Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

pad3 locus from the Col-pad3 mutant to investigate
whether PAD3 is involved in defence responses
mediated by the RPP1 or RPP13 resistance genes.
Material and methods
Isolates of P. parasitica
All isolates of P. parasitica known to be recognised
by RPP1 and RPP13 genes in accession Nd-1 were
used. These isolates are Emco5, waco5, Maks9,
Aswa1, Goco1, Edco1, Emoy2 and Hiks1.
Interaction phenotypes
The seeds were sown according to Holub et al.
(1994). The seedlings were inoculated as described by
Holub et al. (1994). Seven days old seedlings were
inoculated with appropriate isolate of P. parasitica
and incubated in a growth room. The interaction
phenotypes were recorded at 3 and 7 days after
inoculation (dai) according to host response and
pathogen sporulation. Pitting necrosis on the
cotyledon was recorded as "P" and this was always
associated with no pathogen sporulation, thus it was
abbreviated as "PN". Clorotic flecks (abbreviated as
F) were also seen on the host, but sporulation of the
pathogen may occur although very low. In A.
thaliana-P. parasitica research, pathogen growth was
almost always scaled into five categories according to
pathogen sporulation in each cotyledon (Holub et al.,
1994; Tör et al., 1994; Botella et al., 1998; Bittner-
Eddy et al., 1999). These categories are abbreviated to
letters as follows: "N" is for none sporulation, "R" is
for rare (less than 1 conidiophore for per cotyledon),
"L" is for low sporulation (1-5 conidiophores per
cotyledon), "M" is for medium sporulation (5-20
conidiophores per cotyledon) and "H" is for heavy
sporulation (more than 20 conidiophores per
cotyledon).
Selection of genotypes carrying the RPP1, RPP13
and pad3 loci
The Col-pad3 mutant and the accession Nd-1 plants
were grown to flowering and cross-pollination was
processed. F1 was back-crossed to parent Nd-1 to
obtain more Nd-1 effect. F1 seeds were grown for F2
seeds. All selections were made from these F2 families.
Camalexin and RPP1 and RPP13 resistance genes 95
The Col-pad3 mutant does not accumulate
camalexin following induction by P. parasitica or
abiotic inducer AgNO3 (Mert-Türk et al., 1998;
Mert-Türk, 2001; Mert-Türk et al., manuscript in
preparation). Therefore, the marker for the allele of
pad3 would be the phenotype that does not
accumulate camalexin. Camalexin extraction and
Thin Layer Chromotography (TLC) were carried out
as described by Glazebrook and Ausubel (1994). F2
genotypes were first screened for camalexin
deficiency. The genotypes that did not accumulate
camalexin were labelled that they carried the pad3
allele. Then these genotypes were tested whether they
carry RPP1 and RPP13 genes as well. As the map
position and primers near to the RPP1 and RPP13
genes are known all selection process were made
using Polymerase Chain Reaction (PCR).
DNA isolation
PhyoPure Plant DNA Extraction Kit (Nucleon
Biosciences, UK) was used for DNA isolation. Leaf
samples were harvested and freeze-dried at-60°C for
2 days 0.02 g dried tissue was added to a milling tube
containing two ball bearings and milled for 10 min.
The powder was transferred to a 1.5 ml sterile
Eppendorf tube and DNA was extracted as
Manufacture’s advice.
Polymerase Chain Reaction (PCR)
The amplification reaction were carried out in a
thermocycler. Two specific primers, one from each
end of the DNA fragment to be amplified, were used
for selective amplification. The primers
encompassing the RPP1 and RPP13 resistance genes
are shown in Table 1.
PCR reactions were carried out in 25 µl volumes
that contained 2.5 µl 10X PCR buffer, 1 µl of 5 mM
dNTP (equal mixture of four), 1 µl of 50 mM MgCl2,
1 µl of each of 10 µM primers and 1 µl of DNA which
is about 25-50 ng and 0.2 µl Taq DNA polymerase.
Conditions for amplification were as follows: 1 min at
95°C for 1 cycle, denaturation at 95°C for 30 seconds,
annealing 30 seconds at 50-58°C dependent on the
primers used, and polymerisation at 72°C for 2 min.
This cycle was repeated 35 times. A final cycle was 2
min at 72°C for the uncompleted cycles. The
reactions were stopped by chilling to 4°C.