ATMT of new fungi


Experimental setup
ATMT of new fungi
Molecular biology
Growth media


  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

  1. 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. 
  2. 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.


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.


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.


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.


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.


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).




Home | Experimental setup | ATMT of new fungi | Molecular biology | Growth media

Dette sted blev sidst opdateret 12. July 2010