and RNA gel blots the complex expression pattern during plant development is documented. In this
way several decisive promoter regions could be identified.
Due to its pathogen inducible activation pattern the promoter may be well suited to control
expression of genes with anti-pathogenic pro- perties in transgenic crops.
2. Methods
2
.
1
. Plasmid constructions All DNA manipulations, including restriction
digests, agarose gel electrophoresis, ligation and transformation to Eschericha coli DH5a were car-
ried out according to Sambrook et al. [4]. The genomic clone harbouring a 4027 bp fragment of
the A. thaliana ppyk20 [3,1] was used for engineer- ing seven different promoter::uidA constructs A –
G. All ppyk20 fragments were produced by PCR using oligonucleotide primers including a Xho I
site 3 for the reverse primers and a Sma I site 5 for the forward primers Table 1. After PCR
with the Pfu thermostable DNA polymerase Stratagene GmbH, Heidelberg, Germany, the
PCR products were restricted with Xho I and Sma I Promega, GmbH, Mannheim, Germany and
cloned into the binary pMOG819 vector [3] be- tween the uidA gene and the left border of the
T-DNA at the corresponding sites.
2
.
2
. A. thaliana transformation The ppyk20::uidA constructs A – G in the bi-
nary vector pMOG819 were mobilized from E. coli DH5a into the A. tumefaciens strain MOG101
[5] by triparental mating, using the helper plasmid pRK2013 in E. coli DH5a. Roots of A. thaliana
ecotype C-24 were transformed, regenerated and selected according to Valvekens et al. [6]. For each
construct at least 25 independent transgenic A. thaliana plants were regenerated.
2
.
3
. Histochemical localisation of GUS acti6ity GUS activity was histochemically detected by
staining following the method as described [7] using a solution of 2 mM 5-bromo-4-chloro-3-in-
dolyl-b-
D
-glucuronid acid
Biomol, Hamburg,
Germany in 0.1 M sodium phosphate pH 7.0, 0.1 Triton-X 100, 0.5 mM K
3
[FeCN
6
], 0.5 mM K
4
[FeCN
6
], 10 mM Na
2
EDTA and incubated overnight at 37°C. After staining, chlorophyll was
extracted from photosynthetic tissues with 70 vv ethanol. The GUS expression was detected
microscopically by the distinct blue colour which results from the enzymatic cleavage of X-gluc. Of
the 25 produced transgenic lines one representative was selected and analysed optically in detail.
2
.
4
. Fluorometric determination of GUS acti6ity Sixty individuals of the selected transgenic
plants line were were grown in three Petri dishes and harvested as one sample. Proteins were ex-
tracted by grinding material in extraction buffer 50 mM NaH
2
PO
4
pH 7.0, 10 mM EDTA, 0.1 Triton-X 100, 0.1 sarcosyl, 10 mM b-mercap-
toethanol. The protein concentration was esti- mated by the Bradford method [8]. GUS enzyme
activity was determined according to the proce- dure of Jefferson et al. [9] by measuring the
fluorescence emitted by 4-methylumbelliferone, a GUS cleavage product from methyl-4-umbel-
liferyl-b-
D
-glucuronide. The specific activity of GUS enzyme in the extracts was calculated as
nanomoles of 4-methylumbelliferone produced per minute per mg total protein.
Table 1 Primers used for the creation of the ppyk20 constructs A–G
Primer
a
Sequences 5 3 GAATTCTCGAGAAGACATGAGACA-
Pyk20-A REV
AT CTCTCCCGGGCACTCACAATTCACA
Pyk20-A FOR
GTTACTCGAGGTATCACGAAAATGT Pyk20-B REV
Pyk20-B FOR
CTCTCCCGGGCACTCACAATTCACA TAAGCTCGAGCGTAGTTGCATTTTA
Pyk20-C REV Pyk20-C FOR
CTCTCCCGGGCACTCACAATTCACA CATTCTCGAGGTTGGACCGGCTCTGT
Pyk20-D REV
G CTCTCCCGGGCACTCACAATTCACA
Pyk20-D FOR Pyk20-E REV
GTTACTCGAGGTATCACGAAAATGT ACGACCCGGGTCAATAAAACAAACC
Pyk20-E FOR
CAC
Pyk20-F REV TAAGCTCGAGCGTAGTTGCATTTTA
Pyk20-F FOR ACGACCCGGGTCAATAAAACAAACC
CAC TAAGCTCGAGCGTAGTTGCATTTTA
Pyk20-G REV Pyk20-G FOR GTTTCCCGGGTACTAATCGAGAAACA
a
REV, the reverse primers; FOR, the forward primers.
