padova, Italy.
Roberto
R
Moscatiello
Simone
S
Sello
Mara
M
Novero
Alessandro
A
Negro
Paola
P
Bonfante
Lorella
L
Navazio
D000331:Aequorin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D004777:Environment; D018014:Gene Transfer Techniques; D055137:Glomeromycota / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D025301:Hyphae / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007783:Lactones / Q000494:pharmacology; D008163:Luminescent Measurements; D038821:Mycorrhizae / Q000187:drug effects / Q000502:physiology*; D010455:Peptides / Q000378:metabolism; D013559:Symbiosis / Q000187:drug effects / Q000502:physiology*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
C507382:GR24 compound; D007783:Lactones; D010455:Peptides; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D000331:Aequorin; D002118:Calcium
Gigaspora margarita; TAT-GFP; TAT-aequorin; arbuscular mycorrhizal (AM) fungi; calcium; cell-penetrating peptides; strigolactones