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
3
.
1
. Structure of the ppyk
20
Using a promoter-tagging approach we previ- ously identified a nematode responsive gene called
pyk
20
[1]. In order to characterise the correspond- ing promoter, ppyk20, 4027 bp 5 to the ATG start
codon sequence of the pyk
20
gene were subdivided into six different regions I – VI. Two of these
promoter regions I and II were characterised earlier for its expression pattern [3].
A database search for cis-elements in the ppyk20 sequence revealed several putative regula-
Fig. 1. Schematic map of ppyk20 with subcloned regions I – VI and putative promoter elements. TSP-transcription start point. CANNTG-box — nematode responsive box [23]. AS-1 — box of the 35SCaMV promoter [21]. Wun-box — wounding-respon-
sive element [16,17]. ABA-box — ABA responsive elements [18,20]. Poly-T — box a stress-responsive element [13,14]. IAA-box — IAA responsive elements [12].
tory sequences homologous to that of known pro- moters Fig. 1.The region with the highest homol-
ogy to
a TATA-box
consensus sequence
5-TATAA-3 starts at the position 457 bp up- stream of the ATG and 169 bp upstream of the
mapped transcription start TSP [1]. DNA se- quence analysis of the 170 bp, between the puta-
tive TATA-box and the transcription start point comprised 80 pyrimidine nucleotides CT. In
addition, no obvious CAAT box was found to be located close to the TATA-box, even on the oppo-
site strand. Twenty-nine sequence motifs with ho- mology to auxin-inducible elements TCTC or
TGTC were located within the promoter [12]. A sequence with homology to the ‘poly-T box’, a
stress-responsive element [13,14], was identified at position – 270 related to the TSP. Six elements
similar to wounding responsive elements [15] were found in regions I, II and IV Fig. 1. Moreover,
in
region V
a sequence
was found
5- TCATCTTCTT-3 that is identical to the TCA
motif of wound- and pathogen-inducible pro- moters [16,17]. Ppyk20 also contains 2 domains
ACGTG with similarities to the ABA responsive promoter domain of the Osem gene from rice [18].
The ACGT element was detected ten times in the ppyk20. This element was identified in many plant
genes and is induced by diverse environmental and physiological factors like ABA treatment [19,20].
On the opposite strand, we found two domains which are complementary to a TGACG domain.
This motif is also included in the As1 domain of the CaMV 35S promoter and is responsible for the
expression in seedlings and in the roots [21,22]. Twelve CANNTG motifs, which are known to
bind proteins belonging to the superfamily of the helix-loop-helix bHLH transcriptional regulators
[23,24] were found in promoter regions I, III and IV. The significance of all these boxes has to be
established experimentally.
3
.
2
. Histochemical localisation of ppyk
20
promoter acti6ity Seven different 5- and 3-deletions of ppyk20
were made by PCR. The resulting constructs are shown in Fig. 2. Each of these constructs contains
several regions of the ppyk20 fused to the uidA reporter gene. For each construct more than 25
independent transformed lines were tested for GUS expression. The variability of gus activity
between individual lines was evaluated optically and no obvious differences in the quality and
quantity of GUS expression could be observed between the lines. Detailed analyses were per-
formed with one selected line per construct. The GUS assay was performed at 2, 5, 14 and 21 days
after germination dag.
At 2 dag, seedlings of all lines A – G showed GUS expression in cotyledons and in hypocotyl
Fig. 3A. In seedlings of the lines A, B, C, D and E the expression was detected in the entire
hypocotyl, whereas in line F and G only the upper part of the hypocotyl near the apical meristem
showed staining. In the hypocotyls A – E GUS was expressed in cortical and the vascular tissues
but not in epidermal cells. GUS staining also appeared in the root tip in seedlings of line A, B,
C and D Fig. 3A.
At 5 dag, plants lines A, B, C, D, E and F showed GUS expression in the vascular tissue of
root. Moreover, seedlings of lines A, B, C and D expressed GUS in the root tip Fig. 3B. Line G
showed no expression in the root. In all lines, staining was observed in the vascular tissue of
leaves, in mesophyll cells of cotyledons and in apical meristem Fig. 3C. In the hypocotyl, stain-
ing was quite strong in the vascular tissue.
At 14 dag, all lines stained at different intensity in the mesophyll, vascular tissue and epidermal
cell layer of leaves. Lines D and G showed only very faint expression in tissues of younger shoots
and leaf primordia. In addition, we observed GUS expression in young shoot buds in plant line A
Fig. 3D. GUS was also seen in hypocotyl of different intensities in all tested constructs. In
roots of the A, B, C, D, E and F plants GUS staining was detected in vascular tissue and in root
tips. In plants of line A the staining was seen also in lateral root formation tissue. Plants with con-
struct G expressed no GUS in the roots.
GUS expression in flowers was examined at several developmental stages, from developing
buds in which corolla was not yet visible, through several intermediate stages up to mature open
flowers. However, only plants containing con- structs A, B, C and D exhibited a characteristic
pattern of GUS staining in the stamina between two pollen sacks Fig. 3E, in stigmatic tissue of
gynoecium Fig. 3F and faint in the base and top
Fig. 2. Promoter deletion constructs. Deletions were made using PCR as described in Methods. I – VI indicate the promoter regions. Numbers indicate the length in bp of the respective promoter regions. TSP-transcription start point. A – G — promoter
deletion constructs and corresponding transgenic lines.