2
.
5
. Nematode and nematode infection assays Second-stage juveniles J
2
of H. schachtii cul- tures were harvested from in vitro stock cultures
on mustard Sinapis alba roots on 0.2 Knop medium [10]. Hatching was stimulated by soaking
cysts on a 100 mm nylon sieve in 3 mM ZnCl
2
. The J
2
juveniles were washed for four times in sterile H
2
and resuspend
in 0.5
Gelrite before
inoculation. Ten-day-old plant roots were inoculated under
axenic conditions with a batch of 30 4-day-old hatched J
2
juveniles of H. schachtii. The plants were examined for the presence of GUS activity 2,
4, 7 and 12 days after inoculation.
2
.
6
. Wounding The leaves of the 21 days old transgenic A.
thaliana plants and wild type plants were wounded by scissors and by pipette tips. Wounded trans-
genic plants were harvested after 1 or 5 h and submitted to histochemical GUS staining as de-
scribed above.
2
.
7
. Plant hormone treatments A. thaliana plants were grown under normal
growth and light conditions before hormonal treatment. For ABA and IAA, 20 in-vitro grown
A. thaliana plants were sprayed with 2 ml of 50 mM ABA Sigma, Deisenhofen, Germany or 50
mM IAA Sigma in water, respectively. As a control, A. thaliana plants were sprayed with 2 ml
water. 8 h after initiation of these treatments, A. thaliana plants were sampled, frozen in liquid ni-
trogen and stored at − 80°C until further use.
2
.
8
. RNA isolation and RNA gel blot analysis Total RNA from A. thaliana plants was isolated
as described by Gurr and McPherson [11] with an additional chloroform extraction step. For North-
ern hybridisation, 30 mg of total RNA was dena- tured,
fractionated on
a 1.5
agarose – formaldehyde gel, and blotted onto Qiabrane
®
membrane Qiagen GmbH, Hilden, Germany ac- cording to the manufacturer’s instructions. The
pyk20 cDNA clone [1] was used as a probe. To standardize the amounts of RNA loaded, 30 mg of
total RNA was short fractionated as described above and then hybridized with 18s rDNA from
sugar-beet [1]. The probes were labelled using ran- dom primers and [a-32P] dATP and [a-32P] dCTP
Amersham Buchler GmbH Co. KG, Braun- schweig, Germany. Immobilized nucleic acids
were prehybridized in solution containing 5 vv Denhart’s solution, 5 vv SSPE, 0.2 wv
SDS, 100 mgml denatured herring sperm DNA at 50°C for 6 h. The random-primed probe was
added to the prehybridisation solution and incu- bated for 14 – 18 h at 50°C in a hybridisation oven.
The membranes were washed once at 50°C for 20 min in 4 × SSC, 0.1 SDS, then twice for 15 min
in 1 × SSC, 0.1 SDS, and exposed to Hyperfilm MP
®
Amersham.
2
.
9
. RT-PCR Total RNA ca. 2 mg of wounded leaves were
used in a RT reaction according to the manufac- turer’s instructions Promega. 50 ng Oligo dT-
Primer and 20 U M-MLV reverse transcriptase Promega in a total volume of 20 ml were incu-
bated for 60 min at 42°C. Ten microliters were used in a PCR reaction containing 2 mM MgCl
2
, 10 mM Tris – HCl, pH 8.3, 33 mM KCl, 0.2 mM
dNTPs, 50 nM of each primer and 2.5 U Taq – Polymerase Promega. The following primers
were used for the reaction: Pyk20-1: 5 – CACAG- CATGATCAGAGGA-3;
Pyk20-2: 5-TAC-
CATTGGTGTAGGCAT-3. The following PCR conditions were employed: 35 times 45 s at 95°C,
45 s by 55°C, 1 min 74°C. Ten microliters of the PCR products were separated on a 1.5 agarose
gel.
3. Results
