Distribution and sub-cellular localization of the aflatoxin enzyme versicolorin B synthase in time-fractionated colonies of Aspergillus parasiticus.

Aflatoxins are highly toxic and carcinogenic fungal secondary metabolites. At least 18 enzyme activities are required for aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus. One of these enzymes, versicolorin B synthase (VBS), catalyzes bisfuran ring closure in versiconal hemiacetal (a reaction near the middle of the pathway) to form versicolorin B. This reaction is required for the subsequent activation to aflatoxin B1-8,9 epoxide, a highly reactive and toxic aflatoxin metabolite, and is important for aflatoxin toxicity. We analyzed the localization of VBS in the aflatoxin-producing strain A. parasiticus SU-1 grown on solid media using a colony fractionation technique developed previously. A highly specific polyclonal antibody, raised against a maltose-binding protein-VBS fusion protein synthesized in Escherichia coli, was used to detect VBS in SU-1 grown on a rich solid medium via immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold transmission electron microscopy (TEM). VBS was detected in both vegetative hyphae and in asexual developmental structures, called conidiophores. Western blot and CLSM analyses demonstrated the highest abundance of VBS in colony fraction S2 consisting of cells that had grown for 24-48 h; this fraction also contained the highest levels of newly developed conidiophores and the highest abundance of aflatoxin B1, consistent with VBS abundance. At the subcellular level, CLSM and TEM detected VBS distributed throughout the cytoplasm and concentrated in ring-like structures surrounding nuclei. It is uncertain whether enzymatically active VBS is present in either or both locations.

Fluorescence shell: a novel view of sclereid morphology with the Confocal Laser Scanning Microscope.

Confocal Laser Scanning Microscopy (CLSM) was used to observe sclereids from stems of Avicennia germinans and from fruits of two species of pear (Pyrus calleryana "Bradford" and P. communis "Red Bartlett"). The images obtained from thick (25 to 100 microm) free-hand sections were, in certain respects, far superior to those obtained by other, more invasive and time-consuming microscopic techniques upon which previous reports of sclereid morphology were based. The cell wall surfaces, including the "internal" surfaces of the branched pit canals and cell lumens, were much accentuated with the techniques we describe, resulting in a "fluorescence shell" image, meaning the cell wall did not stain all the way through but instead only at the inner and outer wall surfaces, including the edges of ramiform pits. By controlling the time of staining with 1% aqueous Safranin O, or by changing the number of optical sections used in extended focus images, it was possible to get either a conventional view of the cell wall structure or a unique, three-dimensional view of the elaborate cell interconnections. Similar fluorescence shell images of sclereids were also obtained using a periodic-Schiff (PAS) staining system, but the stain was not as specific to sclereid cell walls as was the Safranin O stain. Particularly with the use of a narrow range band pass emission filter of 505-530 nm, the Safranin O staining may be more specific to lignin than reported in the literature.