Biomedical Engineering Reference
In-Depth Information
at 28°C at 200 rpm on an orbital shaker. Bacterial cells and the conidial
suspension of the fungus were mixed (1:1) and 200 µl of this solution
was plated on nitrocellulose membrane in the co-cultivation medium
(CM). After 36 to 48 hr of incubation in the dark at 28°C, the membranes
were transferred to PDA (for
V. dahliae
) or Aspergillus minimal medium
(for
M. oryzae
) containing cefotoxime and hygromycin and incubated at
room temperature. Typically, transformants appeared after 5-7 days of
incubation. Conidia of the individual transformants were harvested and
re-suspended in sterile distilled water (SDW) and plated on water agar
to obtain mono-conidial cultures by transferring a single germinating
spore into a Petri plate containing PDA or oatmeal agar (OMA) media.
Transformants were maintained on PDA (for
V. dahliae
) or OMA (for
M.
oryzae
) for use in ongoing work. The transformation effi ciency or the copy
number of T-DNA in individual transformants varied among these two
fungal species (Table 1). Duration of the co-cultivation period also differed
between these two fungi in relation to the transformation effi ciency.
The T-DNA insertion sites in
M. oryzae
and
V. dahliae
transformants
were identifi ed either by thermal asymmetrical interlaced PCR (TAIL-PCR)
or by inverse PCR (iPCR) (Mullins et al. 2001, Choi et al. 2007, Ochman et
al. 1988, Maruthachalam et al. 2011a). For TAIL-PCR, the T-DNA border-
specifi c primers (left border (LB) and right border (RB)) and arbitrary
degenerate (AD) primers were used. But for iPCR amplifi cation, RB3 and
RBn1 primers were used. After PCR amplifi cation, products were treated
with EXOSAP-IT to eliminate unincorporated primers and sequenced using
border specifi c primers (LB3 or RB3). Obtained sequences were used as
queries to search the Verticillium Group and
Magnaporthe grisea
databases at
the Broad Institute (
http://www.broadinstitute.org/annotation/genome/
verticillium_dahliae), (
http://www.broad.mit.edu/annotation/fungi/
magnaporthe) and the National Center for Biotechnology Information
(NCBI) websites (
www.ncbi.nlm.nih.gov/Genbank/)
via the Blastn and
Blastx algorithms.
Table 1.
Agrobacterium tumefaciens
transformation in
Magnaporthe oryzae
and
Verticillium dahliae.
Fungal
species
Agrobacterium
strains
Hygromycin
concn.
Suitable
co-
cultivation
time (hr)
Percent of
single copy
T-DNA
insertion
Reference
M. oryzae
AGL-1
200 µg/ml
48
60
Rho et al.
2001
V. dahliae
EHA105
50 µg/ml
36
69
Maruthachalam
et al. 2011a
