plant surface microbiology.pdf

hiza. Cells with well-preserved cytoskeletal structures can then be examined
either by a regular fluorescence microscope or by a laser scanning confocal
microscope.
In ectomycorrhiza, the thick hydrophobic sheath around the root formed
by fungal hyphae inhibits the penetration of the fixative or rapid freezing of
the fungal and plant cells. The fixation of ectomycorrhizal root in situ under
vacuum facilitates and speeds up the penetration of the fixative into the ectomycorrhizal
root (Timonen et al. 1993; Niini and Raudaskoski 1998). In ectomycorrhiza
it is necessary to prepare sections from the fixed roots and until
now, the best results in immunolocalization of cytoskeletal elements have
been achieved by using cryosections (Fig. 1A, B; Niini and Raudaskoski 1998).
No success in the visualization of MTs or MFs has yet been achieved by
embedding the Pinus short roots or ectomycorrhizal roots in wax.
9.2 Microinjection Method
Microinjection of fluorescent-labeled phalloidin or tubulin has been used to
visualize MFs (Schmit and Lambert 1990; Zhang et al. 1993; Cleary 1995; Kim
et al. 1995; Miller et al.1996) and MTs (Zhang et al. 1990; Yuan et al. 1994) in
living plant cells. However, the visualization of cytoskeletal elements succeeds
with microinjection only in a few plant cell types, such as stamen hair cells of
Tradescantia (Zhang et al. 1993), or epidermal cells of leaves (Yuan et al. 1994),
while cytoskeletal elements in narrow fungal hyphae are not easily studied
with this method.
9.3 Green Fluorescence Protein Technique
18 Mycorrhizal Development and Cytoskeleton 317
The MFs and MTs in plant and fungal cells have been successfully visualized
with the help of green fluorescence protein (GFP) fused to actin, tubulin or to
a cytoskeleton-associated protein. In plant cells, tubulin-GFP fusion protein
has been used to visualize MTs in different Arabidopsis tissues (Ueda et al.
1999; Whittington et al. 2001). Transient expression of the MT binding
domain of mammalian MAP4 labeled with GFP and transgenic plants with
the same construct have been used for visualization of the MT organization in
epidermal cells of fava bean (Marc et al.1998) and in Arabidopsis root cells
(Bao et al. 2001). Fusion of GFP to the actin-binding domain of talin has led to
visualization of actin cytoskeleton transiently in tobacco BY-2 suspension
cells (Kost et al. 1998) and pollen tubes (Kost et al. 1998, 1999a, b; Fu et al.
2001). Constitutive expression of the same construct visualized actin
cytoskeleton in different tissues of Arabidopsis including root hairs (Kost et
al. 1998; Baluška et al. 2000; Baluška and Volkmann 2002). Recently, both MTs
and MFs were visualized in living onion epidermal cells by using the MT