plant surface microbiology.pdf

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Thomas F.C. Chin-A-Woeng et al.
tors have been described that enable routine production of GFP,YFP and CFP
fusions in Gram-positive bacteria.
One disadvantage of the use of fluorescent proteins is the maturation time
of the protein, particularly that of DsRed. Although EGFP requires ~ 4 h for
efficient microscopic visualisation, visualisation of DsRed requires longer
periods. This delay is not due to inefficient expression of the DsRed protein
since the protein can be detected in high quantities very soon, but it is rather
due to an extended maturation time of the protein (20–48 h). DsRed is in fact
brighter than first reported, but the fluorescence matures very slowly and the
protein naturally forms a tetramer. More rapidly maturing and soluble variants
of DsRed have been generated by mutagenesis (Brooke and Glick 2002).
Furthermore, E. coli cells expressing DsRed protein are in general smaller
than cells expressing EGFP or untransformed bacteria, indicating that DsRed
might have a toxic effect. Another problem with the use of fluorescent proteins
is the variability of expression in different bacterial species. GFP
expressed from the same constructs is two to ten times higher expressed in E.
coli than in pseudomonads. Interference by other fluorescent particles, bacteria,
or root autofluorescence may also introduce artefacts or complicate the
observations.
5.3 Confocal Laser Scanning Microscopy of Rhizosphere Interactions
The advent of fluorescent proteins offers a broad range of applications to
track bacteria and study gene expression in the rhizosphere. By labelling different
strains with different flavours of fluorescent proteins such as green, red,
blue, or yellow fluorescent protein, multiple bacterial strains and their interactions
with pathogens can be tracked simultaneously in the rhizosphere.
To express gfpin F.o.r.l., pGFDGFP on which the sgfp gene is cloned between
the A. nidulans gpdA promoter and the trpC terminator sequences was transformed
to F.o.r.l.. The fungus was transformed by the previously described
polyethyleneglycol/CaCl 2 -mediated transformation of protoplasts in the presence
of pAN7–1, which allows selection for hygromycin B resistance
(100 mg/ml). The level of gfp expression was high in the mycelium, micro- and
macroconidia, and chlamydospores. The labelled isolates were equally pathogenic
to tomato as the wild type. The marked fungus was introduced into the
gnotobiotic sand system by mixing spores with sand. First, the interactions
between fungal pathogens and the tomato root were studied. CLSM observations
show that after 2 days the main root is surrounded by hyphae, which are
interwoven with the root hairs. The contact between hyphae and the root was
initiated at or via the root hairs.After 3 days,spot attachments of hyphae to the
root surface are observed, predominantly at the crown and hyphae grow along
the junctions of the epidermal cells after attachment. The first infection events
take place 4 days after inoculation, as observed by penetration of epidermal