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When setting up
Agrobacterium mediated transformation for a fungus for which no protocol
exists, the following check list most likely will help the process
Initial tests
- Establish whether the target fungus is susceptible to the antibiotic
(or other selection marker) you are planning to use and at what concentrations the antibiotic is
active. It is a good idea to use the same growth medium that you plan to
use during the different selection rounds during ATMT, as the activity of
many antibiotics depend on the used medium.
- Test that the filter papers you plan to use does not inhibit fungal
growth and that the fungus is able to grow though the filters and into the
underlying solid medium.
Transformation conditions
Many factors can affect the transformation frequency, ranging from
co-cultivation period, temperature, Agrobacterium tumefaciens
strain, vector, selection marker gene, promoter that drives the selection
marker, co-cultivation medium, acetosyringone concentration (inducer of
virulence system in A. tumefaciens) and the fungal
material you are trying to transform.
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Starting material: One
of the great advantages with ATMT is that it can be performed on most types of
cellular material, ranging from sexual spores, asexual spores, germinating
spores, protoplasts, fragmented and intact mycelium. This means that the
technique can be used for most culturable fungi. In most cases spores are
preferred as they generally are easy to handle and quantify, however it of
cause depends on the system one is working with. The number of cells
used for the co-cultivation can affect the growth rate and the germination
frequency of the fungal cells, as high spore densities
in many
cases decrease the germination frequency. The developmental and growth
stage of the used cells can also affect the transformation frequency and
a short pregermination (few hours)
of the spores is often an advantage. |
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Agrobacterium strains:
The different available vectors are generally designed to work with a
specific Agrobacterium strain, however sometimes trying new combinations
of vectors and strains will yield higher transformation frequencies. The
standard At strains LBA4404, AGL-1 and EHA105 is in most instances sufficient for standard transformation of fungi, however newer strains with
multiple copies of the significant virulence genes have been developed for plant
transformation and can also be used for fungi. |
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Selection marker and
promoter: It is of course very important that the selection marker gene
you use is able to protect the resulting transformants against the
antibiotic you are using for killing the non-transformed fungal cells. It
is of equally importance that the promoter and terminator used for
controlling expression of the selection marker gene is functional in the
fungus type you are trying to transform. The cis acting elements of ascomycetes and basidomycetes can vary greatly,
and in some cases it can be necessary to stabilize the transcript by
adding an intron. All available vectors (that I am aware of)
depends on a constitutive promoter for driving expression of the selection
marker gene, however this can have a pronounced effect on the fitness (growth
rate) of the resulting transformants due to the energy that goes into
constructing and maintaining the resistance machinery. Why it is always
advisable to use a "wild type" (non-deletion strain) that expresses the
selection marker gene for comparative studies, or to remove the selection
marker gene from the deletion strains following confirmation of the strain.
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Co-cultivation time: The
co-cultivation time can have a huge impact on the transformation frequency
from fungus to fungus. This is most likely due to the difference in the
speed by which the fungal material germinates and proliferates. For
Fusarium sp. we normally co-cultivate between 48-60 hours, we have not
experienced that prolonging the co-cultivation period has had a negative
effect on the transformation efficiency, however this has been reported for
other systems. |
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Co-cultivation temperature:
The co-cultivation step is a trade-off between providing the best
conditions for the bacterium to poliferate and form the conjugation pilus
and the best conditions for growth of the target fungus. For Fusarium
I normally incubate the plates at temperatures between 26 and 28
oC.
The growth optimum for most A. tumefaciens strains is 28 oC, while
Fusarium grow best at 25 oC. |
If you are not successfull in transforming your target fungus with a
non-targeted vector construct (the empty vector), which will depend on
integration by non-homologous end-joining DNA repair pathway, a possible
explanation could be that the fungus you are working with is impaired in
this pathway. This situation has been described for Fusarium fujikuroi,
where transformants can only be obtained if the vectors includes a piece of
the hosts DNA. We have experienced a similar effect in Fusarium
graminearum, but not in Fusarium culmorum or Fusarium
pseudograminearum. In this situation you should try to use a vector
constructed for targeted integration into the genome (remember not to target
an essential gene).
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