The
ability of Agrobacterium to transform plants and other
organisms is under highly regulated genetic control. Two Virulence (Vir)
proteins, VirA and VirG, function as a two-component regulatory system to sense
particular phenolic compounds synthesized by wounded plant tissues. Induction
by these phenolic compounds, in the presence of certain neutral or acid sugars,
results in activation of other vir genes, leading to the
processing of T-DNA from the Ti-plasmid and transfer of T-DNA to recipient host
cells. Many plants, and most nonplant, species do not provide sufficient
quantities of the correct phenolic compounds to permit efficient Agrobacterium-mediated
genetic transformation to occur. In order to transform these species, phenolic
inducing compounds must be added to agrobacteria before and/or
during cocultivation of recipient cells with the bacteria.
The discovery that the host range of A. tumefaciens could
be extended to include fungi provided an efficient transformation tool for
species in which it was previously impossible to conduct molecular genetics
experiments. ATMT experiments can be divided into three groups: i) Forward
genetics (i.e., random mutagenesis), ii) Reverse genetics (i.e., targeted
genome modification and random integration) and iii) the introduction of
reporter genes (e.g., GFP, RFP and GUS) that allow in situ monitoring of the
fungus. The use of ATMT for forward genetics experiments has primarily included
classic random insertional inactivation strategies to obtain loss-of-function
mutants. For reverse genetics experiments, ATMT has been used to introduce
targeted genome modifications (e.g., disruptions, replacements, overexpression
and complementation) and to generate random integrations for complementation,
heterologous expression, and expression of transcriptional and translational
fusion reporters and RNAi-mediated down-regulation of gene expression.