Biomedical Engineering Reference
In-Depth Information
based on homologous recombination to achieve gene disruption and gene
replacement with greater precision and effi ciency. In addition, utilization
of transformation methods for molecular characterization of genes and
availability of classical genetics studies placed many species of ascomycetes
including
Neurospora crassa
,
Aspergillus nidulans
and
Magnaporthe oryzae
as
model organisms. Several transformation methods such as protoplast with
polyethylene glycol (PEG) (Meyer et al. 2003), electroporation (Robinson
and Sharon 1999) and restriction enzyme-mediated integration (REMI)
(Tanaka et al. 1999, Balhadere et al. 1999, Redman et al. 1999, Thon et al.
2000) are being employed to understand the genetic basis of pathogenicity
of fungal plant pathogens (Olmedo-Monfi l et al. 2004, Michielse et al. 2005).
In contrast to other insertional mutagenesis techniques such as REMI,
Agrobacterium tumefaciens
-mediated transformation (ATMT) does not require
protoplasts and has a choice of starting materials such as conidia, hyphae
and blocks of mycelia from fruiting body (Chen et al. 2000, Stone
et al.
2000, Amey et al. 2002, Meyer et al. 2003, Michielse et al. 2005) to choose
for transformation. In addition, ATMT method has higher transformation
effi ciency than other transformation techniques described so far.
Agrobacterium tumefaciens
-mediated transformation has long been used
to produce transgenic plants. An array of transgenic plants have been
generated in
Arabidopsis thaliana
(Ostergaard and Yanofsky 2004) as also in
many agriculturally important crops including rice (Hirochika et al. 2004)
for functional genomic studies. With the demonstration of the expansion
of host range of
A. tumefaciens
to include the budding yeast,
Saccharomyces
cerevisiae
to create random insertional mutagenesis (Piers 1996), the utility
of this technique was subsequently extended to other fi lamentous fungi
including many phytopathogenic fungi (Bundock et al. 1995, de Groot et
al. 1998, Chen et al. 2000, Rho et al. 2001, Mullins et al. 2001, Michielse et al.
2005). ATMT also has been widely used for targeted mutagenesis in fungi
(Michielse et al. 2005, Bhadauria et al. 2009). Furthermore, with targeted gene
deletion experiments, high frequency of homologous recombination was
produced, indicating the effectiveness of ATMT for targeted mutagenesis
(Michielse et al. 2005, Bhadauria et al. 2009).
Verticillium
spp. cause Verticillium wilt in many economically important
crops worldwide and cause billions of dollars crop losses annually. So far
more than 400 plant species have been reported as hosts for this
Verticillium
spp. In
Verticillium
, conidia or microsclerotia germinate and initiate the
Verticillium wilt disease cycle. In the absence of suitable environment
or host, the microsclerotia can stay in the soil for up to 14 years (Pegg
and Brady 2001). When environmental conditions are favorable, the
microsclerotia germinate and produce hyphae that colonize the host roots
that subsequently leads to symptom development
(Fig. 1).
