A key role for vesicles in fungal secondary metabolism

doi:10.1073/pnas.0907416106

A key role for vesicles in fungal secondary metabolism

^aDepartment of Food Science and Human Nutrition,
^dNational Food Safety and Toxicology Center, and
^eDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824;
^bCenter for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521; and
^cDepartment of Biological Sciences, Northern Illinois University, DeKalb, IL 60115

↵¹Present address: International Environmental Research Center, Gwangju Institute of Science and Technology, Gwangju, South Korea.

Edited by Joan Wennstrom Bennett, Rutgers University, New Brunswick, NJ, and approved September 28, 2009 (received for review July 6, 2009)

Abstract

Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B₁; these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.

Footnotes

²To whom correspondence should be addressed. E-mail: jlinz{at}msu.edu
Author contributions: A.C., L.V.R., and J.E.L. designed research; A.C., L.V.R., S.K., and K.A.A. performed research; G.R.H., N.V.R., and A.M.C. contributed new reagents/analytic tools; A.C., L.V.R., and J.E.L. analyzed data; and A.C., L.V.R., and J.E.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The nucleotide sequence for Aspergillus parasiticus vb1 (avaA) has been deposited in GenBank (accession no. AY52045).
This article contains supporting information online at www.pnas.org/cgi/content/full/0907416106/DCSupplemental.