Fig. 3. Histochemical localisation of GUS activity in transgenic A. thaliana. A GUS expression in 2-day-old seedlings construct C. Bar = 4 mm. B GUS expression in vascular tissue and in root tip 5 days after germination dag construct C. Bar = 100
mm. C GUS expression in cotyledons 5 dag construct C. Bar = 1 mm. D GUS expression in young shoot buds of 14-day-old plants construct A. Bar = 250 mm. E GUS expression between two pollen sacks construct C. Bar = 10 mm. F GUS
expression in flowers construct C. Bar = 250 mm. G GUS expression in abscission zone of the fully elongated siliques construct C. Bar = 1 mm. H GUS expression in NFS, 7 days after inoculation with infective juveniles of H. schachtii construct C.
Bar = 100 mm. I GUS expression in leaf 5 h after wounding construct C. Bar = 1 mm.
of sepals Fig. 3F. In addition, GUS expression was also found in the abscission zone of the fully
elongated siliques Fig. 3G. The fluorometric GUS assay on protein extracts
from whole 21 days old transgenic A. thaliana plants is shown in Fig. 4A. The highest level of
GUS activity was observed in line C. However, no significant differences in the GUS activity could be
seen in lines A – D, whereas expression levels in lines E – G were significantly reduced.
To confirm these results the transcription of the pyk
20
gene in different plant tissues was moni-
tored by Northern blot analysis. Equal amounts of total-RNA
from silique,
flower, stem,
root, rosette, and leaf of the wild type A. thaliana were
probed with the pyk
20
cDNA clone. High levels of pyk
20
mRNA were found in the rosette and stem tissues Fig. 5.
3
.
3
. Histochemical localisation of ppyk
20
acti6ity after nematode infection
The number of GUS expressing NFS nematode feeding structures was scored 2, 4, 7 and 12 days
after inoculation dai with J
2
juveniles of H. schachtii. The highest number of GUS expressing
NFS was observed 7 dag in all lines Table 2 Fig. 3H. Cross sections of the 7-day-old NFS showed
strong GUS staining inside the NFS. From all tested lines the highest percentage of GUS positive
NFS was detected in plants harbouring construct C Table 2.
After nematode infection, no changes in the GUS expression was observed in the histological
Fig. 4. Fluorometric GUS assay of the 21-day-old A. thaliana plants containing the seven different ppyk20::uidA constructs A – G. A Control plants after treatment with 2 ml water. B After treatment with 2 ml of 50 mM ABA solution. C After treatment
with 2 ml of 50 mM IAA.
Fig. 5. Comparison of the expression of the pyk
20
gene in different organs of A. thaliana wildtype C-24. Total RNA 5 mg from silique lane 1, flowers lane 2, stem lane 3, roots lane 4, rosette lane 5 and leaves lane 6 was examined by gel blot analysis.
The gel blot was probed with the
32
P-labelled cDNA clone of the pyk
20
gene. Control hybridisation to standardize the amounts of RNA loaded were performed with an rDNA.
Table 2 Number of NFS showing GUS expression driven by the ppyk20 constructs
No. of GUS − Total number of NFS
Construct GUS + NFS
No. of GUS + NFS
a
NFS
b
17 A
93 76
81.7 19
99 80
80.8 B
692 C
153 835
81.9 1149
1853 D
38.0 704
62 234
172 73.5
E 320
F 219
539 59.4
78 G
176 98
55.7
a
Number of NFS expressed GUS.
b
Number of NFS without GUS expression.
and the fluorometric assay in whole plants. Al- though we did not quantify GUS expression in
NFS, in line G the GUS staining was obviously lower than in all other lines.
3
.
4
. Effect of wounding, IAA and ABA on ppyk
20
acti6ity In order to determine the effect of mechanical
wounding lines A – G were wounded with scissors and pipette tips. Only in lines A, B and C blue
staining was observed around the wounded leaf tissue 1 and 5 h after wounding Fig. 3I. In plants
containing other promoter::uidA constructs no re- sponse was observed. In order to confirm these
results, we also tested induction of pyk
20
gene by RT-PCR and Northern blots. Pyk
20
transcripts were detected 1 and 5 h after wounding using
RT-PCR Fig. 6 and Northern blot analysis. We demonstrated previously that transcription
of pyk
20
gene is specifically up-regulated by IAA and down-regulated by ABA treatment [1]. In
order to determine the ppyk20 regions responsible for IAA- and ABA-responsiveness, 21-day-old
plants of lines A – G were sprayed with 50 mM ABA or 50 mM IAA solutions. After 8 h in the case of
ABA treatment lines A – D showed down-regula- tion of the GUS expression Fig. 4B. Lines A – C
responded to IAA treatment with a clear up-regula- tion of GUS expression, while lines D – G did not
alter GUS levels compared to the control Fig. 4C.
4. Discussion
