Book of Abstracts (PDF) - International Mycological Association

IMC7 Thursday August 15th Lectures
to monitor dynamics of organelles and the cytoskeleton
itself in Neurospora crassa and Ustilago maydis. These
experiments have revealed remarkable levels of motility
within the fungal cell and it has become obvious that
intracellular motions are essential for spatial organization,
growth and morphogenesis of fungi.
268 - Using light and electron microscopy to explore
hyphal cytoplasmic order, behavior and mutation mode
of action
R.W. Roberson 1* & S. Bartnicki-García 2
1
Arizona State University, Department of Plant Biology
2
Tempe, Arizona 85287-1601, U.S.A. - Centro de
Investigación Científica y de Educación Superior de
Ensenada, 22830, Baja California, Mexico. - E-mail:
robert.roberson@asu.edu
To assess the impact of single mutations on cell
morphogenesis, it is essential to explore the effect of the
mutation on cytoplasmic organization and behavior. Video
microscopy provides a real time record of organelle
behavior and morphological changes in living cells. Such
images can provide clues about the mode of action of a
mutation that can be tested by transmission electron
microscopy of sectioned specimens. One good example is
in the study of the ro-1 and nudA mutations in hyphae of
Neurospora crassa and Aspergillus nidulans, respectively.
These genes encode subunits of cytoplasmic dynein. In
wild-type cells a well-defined Spitzenkörper (Spk)
dominated the cytoplasm of the hyphal apex. Vesicles
exhibited active motility in subapical regions. Hyphae
contained abundant microtubules (MT) that were mostly
aligned parallel to the growing axis of the cell.
Mitochondria and nuclei maintained a near constant
position in the advancing cytoplasm. Dynein deficiency
causes disruption of MT organization and function. Beside
the overall perturbation to cytoplasmic organization and
organelle motility, these mutations disrupt the organization
and stability of the Spk, which, in turn, leads to severe
reduction in growth rate and altered morphology. The
combined use of light and electron microscopy has lead to
a more complete understanding of MT disruption and other
cytoplasmic phenotypes that result from dynein deficiency.
269 - Four-dimensional laser scanning microscopy of
fungal plant pathogens expressing fluorescent proteins
R.J. Howard 1* , T.M. Bourett 1 , J.A. Sweigard 2 , K.J.
Czymmek 3 & K.E. Duncan 1
1
DuPont Crop Genetics, Exp Stn, Wilmington, DE 19880-
2
0402, U.S.A. - DuPont Crop Genetics, Delaware
Technology Pk, Newark, DE 19716, U.S.A. - 3 University of
Delaware, Department of Biological Sci, Newark, DE
19716, U.S.A. - E-mail: richard.j.howard@usa.dupont.com
86
Book of Abstracts
The three-dimensional mapping of host-pathogen cell
interfaces over time, or 4D analysis, represents a
challenging but important prerequisite for understanding
pathogenesis. We generated fluorescent transformants of
two different pathogens, Fusarium verticillioides and
Magnaporthe grisea, and used them for real-time imaging
by confocal and multi-photon microscopy. Driven by
strong constitutive fungal promotors, expression of spectral
variants of green fluorescent protein, as well as the recently
identified reef coral fluorescent proteins derived from
several Anthozoa species, had no detectable effect on either
growth rates or abilities to cause disease. Cytoplasmtargeting
of fluorescent proteins, coupled with fast image
capture rates, allowed discrimination of many subcellular
organelles by differential exclusion and facilitated
monitoring of rapid changes in permeability of the nuclear
envelope. Alternatively, use of fluorescent markers as
fusion proteins, for example with tubulin, made it possible
to image specific proteins/structures/organelles during cell
growth, development, and pharmacological treatment. The
intense brightness of some strains expressing fluorescent
proteins in the cytoplasm permitted documentation of
pathogen cells during invasion of plant host tissues.
AmCyan and ZsGreen reef coral fluorescent proteins were
sufficiently excited at 855 and 880 nm, respectively, to
facilitate time-resolved in planta imaging by two-photon
microscopy.
270 - Studying cell biology of arbuscular mycorrhizal
fungi by combined high resolution biochemical,
molecular biology and microscopy techniques
B. Bago 1* , P.E. Pfeffer 2 , W. Zipfel 3 & Y. Shachar-Hill 4
1 CSIC, CIDE, Cami de la Marjal s/n, Albal (Valencia),
Spain. - 2 USDA, ERRC, 600 E. Mermaid Ln. 19038
Wyndmoor, PA, U.S.A. - 3 Cornell University, Clarck Hall,
Cornell University, Ithaca NM, U.S.A. - 4 NMSU, Dept.
Chemistry and Biochemistry, NMSU, 88033 Las Cruces
NM, U.S.A. - E-mail: berta.bago@uv.es
Glomeromycotan or arbuscular mycorrhizal (AM) fungi
are widespread mutualistic symbionts whose taxonomic
position, ecological distribution, reproductive strategies,
not to mention cell biology is still poorly understood. At
the basis of this lack of knowledge is probably the fact that
these fungi are obligate biotrophs, i.e. they cannot complete
their life cycle unless they have successfully colonized a
host plant. Unsuccessful attempts of culturing AM fungi
axenically have been made in the last forty years, this
greatly hindering progress in our knowledge of these
ecologically important fungi. Moreover, the difficulties to
study the intraradical fungal phase withouth disturbing
symbiosis functioning, and the extraradical mycelium
while maintaining intact their soil-growing hyphae requires
of non-destructive, in situ techniques. In this presentation
we will briefly review recent advances in our understaning
of the cellular biology of these symbiotic fungi. This has
been possible by combining techniques such as monoxenic
AM cultures, NMR spectroscopy, image analysis and
multiphoton microscopy.